GRADUATE SCHOOL APPROVAL RECORD

 

NORTHEASTERN UNIVERSITY

Graduate School of Arts and Sciences

 

Thesis Title:                  The Search for Partner Molecules Involved in Qa-2 Signaling

 

Author:             Kenneth E. Domino

 

Department:                  Biology

 

Approved for Thesis Requirements of the Master of Science Degree

 

Thesis Committee

 

 

___________________________________                                 ________________

Carol M. Warner, Chairperson                                                        Date

 

___________________________________                                 ________________

Wendy A. Smith                                                                              Date

 

___________________________________                                 ________________

Rebeca B. Rosengaus                                                                      Date

 

 

 

Head of Department

 

___________________________________                                 ________________

Susan Powers-Lee                                                                          Date

 

 

 

Graduate School Notified of Acceptance

 

___________________________________                                 ________________

Director of the Graduate School                                                      Date

 

 

 

Copy Deposited in Library

 

___________________________________                                 ________________

Signed                                                                                             Date


 

DEPARTMENTAL APPROVAL RECORD

 

NORTHEASTERN UNIVERSITY

Graduate School of Arts and Sciences

 

Thesis Title:                  The Search for Partner Molecules Involved in Qa-2 Signaling

 

Author:             Kenneth E. Domino

 

Department:                  Biology

 

Approved for Thesis Requirements of the Master of Science Degree

 

Thesis Committee

 

 

___________________________________                                 ________________

Carol M. Warner, Chairperson                                                        Date

 

___________________________________                                 ________________

Wendy A. Smith                                                                              Date

 

___________________________________                                 ________________

Rebeca B. Rosengaus                                                                      Date

 

 

 

Head of Department

 

___________________________________                                 ________________

Susan Powers-Lee                                                                          Date

 

 

 

Graduate School Notified of Acceptance

 

___________________________________                                 ________________

Director of the Graduate School                                                      Date


 

 

 

THE SEARCH FOR PARTNER MOLECULES INVOLVED IN QA-2 SIGNALING

 

 

A thesis presented

by

Kenneth E. Domino

to

The Department of Biology

in partial fulfillment of the requirements for the degree of

Master of Science

in the field of

Biology

 

Northeastern University

Boston, Massachusetts

April 2006


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ó 2006

Kenneth E. Domino

ALL RIGHTS RESERVED


 

 

 

THE SEARCH FOR PARTNER MOLECULES INVOLVED IN QA-2 SIGNALING

 

by

 

Kenneth E. Domino

 

 

 

 

 

 

ABSTRACT OF THESIS

 

Submitted in partial fulfillment of the requirements

for the degree of Master of Science in Biology

in the Graduate School of Arts and Sciences of

Northeastern University, April 2006


 

ABSTRACT

 

Qa-2 is an important protein implicated in the development of mouse embryos.  Qa-2 is attached to the outer leaflet of the cell membrane by a glycosylphosphatidylinositol (GPI) linkage.  Although Qa-2 does not contain a cytoplasmic tail, cross-linking Qa-2 protein with anti-Qa-2 antibody, anti-mouse IgG secondary antibody, in the presence of phorbol 12-myristate 13-acetate (PMA) causes T cell proliferation.  This study hypothesizes the existence of a partner molecule for Qa-2 that is membrane spanning, and whose function is to transduce signals internally after Qa-2 ligation.  To test this hypothesis, antibodies directed to a likely partner molecule, CD8, a dimer of either CD8aa or CD8ab, were added to Qa-2 positive cells, and proliferation measured in the presence and absence of all combinations of antibodies for Qa-2, CD3e, CD4, CD8a, mouse IgG secondary antibody, and PMA, in soluble and immobilized formats.  It was found that antibodies for CD3e, CD8a, and Qa-2 were synergistic in causing proliferation of T cells.  Antibodies for CD4 and Qa-2 were antagonistic on the proliferation of T cells in soluble format, but synergistic in immobilized format.  T cells from CD8a-negative mice proliferated using anti-Qa-2, secondary antibody, and PMA.  These data suggest that CD8 may be one, but not the only, partner molecule capable of binding to Qa-2 protein.  To test whether Qa-2 binds to the CD8aa or CD8ab dimer, predictive protein docking was employed.  The results of this study suggest that CD8aa and Qa-2 protein may bind, but that CD8ab and Qa-2 protein may not bind.

DEDICATION

 

To my wife, Zarina G. Memon, M.D., who encouraged and supported me in so many ways.  My desire to understand her profession in medicine and anesthesiology sparked my interest to pursue this degree after being a software engineer for 20 years.

To my father, Edward F. Domino, M.D., who was a model researcher for me.  Currently 81 years of age, he is a Professor Emeritus in Pharmacology at the University of Michigan, has published over 300 peer-reviewed articles, and continues to this day to write grants and publications.  He exemplifies a person who is blessed with love of life and of work.

To my mother Antoinette K. Domino, M.S.  She loved life, and with my father, raised five accomplished children.


 

ACKNOWLEGEMENTS

 

I would like to thank my advisor Dr. Carol M. Warner for the opportunity to do research in her laboratory.  Without her support and patience this thesis would not have been possible.  In addition, I would like to thank my thesis committee for their comments.  Finally, I would like to thank the faculty in the Department of Biology who encouraged my studies.

I would like to thank Dr. Sally De Fazio for her helpful comments on my research and thesis.  Her enthusiasm in her research was exemplary.

I also wish to thank the other members of Dr. Warner’s laboratory, including Mike Byrne, Martina Comiskey, Carmit Goldstein, Gary Laevsky, Paula Lampton, Michele Mammolenti, and Judy Newmark for their encouragement, and help with the methods used in this thesis.


 

TABLE OF CONTENTS

ABSTRACT.................................................................................................................. iii

DEDICATION.............................................................................................................. iv

ACKNOWLEGEMENTS.............................................................................................. v

TABLE OF CONTENTS.............................................................................................. vi

LIST OF ABBREVIATIONS........................................................................................ ix

LIST OF FIGURES...................................................................................................... xii

LIST OF TABLES....................................................................................................... xv

CHAPTER 1.  INTRODUCTION................................................................................. 1

Overview.................................................................................................................... 1

Class I MHC antigens................................................................................................. 2

The structure of the Qa-2 protein................................................................................ 4

The function of the Qa-2 protein.................................................................................. 9

Signal transduction by the Qa-2 protein..................................................................... 11

Hypotheses of Qa-2 signaling.................................................................................... 14

CD8......................................................................................................................... 15

The thesis hypothesis................................................................................................. 18

Testing the hypothesis............................................................................................... 19

CHAPTER 2.  MATERIALS AND METHODS.......................................................... 20

Mice......................................................................................................................... 20

Antibodies................................................................................................................ 20

Purification of antibody from ascites........................................................................... 20

Antibody concentration determination........................................................................ 21

Ficoll-Hypaque isolation............................................................................................ 21

T cell enrichment....................................................................................................... 22

Primary and secondary antibody reactivity................................................................. 22

Medium for T cell activation...................................................................................... 23

T cell activation using primary and secondary antibodies, and PMA............................ 23

T cell activation using immobilized antibodies............................................................. 24

MTT assay for proliferation....................................................................................... 25

Predictive protein docking......................................................................................... 25

CHAPTER 3.  RESULTS............................................................................................ 27

Establishing conditions for the activation of T lymphocytes.......................................... 27

Qa-2 is expressed on CD4 T cells less than on CD8 T cells....................................... 30

Qa-2 induced T cell proliferation using CD4 and CD8 cells........................................ 31

Co-cross-linking Qa-2 and CD8 protein................................................................... 31

Co-cross-linking Qa-2 and CD4 protein................................................................... 32

Co-cross-linking Qa-2 and CD8 protein in an ordered incubation.............................. 33

Co-cross-linking Qa-2 and CD3e protein................................................................. 34

Costimulation using immobilized antibodies................................................................ 34

Predictive protein docking for CD8 and Qa-2........................................................... 36

CHAPTER 4.  DISCUSSION..................................................................................... 38

What is the physical significance of antibody stimulation via antibody binding or cross-linking?            39

Evidence supporting the hypothesis............................................................................ 40

Evidence contradicting the hypothesis........................................................................ 43

Is Qa-2 behaving as a MHC I protein, or as a GPI-anchor protein during signal transduction?            45

Is Lck and/or Fyn involved in Qa-2 signaling?............................................................ 49

CHAPTER 5.  CONCLUSIONS................................................................................. 53

REFERENCES............................................................................................................ 54

FIGURES.................................................................................................................... 74

TABLES.................................................................................................................... 112

BIOGRAPHICAL DATA.......................................................................................... 142

 


 

LIST OF ABBREVIATIONS

 

AAALAC

American Association for the Accreditation of Laboratory Animal Care

APC

antigen-presenting cell

b2m

b2-microglobulin

BSA

bovine serum albumin

CDR

complementary-determining regions

CTL

cytotoxic T lymphocyte

DAF

decay-accelerating factor

DAG

Diacylglycerol

DIG

detergent-insoluble, glycolipid-enriched complexes

DMEM

Dulbecco’s Modified Eagles’s Medium

DMSO

dimethyl sulfoxide

DRM

detergent-resistant microdomains

FasL

Fas ligand

FCS

fetal calf serum

GDNFR-a

glial-cell-line-derived neurotrophic factor receptor

GEM

glycosphingolipid-enriched microdomains

GPI

glycosylphosphatidylinositol

H-2

histocompatibility-2 (mouse MHC)

HH

hereditary hemochromatosis

HLA

human leukocyte antigen (human MHC)

iIEL

intestinal intraepithelial lymphocytes

IPG

inositolphosphoglycan

ITAM

immunoreceptor tyrosine-based activation motif

kDa

kilodalton

LAT

linker for activation of T cells

LRC

leucocyte receptor complex

LPS

lipopolysaccharide

mAb

monoclonal antibody

MbCD

methyl-b-cyclodextran

MAPK

MAP kinase

MICL

membrane inhibitor of complement lysis

MHC

major histocompatibility complex

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NK

natural killer

NKC

natural killer complex

NTNR-a

neurturin receptor

OD

optical density

PBS

phosphate buffered saline

PBSAZ

phosphate buffered saline and 1% BSA and 0.1% NaN3

PDGF

platelet-derived growth factor

Ped

preimplantation embryo development (gene)

PI3K

phosphatidylinositol 3-kinase

PI-PLC

phosphatidylinositol-specific phospholipase C

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

PNH

paroxysmal nocturnal hemoglobinuria

SPR

surface plasmon resonance

TCR

T cell receptor

TAP

transporter for antigen processing

TL

thymus-leukemia antigen

TX-100

Triton X-100

 

 


LIST OF FIGURES

 

Figure 1.  Classical MHC Ia antigen processing. 75

Figure 2.  GPI-anchored Qa-2 and MHC class Ia protein on membrane. 76

Figure 3.  Ribbon representation of Qa-2 and H-2Kb proteins. 77

Figure 4.  Intron and Exon structure of Q6, Q7, Q8, and Q9 genes. 78

Figure 5.  Alternatively spliced transcripts of the Q7 and Q9 genes. 79

Figure 6.  Structure of the GPI anchor. 80

Figure 7.  TCR, CD3, CD4, and CD8 on membrane. 81

Figure 8.  Signal transduction via CD8aa bound to MHC class I. 82

Figure 9.  Reactivity of anti-Qa-2 (1-9-9) primary mAb and FITC-conjugated anti-mouse IgG secondary Ab with T lymphocytes. 83

Figure 10.  Reactivity of anti-Qa-2 (1-1-2) primary mAb and FITC-conjugated anti-mouse IgG secondary Ab with T lymphocytes. 84

Figure 11.  Reactivity of anti-Qa-2 (Qa-m2) primary mAb and FITC-conjugated anti-mouse IgG secondary Ab with T lymphocytes. 85

Figure 12.  Reactivity of anti-CD8a (53.6-7) primary mAb and FITC-conjugated anti-mouse IgG secondary Ab with T lymphocytes. 86

Figure 13.  Reactivity of anti-CD4 (GK1.5) primary mAb and FITC-conjugated anti-mouse IgG secondary Ab with T lymphocytes. 87

Figure 14.  Reactivity of anti-CD3e (145-2C11) primary mAb and FITC-conjugated anti-mouse IgG secondary Ab with T lymphocytes. 88

Figure 15.  Optimization of time of MTT pulse and time of measurement for MTT assay using T lymphocytes stimulated by anti-Qa-2 (1-9-9) primary antibody, anti-mouse IgG secondary antibody, and PMA. 89

Figure 16.  Dependence of anti-Qa-2 (1-9-9) mAb induced proliferation on the concentration of anti-Qa-2 primary antibody and anti-mouse immunoglobulin secondary antibody. 90

Figure 17.  Qa-2 expression on CD4+ and CD8+ T lymphocytes of C57BL/6 mice. 91

Figure 18.  Qa-2 expression on CD4+ and CD8+ T lymphocytes of identical size of B6.K2 mice. 92

Figure 19.  Dependence of anti-Qa-2 (1-9-9) mAb induced proliferation of CD8+ T cells upon the concentration of anti-Qa-2 primary antibody and anti-mouse immunoglobulin secondary antibody. 93

Figure 20.  Proliferation of T cells from CD8a -/- mice using Qa-2 cross-linking and PMA. 94

Figure 21. Effect of co-cross-linked anti-Qa-2 (1-9-9) and anti-CD8α (53.6-7) primary antibodies with PMA on proliferation of T cells. 95

Figure 22.  Antagonism of cross-linked anti-CD4 (GK1.5) and anti-Qa-2 (1-9-9) primary antibodies on proliferation of T cells. 96

Figure 23.  Dependence of the order of exposure to primary antibody on the synergy of cross-linked anti-Qa-2 (1-9-9) and anti-CD8α (53.6-7) primary antibodies on proliferation of T cells. 97

Figure 24.  Synergy of cross-linked anti-CD3e (145-2C11) and anti-Qa-2 (1-9-9) primary antibodies on proliferation of T cells. 98

Figure 25.  Synergy of immobilized anti-Qa-2 (1-9-9) and anti-CD3ε (145-2C11) antibodies on T cell proliferation. 99

Figure 26.  Synergy of immobilized anti-Qa-2 (Qa-m2) and anti-CD3ε (145-2C11) antibodies on T cell proliferation. 100

Figure 27.  Synergy of immobilized anti-Qa-2 (1-1-2) and anti-CD3ε (145-2C11) antibodies on T cell proliferation. 101

Figure 28.  Synergy of immobilized anti-CD8α (53.6-7) and anti-CD3ε (145-2C11) antibodies on T cell proliferation. 102

Figure 29.  Synergy of immobilized anti-Qa-2 (1-9-9), anti-CD8a (53.6-7), and anti-CD3ε (145-2C11) antibodies on T cell proliferation. 103

Figure 30.  Synergy of immobilized anti-Qa-2 (1-9-9), anti-CD4 (GK1.5), and anti-CD3ε (145-2C11) antibodies on T cell proliferation. 104

Figure 31.  Predictive protein docking for Qa-2 and CD8aa. 105

Figure 32.  Predictive protein docking for H-2Kb and CD8aa. 106

Figure 33.  Predictive protein docking for Qa-2 and CD8ab. 107

Figure 34.  Costimulation of T cells using primary antibodies, secondary antibody, and PMA. 108

Figure 35.  Costimulation of T cells using immobilized antibodies. 109

Figure 36.  Model of signal transduction via co-cross-linking of CD8aa and Qa-2 protein. 110

Figure 37.  Model of signal transduction via co-cross-linking of CD4 and Qa-2 protein. 111

 


LIST OF TABLES

Table 1.  MHC class I and associated proteins. 113

Table 2.  Summary of the antibodies used in this study. 117

Table 3.  Studies associated with signaling of MHC class. 118

Table 4.  Some GPI molecules involved in signal transduction. 128

Table 5.  Studies associated with signaling of the Thy-1 GPI-anchored protein. 132

 


 

CHAPTER 1.  INTRODUCTION

Overview

Qa-2 is a protein involved in the development of mouse embryos.  The presence of Qa-2 contributes to faster development of the embryo, higher survival rate of pups to term, and higher birth weight (Warner et al. 1991; Warner et al. 1993).  Phenotype differences due to the presence of Qa-2 extend later into the life of the mouse.  Qa-2-negative mice become heavier, develop elevated systolic blood pressure, and females have a larger lung to body mass ratio than in Qa-2-positive mice (Watkins et al. 2006).  It is unknown how Qa-2 protein confers these pleiotropic effects.  In the case of faster embryo development, one possible explanation is that Qa-2, a glycosylphosphatidylinositol-anchored protein, transmits a signal to the embryonic cell to increase the rate of cleavage (McElhinny and Warner 2000).  Although Qa-2 does not contain a cytoplasmic tail, cross-linking Qa-2 protein with anti-Qa-2 antibody and anti-mouse IgG secondary antibody, in the presence of phorbol 12-myristate 13-acetate (PMA), causes embryos to develop faster.  McElhinny and Warner (2000) hypothesized the existence of a partner molecule for Qa-2 protein that is membrane spanning, and whose function would be to transduce signals internally after ligation of Qa-2 protein.  In this thesis, several possible partner molecules were tested.  CD8aa, a likely partner molecule, was evaluated by co-cross-linking antibodies for Qa-2 and CD8a on resting T cells.  Proliferation was measured in the presence and absence of all combinations of primary mouse IgG antibodies for Qa-2, CD3e, CD4, CD8a, secondary antibody directed to mouse IgG, and PMA.  Both soluble and immobilized formats were used.  A second test of the hypothesis that CD8aa binds to Qa-2 employed predictive protein docking of the known x-ray crystallographic structures of CD8aa and Qa-2 to evaluate the best model of CD8aa and Qa-2 binding.

Chapter 1 of this thesis introduces the major histocompatibility complex (MHC) class I proteins, Qa-2 structure and function, previous studies of the effects of cross-linking Qa-2, hypotheses of Qa-2 signaling, and the hypothesis of this thesis.  Chapter 2 presents the methods used, including T cell enrichment, primary and secondary antibody reactivity, soluble format T cell activation using primary and cross-linking secondary antibodies, immobilized format T cell activation using antibodies, the MTT assay, and the protein docking analysis.  Chapter 3 presents the results of the experiments, some of which are corroborative, and some of which refute the hypothesis.  Chapter 4 presents a discussion of the results, and relates these results to other studies on signal transduction using cross-linking antibodies for other GPI-anchored and MHC I proteins.  Finally, Chapter 5 concludes this thesis, and provides recommendations for further research.

Class I MHC antigens

            The major histocompatibility complex (MHC) class I proteins are associated with immune and non-immune functions.  These proteins are classified into the classical MHC Ia and the non-classical MHC Ib.  The classical MHC Ia proteins function in the adaptive and innate immune systems (Natarajan et al. 1999).  These proteins are polymorphic, part of the immunoglobulin-super family, membrane bound, widely expressed in different types of cells, and consist of trimers of a heavy chain of 44 kDa, the non-covalently associated b2-microglobulin (b2m) protein of 12 kDa, and a non-covalently associated peptide of 8-10 amino acids.

In the adaptive immune system, peptides are presented by classical MHC Ia proteins of an antigen-presenting cell (APC) to cytotoxic T lymphocytes (CTL) (Figure 1).  During an infection, viral and bacterial protein present in an infected cell are processed into peptides, bound to MHC Ia protein, and presented on the surface of the cell.  Once on the surface, peptide-bound MHC Ia heterodimers bind to the CD8 and ab T cell receptor (TCR) proteins of the CTL, causing CTLs to express perforin, granzyme B, and Fas ligand (FasL), which results in the destruction of the infected cell.  The polymorphisms of the classical MHC Ia molecules are concentrated in the peptide-binding groove in the heavy chain, which allows for a wide variety of viral peptides to be displayed on the surface of the infected cell.

In the innate immune system, non-specific peptides are presented to natural killer (NK) cells.  According to the “missing-self” hypothesis, the absence of MHC Ia protein on a target cell causes the NK cell to kill the target cell with perforin, granzyme B, and FasL (Karre et al. 1986; Ljunggren and Karre 1990).  Classical MHC Ia proteins react with NK receptors to inhibit the expression of perforin, granzyme B, and FasL.  NK receptors contain extracellular binding domains of C-type lectins, coded on the natural killer complex (NKC) (Yokoyama and Plougastel 2003), or immunoglobulin domains, encoded on the leucocyte receptor complex (LRC) (Martin et al. 2002; reviewed in Parham 2005).

In comparison, the non-classical MHC Ib proteins function in the innate immune system, and/or non-immune functions, and have no known function in the adaptive immune system.  These proteins are oligomorphic, expressed in low amounts and restricted to certain tissues and cell types.  The low polymorphism in the non-classical MHC Ib proteins could be due to their association with ligands that are limited in genetic variability (Braud et al. 1999).  Although similar to classical MHC Ia proteins in structure, non-classical MHC Ib proteins may not bind to b2-microglobulin, TCR, NK receptors, and/or CD8.  For example, HFE is a non-classical MHC Ib protein associated with iron transport, and its absence or mutation causes hereditary hemochromatosis (HH), a disease of iron overloading (Gaulton and Pratt 1994).  HFE binds to the transferrin receptor (the protein involved in the transport of iron), b2-microglobulin, and the TCR (Yewdell and Hickman-Miller 2005).  However, HFE is incapable of binding peptides for presentation, and CD8 (Rohrlich et al. 2005).  Of particular interest in this thesis is Qa-2 protein, a non-classical MHC Ib protein with both immune and non-immune function.

The structure of the Qa-2 protein

Qa-2 protein is oligomorphic and widely expressed, but in low amounts on most tissues (Ungchusri et al. 2001).  Qa-2 protein is a trimer of a heavy chain of 40-kDa, a non-covalently associated b2-microglobulin protein of 12-kDa, and peptide (Figure 2).  The long chain has three domains, a1 (distal to membrane), a2, and a3 (proximal to membrane), each approximately 90 amino acids in length.  The a1 and a2 domains form a structure with two a-helices spanning a b-pleated sheet.  The a3 and b2-microglobulin each contain two b-sheets connected by a disulfide bond between two cysteines and a packing tryptophan.  The a1 and a2 domains form a structure which binds a diverse set of nonameric peptides with a histidine at position 7 and a hydrophobic amino acid at position 9 (Joyce et al. 1994).

Qa-2 is similar in tertiary structure to the classical MHC Ia proteins, and in particular H-2Kb, a MHC Ia protein (He et al. 2001) (Figure 3).  However, the structure of Qa-2 differs with classical MHC Ia proteins in several ways.  First, peptides in the cleft of Qa-2 form a compact bulge in the central five residues of the peptide, whereas in classical MHC Ia proteins, the bulge is located near the C-terminal of the peptide (He et al. 2001).  Second, Qa-2 undergoes a number of unique post-translational modifications.  Qa-2 protein has N-linked glycosylations in the a1 and a3 domains, and has a glycosylphosphatidylinositol moiety for attachment to the cell membrane (Edidin and Stroynowski 1991).  In classical MHC Ia proteins, N-linked glycosylation occurs in all three a domains, and they have a transmembrane domain.  The locations of the N-linked glycosylations are hypothesized to be oriented between the domains of the MHC I protein and the cell membrane (Mitra et al. 2004).  Finally, although Qa-2 binds a nonameric peptide, b2-microglobulin, and the TCR, Qa-2 binds to an unknown NK receptor.  It is unknown whether Qa-2 binds CD8aa or CD8ab.

 

Qa-2 protein is the product of four genes, Q6, Q7, Q8, and Q9, all located in the Q region of the MHC on chromosome 17 of Qa-2-positive mice, such as C57BL/6 and C57BL/10.  Although all Qa-2 genes are transcribed in lymphocytes, only the Q7 and Q9 genes are expressed by preimplantation embryos (Cai et al. 1996; Wu et al. 1998).  Of the approximately 1012 bases or 334 amino acid residues comprising the first five exons for each the genes, the Q7 and Q9 genes differ by two base pairs, corresponding to one amino acid difference (Cai et al. 1996).  The Q6 and Q8 genes differ from each other in two base pairs, corresponding to two amino acids.  The Q7 and Q9 genes differ from the Q6 and Q8 genes by 60 bases, or 50 residues.  The significance of these differences is unknown, but all four proteins encoded by the four genes are called Qa-2 antigen.  The Qa-2 protein used for the x-ray crystallographic structure shown in Figure 3 is the product of the Q9 gene.

All Qa-2 genes are composed of eight exons (Figure 4; Cai et al. 1996).  Exon 1 encodes a leader peptide, used to traffic the protein to the cell membrane.  Exons 2, 3, and 4 encode the a1, a2, and a3 domains of the protein.  Exon 5 encodes the membrane portion, which for Q7 and Q9 is the GPI-anchor.  Exons 6, 7, and 8 encode cytoplasmic portions of Q6 and Q8.  Exon 7 is absent in all transcripts for Q7 and Q9 due to a mutation in a splice site before the exon (Stroynowski and Tabaczewski 1996).  In these genes, the AG dinucleotide, present immediately 5' to the beginning of an exon, is not present 5' of the region (Devlin et al. 1985).

Qa-2 protein is expressed in three isoforms based on alternative splicing (Figure 5; Stroynowski and Tabaczewski 1996), although there is some evidence that other isoforms may also exist (Warner and Goldstein, unpublished).   Isoforms of the Q7 or Q9 genes include the GPI-anchored protein, a soluble, secreted form of 39 kDa (splice S1), and a soluble, secreted form of 25 kDa (splice S2).  Both GPI-anchored and S1 soluble forms bind non-covalently to b2-microglobulin, whereas the soluble S2 form does not bind b2-microglobulin.  Soluble S1 and S2 Qa-2 isoforms are expressed upon T cell activation (Ulker et al. 1990).  The S1 Qa-2 isoform is expressed in the mouse embryo (Comiskey et al. 2003), but the existence of mRNA for other isoforms in mouse embryos is still not certain.

As stated previously, a distinguishing structural feature of Qa-2 protein is the GPI anchorage (Figure 6).  The GPI anchor inserts in the outer leaflet of the cell membrane, and does not extend into the inner leaflet.  The anchor is very flexible and is hypothesized to allow a GPI-anchored protein to lie on its side (reviewed by Sharom and Radeva 2004).  The GPI moiety is attached to the protein during processing in the Golgi by GPI-processing proteins (reviewed by Eisenhaber et al. 2003).  Exon 5 of the Q7 and Q9 genes encodes a conformational epitope that is recognized by GPI-processing proteins, but the sequence pattern is currently not well understood.  Two amino acids found after the point of cleavage are combinations of Ala, Asn, Asp, Gly, and Ser.  In some cases, 10-12 residues after the point of cleavage, there appears to be sequence of hydrophobic residues.  In the case of Qa-2, it is known that a single change in residue Asp after three Val to a Val causes the GPI-processing enzymes to fail, and the protein to be expressed with a transmembrane domain (Ulker et al. 1990).

The GPI tail is important for the expression of the protein on the cell membrane.  Failure of the GPI-processing enzymes can cause disease.  For example, CD59, a GPI-anchored protein, and known as membrane inhibitor of complement lysis (MICL), is an important protein in cells preventing cell lysis.  Failure to express CD59 causes enhanced sensitivity to complement-mediated cell destruction, and the disease paroxysmal nocturnal hemoglobinuria (PNH) (reviewed by Meletis and Terpos 2003).  However, the absence of a GPI-anchored protein does not necessarily result in a disease, for example, as in the case of Qa-2 protein.

Qa-2 is expressed as a GPI-anchored protein in the mouse embryo, but it can also be a transmembrane protein in lymphocytes (Wu et al. 1998).  The treatment of cells with PI-PLC has shown that most Qa-2 expressed on the surface of the cell is cleaved with PI-PLC, with only a small population of Qa-2 positive cells that are resistant to PI-PLC (Mann and Forman 1988; Tian et al. 1992; Stiernberg et al. 1987).  Interestingly, Qa-2 protein on CD8 T cells is resistant to PI-PLC while Qa-2 protein on CD4 T cells is sensitive to PI-PLC (Soloski et al. 1988).  It is unknown why this is so.

An important property of GPI-anchored proteins is their localization to membrane domains known as lipid rafts.  The outer leaflet of rafts contain high concentrations of sphingomyelin, glycosphingolipids, and cholesterol.  However, the composition of the inner leaflet is not known.  As noted, rafts contain GPI-anchored proteins, e.g. Qa-2, but also contain other proteins, including caveolin, G-proteins, Src family kinases, Grb2, Shc, MAP kinase (MAPK), protein kinase C (PKC), the p85 subunit of PI 3-kinase, TRAPs, CD4, and CD8.  Some GPI-linked proteins of T cells include: CD48, CD52, CD55, CD59, CD90 (Thy-1), CD108, CD230 (prion protein), and Ly-6 (Horejsi 2005).  Caveolae, defined as rafts with caveolin, are cave-like structures in the membrane.

As noted previously, rafts contain cholesterol that associates with the sphigolipids, causing the lipid layer to pack into a liquid-ordered phase.  Methyl-beta-cyclodextrin (MbCD), a specific cholesterol-binding agent, disrupts this ordering (Ilangumaran and Hoessli 1998).  In contrast, acyl chains of the lipids outside the raft do not pack as well, and exist in a liquid-disordered phase.  This separation contributes to the differential solubility of the membrane with cold non-ionic detergents such as Triton X-100 (TX-100), which is how rafts were first defined (Simons and van Meer 1988; Simons and Ikonen 1997).  Rafts have low buoyant density, and thus can be separated from the bulk proteins and lipids of the rest of the membrane using equilibrium gradient centrifugation (Brown and Rose 1992).  Estimates on the size of lipid rafts are in the range of 100 nm to 200 nm diameter.  The most important characteristic concerning rafts is that they are known to contain signaling proteins, including Qa-2.

The function of the Qa-2 protein

Relatively little is known about the role of Qa-2.  Two possible functions have been identified.  First, Qa-2 may function as a restriction element for CTL cells in tumors (Ungchusri et al. 2001; Chiang et al. 2002; Chiang et al. 2003; Chiang and Stroynowski 2004; Chiang and Stroynowski 2005).   Tumor peptides extend in a different manner from the binding cleft than self-peptides.  This difference may result in a preference for tumor peptides in presentation by the Qa-2 protein.

A second possible function of Qa-2 protein may be for better survival of pups due to the rapid embryo development phenotype.  Warner and collegues were the first to describe a gene, Ped (preimplnataion embryo development), that regulates the rate of preimplantation embryonic cleavage divisions (Verbanac and Warner 1981).  Early studies associated the Ped gene with the H-2 region of the mouse (Goldbard et al. 1982; Goldbard and Warner 1982; Warner and Spannaus 1984; Goldbard et al. 1985; Warner et al. 1985), and later narrowed the responsible genotype to the Q region of the H-2 complex (Warner 1986).  Further studies suggested that Qa-2 protein, encoded in the Q region, was the protein product of the Ped gene.  Increased levels of expression of the Qa-2 protein was found to be correlated with more rapid embryo development (Warner et al. 1987).  Rapid embryo development was shown to not depend on the maternal uterine environment (Brownell and Warner 1988).  Pups of the B6.K2 mouse strain, which differs only in the Q region from the B6.K1 mouse strain, were found to be heavier at birth then B6.K1, suggesting a reproductive advantage.  Although there are four genes that encode Qa-2, only the GPI-anchored forms coded by the Q7 and Q9 genes were found to be responsible for the phenotype (Tian et al. 1992; Xu et al. 1993; Xu et al. 1994; Cai et al. 1996).  Reverse transcription-polymerase chain reaction (RT-PCR) was used to show that embryos expressed only the mRNA of the Q7 and Q9 genes, with no detectable mRNA found for the Q6 and Q8 genes (Wu et al. 1998).  Transgenic embryos containing the Q7 and Q9 genes derived from Qa-2-negative mice were found to display fast embryo development, confirming earlier studies (Wu et al. 1999).  Although it is unknown how Qa-2 increases embryo development, cross-linking Qa-2 protein on embryos and splenic T cells in the presence of PMA induced faster embryo development and T cell proliferation (McElhinny and Warner 2000).

Signal transduction by the Qa-2 protein

Several studies suggest that Qa-2 positive embryos develop faster due to the transduction of a signal via Qa-2.  Mouse strains without Qa-2 protein, such as B6.K1, do not develop as quickly during the preimplantation period as those with Qa-2 protein, such as B6.K2, C57BL/6, and C57BL/10 (Warner et al. 1993).  When anti-Qa-2 monoclonal antibody is cross-linked with a secondary antibody, the embryos cleave more quickly (McElhinny and Warner 2000).  If Qa-2 is enzymatically removed from the embryonic cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), the embryo cleaves more slowly (Tian et al. 1992).  When the gene for Qa-2 is inserted into Qa-2 deficient mice, the embryos cleave more quickly (Xu et al. 1994).  If Qa-2 is "painted" onto the surface of a mouse strain without the gene, it develops more quickly (McElhinny et al. 2000).   Another reason to suspect that Qa-2 is involved in signal transduction is that GPI anchor proteins transduce a signal when cross-linked (Robinson and Hederer 1994).

The significance of the GPI anchor in signal transductions has been investigated for many years.  Robinson et al. (1989) demonstrated the importance of the GPI tail using synthetic DNA contructs of Qa-2 (Q7 and Q9) and other classical MHC Ia proteins (H-2Ld and H-2Db) to produce chimeric proteins.  Robinson noted that T cells containing modified Qa-2 protein with a transmembrane domain did not undergo proliferation after cross-linking Qa-2.  Other studies have shown similar results, including DAF (Shenoy-Scaria et al. 1992) and CD14 (Pugin et al. 1998).  In particular, Pugin et al. (1998) showed that a transmembrane molecular hybrid of CD14, a receptor on myeloid cells for microbial pathogens, would not localize to rafts and cross-linking the hybrid molecule did not induce Ca+2 mobilization, suggesting that the GPI anchor is essential for signal transduction.  However, other studies suggest that the GPI anchor may not always be required for signal transduction.  Cross-linked T cells with a transmembrane form of the GPI-anchored protein CD73 (Ecto-5’-nucleotidase) induced IL-2 production (Resta et al. 1994).  Cross-linking a MHC Ia protein, which does not have a GPI-tail, induces IL-2 production (Geppert et al. 1989).

The effect of antibody mediated cross-linking of Qa-2 was first reported by Hahn and Soloski (1989) and Robinson et al. (1989).  Hahn and Soloski (1989) found cross-linked Qa-2 induced Ca+2 mobilization, and in the presence of a second signal, PMA, proliferation.  Optimal proliferation, using mouse anti-Qa-2 first antibody, anti-mouse IgG second antibody, in the presence of PMA, was dependent on the concentrations of all three components.  These experiments also indicated a difference in proliferation which depended upon the specificity of the Qa-2 antibody used.  They found greater proliferation with anti-Qa-2 antibody specific for the a3 domain compared to anti-Qa-2 antibody specific for the a1 and/or a2 domains.  Following up on these experiments, Hahn et al. (1992) found that antibodies to Qa-2 α1, α2, or α3 domains have an additive effect on proliferation and Ca+2 release when used in conjunction with sub-optimal doses of anti-CD3 antibody.

Based on the work of Hahn and Soloski (1989), Robinson et al. (1989), and Hahn et al. (1992), others have also studied the effect of cross-linking Qa-2 on lymphocytes using various antibodies to Qa-2 and co-stimulatory agents.  Cook et al. (1992) found that the Fc portion of the antibody is not needed for proliferation following cross-linking with anti-Qa-2.  They also found that ionomycin, IL-2, and IL-4 could augment Qa-2-induced proliferation, but not as strongly as co-stimulation using PMA.  Purified B cells could be stimulated via cross-linking Qa-2, but not to the same extent as T cells, presumably because B-cells display less Qa-2 on their cell surface compared to T cells.

Only one published study has attempted to identify the signaling pathways of Qa-2 induced signaling.  Robinson and Hederer (1994) found a rapid rise in protein tyrosine phosphorylation after cross-linking Qa-2, including an unidentified, non-glycosylated, 110-kDa protein.

Recently, researchers used Qa-2 as a tool to investigate the structure of the cell membrane (Suzuki et al. 2000; Suzuki and Sheetz 2001).  Using laser tweezers surface scanning resistance, beads coated with anti-Qa-2 exhibited resistance to movement at a much greater level and in a unique manner not displayed for phosphatidylethanolamine linked proteins.  Because Qa-2 does not span the membrane, their study suggests that Qa-2 may bind to an unknown transmembrane protein.

Hypotheses of Qa-2 signaling

All GPI-anchored proteins transduce a signal when cross-linked.  In the presence of PMA, cross-linking GPI-anchored proteins induce the proliferation of T cells.  However, the mechanism by which GPI-anchored proteins transduce a signal is unknown because the GPI-anchor does not extend through the plasma membrane.  Several hypotheses have been proposed to explain this phenomenum (reviewed by Horejsi 2005).

The first hypothesis of signal transduction states that the GPI-anchored protein is physically associated with a transmembrane “partner” protein which has signaling capabilities (Robinson 1991).  When the GPI-anchored protein is cross-linked, a signal is transduced by the associated transmembrane molecule, either by a change in configuration of the transmembrane protein, self-phophorylation, or the colocalization of trapped signaling molecules.  This hypothesis is supported with evidence that shows the association of the GPI-anchored protein and a transmembrane protein known to be involved in signaling.  For example, Thy-1 was found to associate with several transmembrane molecules, including the TCR/CD3 (Gunter et al. 1987), and a 100 kDa transmembrane protein (p100) using co-precipitation on CD4 T cells (Lehuen et al. 1992; Lehuen et al. 1995).  Two other similar examples are: (1) the GPI-anchored proteins glial-cell-line-derived neurotrophic factor receptor (GDNFR-a) and neurturin receptor (NTNR-a), which were found to associate with the transmembrane protein Ret (Klein et al. 1997); (2) the GPI-anchored proteins LPS receptor (CD14), FcgRIIIB (CD16b), and the urokinase-type plaminogen activator receptor (CD87), which were found to associate with b2 integrin CR3 (reviewed in Petty and Todd 1996).

            A second hypothesis of signal transduction states that the signal is generated via a GPI-anchor-derived secondary messenger such as inositolphosphoglycan (IPG) or diacylglycerol (DAG), produced via a phospholipase (Gaulton and Pratt 1994; Malek et al. 1994).  However, there has been no evidence to support this hypothesis to date, and it has been generally discounted.

A third hypothesis of signal transduction states that the GPI-anchor itself associates with signaling components within the lipid raft (Harder et al. 1998).   Src PTKs are known to aggregate in lipid rafts, and GPI-anchored proteins are functional only when they localize to the lipid rafts (van den Berg et al. 1995).  As mentioned previously, transmembrane constructions of GPI-anchored Qa-2 require the GPI linkage in order to signal (Robinson et al. 1989).  Rafts subjected to methyl-b-cyclodextran (MbCD) lose GPI-anchored proteins from the plasma membrane and do not signal (Ilangumaran and Hoessli 1998).  These studies suggest that the GPI-anchor directly interacts with Src PTKs.

CD8

CD8 is a transmembrane glycoprotein coreceptor for antigen recognition of the peptide/MHC complex with the T cell receptor (TCR).  CD8 appears to function in two roles, to stabilize the binding of the TCR to MHC I and in signaling by recruiting p56lck (Lck)  (Paul 2003).  CD8 exists as a homodimer (aa) or heterodimer (ab) in the mouse and the human, and additionally as a homodimer of the b-chain in the human (Devine et al. 2000).  A diagram of the structures of CD8aa and CD8ab is shown in Figure 7.

The structure of the human and mouse CD8aa is known from X-ray crystallography (Leahy et al. 1992 and Kern et al. 1998).  More recently, the X-ray crystallographic structure of CD8ab been discovered (Chang et al. 2005).  CD8a of the mouse contains a 30 amino acid cytoplasmic domain, a 21 amino acid transmembrane domain, a 40 amino acid stalk, and an Ig-like V-type domain.  CD8a binds to the tyrosine kinase Lck via a Zinc clasp structure (Kim et al. 2003).  CD8b of the mouse contains a 30 amino acid stalk, an Ig-like V-type domain, and a 19 amino acid cytoplasmic tail (Merry et al. 2003).  The two molecules are covalently bound together in two places in the stalk region (Leahy et al. 1992).

Both CD8aa and CD8ab are known to bind to the a2 and a3 regions of MHC class Ia and b2m, with various affinities (Gao and Jakobsen 2000).   X-ray crystallographic data for CD8aa and two MHC class Ia proteins has been found, specifically H-2Kb (Kern et al. 1998) and HLA-A2 (Gao et al. 1997).  The X-ray crystallographic data for CD8aa and TL has also been found (Liu et al. 2003).  These structures show the binding of residues of the complementary-determining region (CDR) of CD8aa with residues the a3 domain of the MHC class I protein.  Other residues of CD8aa interacting with residues in the a2 domain of the MHC class I protein and b2m to provide additional stability of the structure. However, there is no X-ray crystallographic data for CD8ab and any MHC class I proteins together.  It is believed that CD8ab binds to the MHC class I protein in a similar manner as CD8aa binds to the MHC class I protein (Gao et al. 1997; Devine et al. 1999; Kern et al. 1999; Chang et al. 2005).

The purpose of the two dimers (aa and ab) is currently unknown, but there is evidence that they relate to different lines of differentiation.  The cytoplasmic domain of CD8b is important in the maturation of CD8 T cells.  In mice lacking the CD8b cytoplasmic tail, there is a 2-fold reduction in mature CD8 T cells, and if lacking the entire CD8b gene, there is a 5-fold reduction in mature CD8 T cells (Itano et al. 1994).  CD8ab has been shown to preferentially localize to lipid raft domains, while CD8aa appears to be preferentially excluded (Harder 2004).  CD8aa is related to the development of CD8 memory T cells.  Madakamutil et al. (2004) have shown that CD8aa and thymus-leukemia antigen (TL), a MHC Ib molecule which binds to CD8aa, are induced upon antigenic stimulation.  Thymocytes which express self-reactive T cell receptors normally undergo negative selection.  However, Yamagata et al. (2004) have shown that some cells which express self-reactive T cell receptors can undergo positive selection, and can differentiate into CD8aa T cells.  These cells show rapid effector cytokine response.

CD8aa and CD8ab may not bind some MHC class I proteins.  For example, HFE does not bind to CD8aa nor CD8ab (Bennett et al. 2000; Drakesmith and Townsend 2000).   A summary of the binding properties of MHC class I proteins, including their bind to CD8, is shown in Table 1.

The thesis hypothesis

The hypothesis of this thesis is that Qa-2 signals via a partner molecule, specifically CD8aa (Figure 8).  This hypothesis is based on several studies which suggest that Qa-2 and CD8aa protein may bind to each other.  First, Das et al. 2000 found that intestinal intraepithelial lymphocytes (iIEL) expressing only Qa-2 protein are primarily CD8aa lymphocytes.  In Qa-2-negative mice, few CD8aa lymphocytes are found in iIEL.  One explanation is that Qa-2 and CD8aa protein bind to each other.

Second, it is known that CD8aa is highly flexible, suggesting the possibility that CD8aa could interact with Qa-2 on the same cell.  In order to get crystals of CD8aa for x-ray crystallography, it was necessary to treat the protein with neuraminidase, O-glycosidase, and V8 protease.  Even after treatment, the electron density is not identifiable for the amino acids in the stalk region because the stalk is highly flexible (Figure 8).  This allows for the possibility that Qa-2 and CD8aa protein bind on the same membrane of a cell in a cis configuration, rather than having one molecule on each of two cells in a trans configuration.

Third, it has long been suspected that CD8aa may bind to MHC I molecules in more than one way (Leahy et al. 1992).  Based on single point amino acid mutations and a cell-cell adhesion assay, Giblin et al. (1994) observed the probability for bivalent interactions of CD8aa and MHC I molecules.  Although co-crystallographic data of CD8aa and HLA-A2 (Gao et al. 1997) and of CD8aa and H-2Kb (Kern et al. 1998) suggest the existence of only one binding, more recent data still suggest the contrary.  Jelonek et al. (1998) found, using surface plasmon resonance, that peptides of the CD8a chain could bind to the peptide binding cleft of the MHC class Ia protein H-2Ld.  Finally, Daniels et al. (2001) and later Devine et al. (2004) have suggested multiple bindings for CD8 and MHC Ia based on the increased affinity of MHC I tetramers to CD8 when using certain antibodies for CD8.

Testing the hypothesis

There are several methods to search for partner molecules for Qa-2: yeast 2-hybrid system, immunoprecipitation followed by western blotting and/or mass spectrometry, inhibitors to known signaling pathways, DNA chip analysis of activated (via anti-Qa-2 cross-linking) versus non-activated cells, and costimulation using multiple antibodies.

The hypothesis was tested in three ways: (1) stimulation of splenic T cells purified from CD8-positive and CD8-negative mice, using antibodies for CD3e, CD4, CD8a, and Qa-2, with secondary antibody, in the presense of PMA, and measuring proliferation; (2) stimulation of splenic T cells purified from CD8-positive mice, using immobilized antibodies for CD3e, CD4, CD8a, and Qa-2, and measuring proliferation; (3) predictive protein docking of the X-ray crystal structures of Qa-2, CD8aa, and CD8ab.  The resulting docking complex may help address the question: do  CD8aa and Qa-2 bind?


 

CHAPTER 2.  MATERIALS AND METHODS

Mice

This study used the following mouse strains: C57BL/6 (Qa-2 positive), B6.K1 (Qa-2 negative), B6.K2 (Qa-2 positive), and B6.129S2-Cd8atm1Mak/J (CD8a negative, Qa-2 positive).  All mice used were bred in our facilities, which are approved by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).  The C57BL/6 and B6.129S2-Cd8atm1Mak/J strains were purchased from Jackson Laboratory (Bar Harbor, ME).  The B6.K1 and B6.K2 strains were originally provided by L. Flaherty (Genomics Institute, Wadsworth Center, Jordan Road, Troy, NY).  The CD8a knockout mice were housed in facilities for immunocompromised rodents.  Only male mice of 8 weeks or older were used.

Antibodies

Table 2 describes the antibodies used for this work.  There are no isotype controls for secondary antibodies because they are polyclonal.  Details of the production and specificity of the three anti-Qa-2 monoclonal antibodies are described in Sharrow et al. (1989).

Purification of antibody from ascites

 

The anti-Qa-2 (Qa-m2) primary antibody was supplied in ascites, and was purified using an immobilized Protein A column (catalog number 44667, Pierce, Rockford, IL).  After elution, the antibody was concentrated using a Vivaspin 4 mL ultracentrifugation concentrator with 10,000 MWCO (Vivascience, Edgewood, NY).

Antibody concentration determination

 

The concentration of the antibody after purification was determined using two methods: the Bradford Method using the microassay procedure for microtiter plates, and ultraviolet absorbance. 

In the microassay procedure, samples of 160 µL of the dilution were added to wells of a 96 well flat bottom plate, then 40 µL of Bio-Rad Protein Assay dye added (catalog number 500-0006, Bio-Rad Laboratories, Hercules, CA).  The absorbance at 595 nm was measured.  The antibody concentration was determined by graphing absorbance vs. concentration for the antibody, and fitting the data for the purified and reference antibody to a straight line with minimal R2.

In the ultraviolet absorbance assay, 1 mL of the antibody was placed in a quartz cuvette, and the absorbance at 260 nm measured.  A blank of PBS was measured and subtracted from the absorbance of the antibody.  The concentration was determined by Beer’s law, and an assumed extinction coefficient of 1.36.  After measurement, the antibody was sterilized using a 0.22 mm filter (catalog number SLGV013SL, Fisher Scientific, Hampton, NH).

Ficoll-Hypaque isolation

Three mL of Histopaque, density 1.083 (catalog number 10831, Sigma-Aldrich, St. Louis, MO) were placed into a 15 mL centrifuge tube, and warmed to room temperature.  All fat and connective tissue were trimmed from one or two mouse spleen(s), and placed on a stainless steel screen on top a 50 mL centrifuge tube.  The spleen was mashed with the plunger of a 3 mL syringe.  Dulbecco’s Modified Eagles’s Medium (DMEM) (catalog number MT 15-017-CV, Mediatech/Cellgro, Herndon, VA) at room temperature was used to suspend the cells.  The cells were centrifuged at 500 g for 30 minutes at room temperature, then re-suspended in 1 mL of 1x T cell column buffer for the T cell enrichment column (see following protocol).  The 1x T cell column buffer was prepared from sterilized ddH2O using a 0.22 mm filter (catalog number SLGV013SL, Fisher Scientific, Hampton, NH).  All cell manipulations used sterile procedures.

T cell enrichment

T cells were enriched using a T cell enrichment column (Mouse CD3+ T cell Enrichment Column, Small, MTCC-5, R&D Systems, Minneapolis, MN).  In general, one spleen per column was used.  After enrichment, the cells were re-suspended in 1 mL of DMEM/FCS (see following protocol), counted, and diluted to 4 x 106/mL.  In all experiments, triplicate wells were set up for each experimental condition.

Primary and secondary antibody reactivity

In order to determine the reactivity of primary and secondary antibodies, the secondary antibody was conjugated to FITC using the FluoReporter® FITC Protein Labeling Kit (catalog number F6434, Molecular Probes, Eugene, OR).  Whole spleen cells were isolated from C57BL/6 mice.  1 x 106 cells in 100 mL were reacted with 5 mL Fc receptor block (catalog number 16-0161, e-Bioscience, San Diego, CA) for 15 minutes, then reacted with 3 mL primary antibody for 25 minutes, and washed three times with 200 mL PBSAZ.  Finally, the cells were reacted with 4 mL of FITC-conjugated secondary antibody for 25 minutes, and washed three times with 200 mL PBSAZ.  Immunofluorescence levels were measured on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).  Lymphocytes were selected by side and forward-scatter gating and analyzed for antibody reactivity.  The results were analyzed using FlowJo for Windows software (TreeStar, Ashland, OR).

Medium for T cell activation

Medium for T cell activation was DMEM supplemented with 10% heat inactivated fetal calf serum (FCS) (catalog number F2442, Sigma-Aldrich), 2 mM L-glutamine (catalog number G7513, Sigma-Aldrich), 50 μM 2-mercaptoethanol, 0.01 mg/mL gentamicin (catalog number G1272, Sigma-Aldrich), and 2 mM sodium pyruvate.

T cell activation using primary and secondary antibodies, and PMA

50 µL of cell suspension (2 x 105 cells) were placed in each well of a 96 well round bottom plate (catalog number 3799, Fisher Scientific).  Serial dilutions of anti-Qa-2, anti-CD3e, anti-CD4, and anti-CD8a primary antibodies, cross-linking rabbit anti-mouse IgG secondary antibody, isotype control antibody, and PMA in DMEM/FCS complete medium were prepared.  Stock PMA was prepared from 1 mg of PMA (catalog number P1585, Sigma-Aldrich) dissolved in 1 mL of dimethyl sulfoxide (DMSO) (catalog number D128-500, Fisher Scientific), and divided into 20 µL aliquots, and store at -20 şC.  50 µL of diluted anti-Qa-2 antibody or isotype control for primary antibody were added to each well, and incubated at room temperature for 30 minutes.  100 µL of diluted cross-linking secondary antibody, with PMA, were added to each well.  Cells were incubated at 37°C and 7.5% CO2 for 72 h.  The concentration of the primary antibody(-ies) shown in the Results section is the value after incubation with the cells, but before further dilution with secondary antibody.  The concentration of the secondary antibody shown in the Results section is the value after incubation with the cells, and primary antibody.

T cell activation using immobilized antibodies

Serial dilutions of anti-Qa-2, anti-CD3e, anti-CD4, and anti-CD8a antibodies in PBS were prepared.  50 µL of diluted antibodies were added to each well of a 96 well flat bottom plate (catalog number 3799, Fisher Scientific) and the plates were refrigerated for 24 h at 4°C.  After incubation, the plates were washed three times with 200 mL PBS.  50 µL of cell solution (2 x 105 cells) were placed in each well.  Cells were incubated at 37°C and 7.5% CO2 for 48 hours prior to the start of the MTT assay, and a further 24 hours for the MTT assay incubation time.  The total incubation time was 72 hours.

MTT assay for proliferation

In order to measure the effect of stimulation of T cells by anti-Qa-2 monoclonal antibody, proliferation was measured using the MTT assay (Mosmann 1983, Gerlier and Thomasset 1986, Hansen et al. 1989).  In this assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, catalog number M2128, Sigma-Aldrich) is cleaved to form a formazan by mitochondria in cells, which appear in solution as dark blue crystals.  These crystals are then mixed in acidified isopropanol in order to dissolve the crystals for photometric measurement.  At 48 h, wells were pulsed with 20 µL of 2 mg/mL MTT.  At 72 h, 125 µL of the supernatant were removed from each well, 50 µL of PBS and 150 µL of 0.1 N HCl/isopropanol added, and mixed.  The absorbance at 565 nm was measured using a Synergy™ HT Multi-Detection Microplate Reader (Bio-Tek, Winooski, Vermont) and KC4™ Data Analysis Software.  The data was analyzed using GraphPad (GraphPad Software, San Diego, CA).

Predictive protein docking

In order to test the best models of Qa-2 and CD8aa complexes, predictive protein docking was performed using ClusPro (Comeau et al. 2004a).  Predictive protein docking is a computational method which derives the binding of two proteins based on the three-dimensional coordinates of the two independently crystallized proteins.  The method searches for all possible complex structures which have favorable surface complementary, desolvation, and electrostatic energies, and ranks them accordingly.  The PDB model for Qa-2 was obtained from the Protein Data Bank, http://www.pdb.org, PDB ID 1K8D.  The PDB model for CD8aa was obtained from subsetting the CD8a chains from the PDB model for TL/CD8aa data, PDB ID 1NEZ.  The TL/CD8aa complex was chosen because it was the only model which provided CD8aa dimmer model data.  The two models were inputed to the ClusPro Server at http://structure.bu.edu (Comeau et al. 2004b; Comeau et al. 2004a).  The algorithm selected to perform the docking was DOT, a fast fourier transform method which evaluates approximately 1010 putative complexes, retaining 20,000 of the best surface complementarities.  The computed values for  electrostatic and free desolvation free energies are not included in the output from the server.  As a control, the experiment was repeated using the structures for CD8ab (2ATP) and H-2Kb (1LK2).


 

CHAPTER 3.  RESULTS

 

Establishing conditions for the activation of T lymphocytes

The conditions for the activation of T lymphocytes were established to resolve several unknowns in the methods.  First, the reactivity of the secondary anti-mouse IgG antibody to the primary antibodies was unknown, because many of the primary antibodies were not of mouse isotype.  In particular, anti-CD8a (53.6-7) and anti-CD3e (145-2C11) are rat and hamster isotype, respectively.  In order to activate T cells, the anti-mouse IgG secondary antibody must react with each of the primary antibodies.  Second, the MTT assay used to measure T cell proliferation required optimization of the conditions for culturing.  This required tests to establish the best concentration of FCS for proliferation, the best concentration of CO2 for incubation, whether to use of round or flat bottom wells in the incubation, the range of concentrations for primary and secondary antibodies, the time at which the cells are pulsed with MTT, and the time at which the plates are read for absorbance.

To test the reactivity of the antibodies, whole mouse splenocytes were incubated with the primary antibody and the secondary antibody in FITC-conjugated form, then analyzed using flow cytometry.  The results for these experiments are shown in Figure 9 through Figure 14.  For each anti-Qa-2 antibody, nearly all lymphocytes were Qa-2 positive (>94% of total cells).  The FITC-conjugated secondary antibody incubated alone with the splenocytes reacted minimally with the cells (Figure 9 through Figure 11).  Using anti-CD8a (Figure 12) and anti-CD4 (Figure 13), two sub-populations were identified, CD8 positive (44%) and CD4 positive (41%), accounting for over 85% of all T lymphocytes.  Using anti-CD3e (Figure 14), nearly all lymphocytes labeled were CD3e positive (>91%).  These results indicate that the anti-mouse IgG secondary antibody reacted with each of the primary antibodies.

Optimal conditions of FCS and CO2 concentrations, and the use of round vs. flat-bottomed well plates for T cell proliferation, were found for 2 x 105 cells/well using anti-Qa-2 to induce stimuation.  T lymphocytes from C57BL/6 mice were incubated with anti-Qa-2 (1-9-9) primary antibody (5 mg/mL), anti-mouse IgG secondary antibody (50 mg/mL), and PMA (5 ng/mL) for 72 h.  MTT was added to each well for the last 24h of incubation[KD1] .  Optimal T cell proliferation was found at 10% FCS and 7.5% CO2 (data not shown).  There was no statistical difference in proliferation between round vs. flat bottom welled plates (data not shown).

To optimize the MTT assay, initial trials for the times of incubation prior to and after the addition of MTT were determined from data suggested in other studies using T cells (Ogata et al. 1998; Svensson et al. 2001; Joo 2003).  Purified T cells (2x105 cells/well) were stimulated with anti-Qa-2 (1-9-9) primary antibody (5 mg/mL), anti-mouse IgG secondary antibody (50 mg/mL), and PMA (5 ng/mL) for various incubation times.  T cells were pulsed with MTT at 4, 8, or 24 h prior to reading the absorbance.  The absorbances were read at 72, 76, 80, or 84 h.  These results are show in Figure 15.  The absorbance measured for T cells pulsed 4 h prior to measurement rose to maximum absorbance at 80 hours.  Interestingly, the absorbance decreased with longer pre-incubation periods, possibly suggesting cell death.  However, absorbance for T cells pulsed 8 h prior to measurement rose to a maximum at 72 hours, and declined.  The maximum absorbance was observed with the MTT pulse addition 24 hours before reading the absorbance at 72 h.  Consequently, these times for the MTT assay were chosen for the remainder of the experiments.

The range of concentrations of anti-Qa-2 (1-9-9) primary antibody, secondary antibody, and PMA on T cell proliferation were found using T lymphocytes (2 x 105 cells/well) from C57BL/6 mice.  T cells were stimulated using varying concentrations of anti-Qa-2 (1-9-9) primary antibody, varing concentrations of anti-mouse IgG secondary antibody, and PMA (5 ng/mL) for 72 h.  Figure 16 shows the titration of anti-Qa-2 between 0.16 mg/mL and 10u/ml, and secondary cross-linking antibody between 0 mg/mL and 100 mg/mL.  These results indicated T cell proliferation was optimal at 2.5 mg/mL for 1-9-9 primary antibody, and 100 mg/mL secondary antibody.  However, near optimal conditions also occured at 2.5 mg/mL for 1-9-9 primary antibody, and 50 mg/mL secondary antibody, which is advantageous because less secondary antibody is required.  These results provide a lower optimal concentration of the anti-Qa-2 antibody compared to a previous study (5 mg/mL, McElhinny and Warner 2000).  It is possible that this is due to the use of 2-ME in this study.  Therefore, 2.5 mg/ml of 1-9-9 primary antibody and 50 mg/ml of secondary antibody were used for subsequent experiments.

Qa-2 is expressed on CD4 T cells less than on CD8 T cells

The previous study by Hahn et al. (1992) stated that there was no difference in the expression of Qa-2 between CD4 and CD8 T cells.  However, careful examination of the data presented in their paper suggests otherwise, namely that CD8 T cells express more Qa-2 antigen than CD4 Tcells.  Since it is important to understand the level of expression of Qa-2 between CD4 and CD8 T cells in order to compare proliferation data, the level of Qa-2 was measured using flow cytometry.  T cells from C57BL/6 and B6.K2 were stained with fluorescence (FITC, PE, or Alexa Fluor® 647/biotin) conjugated antibodies for Qa-2, CD3e, and CD4, or Qa-2, CD3e, and CD8a.  The results are shown in Figure 17 and Figure 18.  These data show a small difference in the levels of Qa-2 expressed between CD4 and CD8 T cells, with CD8 T cells expressing more Qa-2 than CD4 T cells.  The geometric mean of the peak fluorescence for Qa-2 was greater for CD8 T cells (mean = 330 in C57BL/6; mean = 113 in B6.K2) than for CD4 T cells (mean = 230 in C57BL/6; mean = 85 in B6.K2), in both mouse strains.  The significance of this difference in C57BL/6 was determined by Student’s t test (P = 0.08).  The forward scatter plots shown in panel (b) of Figure 17 and Figure 18 indicate that this difference is not due to the size of the CD4 and CD8 T cells, since the forward scatter plots of CD4 and CD8 cells were similar.

Qa-2 induced T cell proliferation using CD4 and CD8 cells

The hypothesis of this thesis is that CD8, and in particular CD8aa, is a partner molecule for signaling in anti-Qa-2 induced proliferation.  To begin to test this hypothesis, purified T cells from CD8a-negative mice (CD4+ CD8-) were subjected to anti-Qa-2 induced proliferation using varying concentrations of anti-Qa-2 (1-9-9) primary antibody, varying concentrations of anti-mouse IgG secondary antibody (50 mg/mL), and PMA (5 ng/mL).  As a control, purified CD8 T cells from C57BL/6 mice were stimulated using identical conditions.  These results are shown in Figure 19 and Figure 20.

This study found that T cells from both CD8- (CD4+ CD8-) and CD8+ (CD4+ CD8+) proliferate using anti-Qa-2 antibody, cross-linking secondary antibody, in the presence of PMA.  These data indicate that CD4+ T cells proliferate in the absence of CD8+ T cells.  Therefore, there must be different signaling partner in CD4 T cells than CD8.  However, these data do not answer the question of whether CD8 is a signal partner in CD8 Tcells.

Co-cross-linking Qa-2 and CD8 protein

               To further test the possibility that CD8aa is involved in Qa-2 signaling, purified T cells were stimulated using anti-Qa-2 (1-9-9) and anti-CD8a (56.6-7) antibodies in equal varying concentrations, anti-mouse IgG secondary antibody (50 mg/mL), and PMA (5 ng/mL).  The results of this experiment are shown in Figure 21.  This experiment was repeated a total of four times.

               Compared to cross-linked Qa-2 using anti-Qa-2 primary antibody, anti-mouse IgG secondary antibody, and PMA alone, co-cross-linking Qa-2 and CD8 using anti-Qa-2 and anti-CD8a, anti-mouse IgG secondary antibody, and PMA induced greater T cell proliferation.  The synergy of co-cross-linking Qa-2 and CD8 was statistically significant and largest at 0.75 mg/mL anti-Qa-2 and anti-CD8a antibody.  At this concentration, the ratio R of the mean absorbance with co-cross-linked stimulation vs. the mean absorbance with cross-linked stimulation [= ((Mean Co-cross-link abs - Control abs) / (Mean Cross-link Qa-2 abs - Control abs))] for four experiments was 1.23, 1.27, 1.33, 1.22 (mean = 1.26, stdev = 0.05).  The significance of this difference was determined by Student’s t test (P = 0.09, 0.10, 0.01, 0.09).  The difference in proliferation was significant for other concentrations as well:  at 0.375 mg/mL, R = 1.30, 1.45, 1.58, 1.18 with a mean = 1.26, SD = 0.15, and P = 0.03, 0.06, 0.06, 0.04 for four repetitions of the experiment.  This synergy was smaller for lower concentrations of anti-CD8a and anti-Qa-2.  The primary antibodies became cytotoxic for concentrations greater than 3 mg/mL.  Interestingly, cross-linking CD8 using anti-CD8a antibody, anti-mouse IgG secondary antibody, and PMA did not produce any proliferation.  These results indicate that the signal provided by cross-linking CD8 and PMA is not sufficient for proliferation.

Co-cross-linking Qa-2 and CD4 protein

               CD4 is known to function in a similar manner as CD8, except CD4 binds to MHC class II molecules whereas CD8 binds to MHC class I molecules.  Both coreceptors bind Lck.  Therefore, as a control, purified T cells were stimulated using anti-Qa-2 (1-9-9) and anti-CD4 antibodies in equal varying concentrations, anti-mouse IgG secondary antibody (50 mg/mL), and PMA (5 ng/mL).  The results of this experiment are shown in Figure 22.  For comparasion, stimulation with anti-Qa-2 and anti-CD8 was performed.  This experiment was repeated a total of three times.

               Compared to cross-linked Qa-2 using anti-Qa-2 primary antibody, anti-mouse IgG secondary antibody, and PMA alone, co-cross-linking Qa-2 and CD4 using anti-Qa-2 and anti-CD4, anti-mouse IgG secondary antibody, and PMA induced less T cell proliferation.  The antagonism of co-cross-linking Qa-2 and CD4 was statistically significant and largest at 1.5 mg/mL.  At this concentration, the ratio R of the mean absorbance with co-cross-linked stimulation vs. the mean absorbance with cross-linked stimulation [= ((Mean Co-cross-link abs - Control abs) / (Mean Cross-link Qa-2 abs - Control abs))], R = 0.56, 0.35, 0.42 (mean = 0.46, stdev = 0.11), and P = 0.003, 0.02, 0.01.  As with cross-linking CD8aa above, cross-linking CD4 alone did not produce any proliferation.

Co-cross-linking Qa-2 and CD8 protein in an ordered incubation

It has been reported in other studies that stimulation of T cells with anti-CD8 and anti-CD3 is order dependent (Moldwin et al. 1987).  Therefore, it was important to understand the difference in proliferation of T cells using co-cross-linking anti-CD8 and anti-Qa-2 antibodies, but in a different order.  Anti-CD8a was added to the T cells before adding anti-Qa-2, followed by co-cross-linking both antibodies using anti-mouse IgG secondary antibody (50 mg/mL), in the presence of PMA (5 ng/mL) (Figure 23).  This experiment was performed three times with similar results.  These results show an antagonistic effect of anti-CD8a on proliferation when added before the addition of anti-Qa-2 antibody, in contrast to the enhancing effect of anti-CD8a seen when both anti-CD8a and anti-Qa-2 were added simultaneously.

Co-cross-linking Qa-2 and CD3e protein

It is known that cross-linking the CD3e unit of the TCR can induce proliferation in T cells (Geppert and Lipsky 1986; Geppert and Lipsky 1987; Geppert and Lipsky 1988).  Further, costimulation of T cells using immobilized anti-CD3e and anti-Qa-2 primary antibodies can cause greater proliferation in T cells than immobilized anti-CD3e alone (Hahn et al. 1992).  For comparison, costimulation of T cells using the soluble format was tested.  Purified T cells were stimulated using anti-Qa-2 (1-9-9) and anti-CD3e (145-2C11) antibodies in equal varying concentrations, anti-mouse IgG secondary antibody (50 mg/mL), and PMA (5 ng/mL).  These results are shown in Figure 24.  These results show greater T cell proliferation when co-cross-linking CD3e and Qa-2 than cross-linking either separately.

Costimulation using immobilized antibodies

It has been reported that T cell stimulation using anti-Qa-2 antibody in either soluble or immobilized format requires PMA (Hahn and Soloski 1989; Cook et al. 1992; Hahn et al. 1992).  Further, costimulation of T cells using immobilized anti-CD3e and anti-Qa-2 primary antibodies can cause greater proliferation in T cells than anti-CD3e alone (Hahn et al. 1992).  For a comparison with these studies, and with the experiments performed in soluble format, we tested the effects of immobilized anti-Qa-2, anti-CD3e, anti-CD4, and anti-CD8a primary antibodies, all without PMA, on T cell proliferation.  Serial dilutions of various combinations of the antibodies were used.  These results are shown in Figure 25 through Figure 30.  Confirming the results of Cook et al. (1992) and Hahn et al. (1992), there is no T cell proliferation using anti-Qa-2 antibody without PMA (Figure 25; Figure 26; Figure 27).  This is true for three different anti-Qa-2 antibodies, 1-9-9 (Figure 25), Qa-m2 (Figure 26), and 1-1-2 (Figure 27).  Further, immobilized anti-CD3e and anti-Qa-2 primary antibodies causes greater stimulation of T cells than immobilized anti-CD3e alone, confirming the results of Hahn et al. (1992).  In comparison with the results of the experiments using the soluble format, immobilized antibody stimulation gives some similar and some different results.  A first  similarity is that costimulation using anti-CD3e and anti-Qa-2 primary antibodies causes greater stimulation of T cells than costimulation of either one separately, whether in soluble format (Figure 24) or immobilized format (Figure 25; Figure 26; Figure 27).  A second similarity is that the addition of anti-CD8a primary antibody enhances anti-Qa-2-induced proliferation, whether in soluble format (Figure 21) or in immobilized format (Figure 28 and Figure 29).  There was one difference observed in Qa-2-induced proliferation between the immobilized format and soluble format.  The addition of anti-CD4 primary antibody enhances T cell proliferation in the immobilized format using anti-CD3e and anti-Qa-2 (Figure 30); the addition of anti-CD4 primary antibody antagonizes T cell proliferation in the soluble format using anti-Qa-2 primary antibody (Figure 22).

Predictive protein docking for CD8 and Qa-2

An important assumption regarding the hypothesis is that CD8, and in particular CD8aa, and Qa-2 bind.  While there is limited data to suggest that CD8aa and Qa-2 do interact in some manner, there is no direct proof that this is so.  Therefore, predictive protein docking was used to determine the best binding of these proteins.  Predictive protein docking calculations were performed using ClusPro.  X-ray crystallography data of CD8aa, CD8ab, Qa-2, and H-2Kb proteins used were from the Protein Data Bank: 1NEZ for CD8aa; 2ATP for CD8ab; 1K8D for Qa-2; and 1LK2 for H-2Kb.  The ten best complexes were computed.  Because the computations do not include constraints between the proteins due to interference with the GPI linkage, the CD8 stalk, and orientation of the MHC class I protein on the surface of the cell, all bindings deemed invalid were discounted.

The best predicted binding for CD8aa and Qa-2 is shown in Figure 31.  This configuration suggests this binding is almost identical to the known binding of CD8aa and the MHC class Ia protein H-2Kb (Kern et al. 1998) and the non-classical MHC Ib protein TL (Liu et al. 2003).  As a control, the binding for CD8aa and H-2Kb was computed, and is shown in Figure 32.  This result agrees with the known X-ray crystallographic data of the structure of H-2Kb and CD8aa (Kern et al. 1998), and supporting the validity of the method.

For a comparison, the bindings of CD8ab with Qa-2 and H-2Kb were computed.  The best predicted binding of CD8ab and Qa-2 is shown in Figure 33.  A similar binding was found for CD8ab and H-2Kb.  These results suggest that CD8ab and Qa-2 do not bind.


 

CHAPTER 4.  DISCUSSION

 

This thesis poses that CD8aa, a transmembrane protein, binds to Qa-2 and transmits a signal when Qa-2 is cross-linked.  This hypothesis was tested using costimulation of T cells with antibodies directed against Qa-2, CD8a, CD4, and CD3e, with and without secondary cross-linking antibody, in soluble and immobilized formats.  PMA was added as a second signal to support proliferation in the soluble format, but not in the immobilized format.  In addition, predictive protein docking was used to determine the best binding of CD8aa, and CD8ab, and Qa-2 from x-ray crystallographic data.  The results found in this study provide some support for this hypothesis.  However, other results do not.

This study found that co-cross-linking anti-Qa-2 and anti-CD8a modestly enhanced proliferation of T cells in comparison to cross-linking with anti-Qa-2 alone.  This result provides support for the hypothesis.  This study confirmed a previous study (Bushkin et al. 1990) that cross-linking CD8 alone in the presence of PMA does not induce proliferation in T cells, which tends to not support the hypothesis.  In addition, cross-linking antibodies for Qa-2 in the presence of PMA on T cells from CD8 knockout mice induces proliferation, which indicates that a CD8-Qa-2 interaction is not required for signaling via Qa-2.  While it may be argued that these results do not contradict the hypothesis, research by others in GPI and MHC Ia signaling suggest alternative hypotheses for Qa-2 signaling.

In view of these results, we will discuss the use of antibodies in stimulation, the present data in support of, or contradicting the hypothesis, examine research concerning the signaling of MHC class I and GPI-anchored proteins, and propose alternative models for Qa-2 signaling.  However, first the nature of T cell stimulation via antibody binding will be discussed.

What is the physical significance of antibody stimulation via antibody binding or cross-linking?

            In many experiments, researchers have used these methods to mimic the binding of naturally occurring ligands to receptors.  Three methods have been developed, the later two of which were used in this thesis: (1) exposure of cells to primary antibodies in solution (Fab, F(ab′)2, or whole antibody); (2) reaction of cells with primary antibodies which are then cross-linked with secondary antibodies in solution (“soluble format”) (Figure 34); (3) letting cells settle onto primary antibodies bound to a solid surface (“immobilized format”) (Figure 35).  In the immobilized format, neither secondary antibody nor PMA was used.  Each of these methods mimic a different manner of ligand binding to a receptor.  For example, because they would not promote antigen cross-linking, Fab fragments could function to interfere with the binding of a ligand and receptor.  Deliberate cross-linking with antibody is often stimulatory.  For example, antibody to the platelet-derived growth factor (PDGF) receptor induces dimerization and phosphorylation of the chains of the PDGF receptor, and mimics the binding of the chemokine PDGF to the PDGF receptor (Heldin et al. 1989).  In a particularily relevant example, co-cross-linking antibodies for the CD8 or CD4 coreceptors and the T cell receptor allow Lck to phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAM) of the z chain of the CD3 portion of the TCR, thereby inducing T cell proliferation (Barber et al. 1989; reviewed in Germain and Stefanova 1999).  This example mimics the binding of MHC I to the TCR and CD8 (Emmrich et al. 1986; Boyce et al. 1988; Eichmann and Emmrich 1988; Janeway 1992).  Finally, immobilized or cross-linked antibodies for CD3e are known to induce T cell proliferation (Geppert and Lipsky 1986; Geppert and Lipsky 1987; Geppert and Lipsky 1988).  It is assumed that this arrangement mimics the display of antigen bound MHC I or II protein of an APC (Geppert and Lipsky 1986; Geppert and Lipsky 1988).  These three methods can be used singly or in conjunction in various combinations.

It is currently unknown if exposure to cross-linking antibodies or immobilized antibodies for Qa-2 mimics the binding of ligand, as yet unknown, to Qa-2, or is the produce of an artificial method.  Although the ligands for several GPI-anchored proteins are known, antibody induced stimulation appears to mimic the binding of ligand for one GPI-anchored protein: cross-linking antibodies for CD48 mimics the effect of ligand binding (CD244) on proliferation (reviewed in Assarsson et al. 2005).

Evidence supporting the hypothesis

The thesis hypothesis is supported by several results found in this study.  First, co-cross-linking anti-CD8a and anti-Qa-2 antibodies, in the presence of PMA, induced greater proliferation of T cells than cross-linking anti-Qa-2 alone.  Co-cross-linking antibodies to CD8a and Qa-2 mimics a more efficient binding of CD8 and Qa-2 than a hypothesized weak association between CD8 and Qa-2.  Second, antibodies for CD8a and Qa-2 in immobilized and co-cross-linked formats enhance T cell proliferation using antibodies for CD3e.  Co-cross-linking antibodies for Qa-2 mimics the colocalization of CD3e and CD8, and the juxtaposition of CD8-associated Lck with CD3e.  Third, T cell proliferation was seen to be dependent on the order of the application the antibodies.  When anti-CD8a antibody was incubated prior to anti-Qa-2 antibody, an antagonist effect on the proliferation of T cells was observed.  However, when anti-CD8a was incubated simultaneously with anti-Qa-2, the effect was synergistic.  This effect is consistent with our model because the application of anti-CD8a prior to the application of anti-Qa-2 might be expected to cause CD8 to distribute away from Qa-2, thus sequestering Lck, and reducing proliferation (Harder et al. 1998).

Additional studies in the Warner laboratory offer further support for the hypothesis.  PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) is a potent inhibitor of Src kinases, including Lck (Zhu et al. 1999).  PP2 reduces Qa-2-induced T cell proliferation in a dose-dependent manner (De Fazio and Warner, unpublished).  Further, cross-linking Qa-2 on T cells promotes the activation of PI 3-kinase and Akt, signaling molecules of downstream of Lck activation (De Fazio et al., submitted for publication).

Studies by other workers lend support to the hypothesis.  As noted previously, the presence of CD8aa is associated with the presence of Qa-2 in intestinal intraepithelial lymphocytes (Das et al. 2000).  Das et al.  found that CD8aa/TCRab cells failed to develop in Qa-2-negative mice suggesting Qa-2 mediated selection and a possible interaction.  They found that when a Qa-2-transgenic mouse is made from the Qa-2-negative strain, the presence of CD8aa/TCRab cells is restored.

Researchers have long been puzzled by the response of cross-linking antibodies to MHC class Ia proteins (Geppert et al. 1988; Geppert et al. 1989).  Unexpectedly, removal of the cytoplasmic tail of an MHC class Ia protein does not ablate signaling when it is cross-linked (Gur et al. 1990) suggesting that signal transduction is due to an extracellular, transmembrane partner molecule interacting with the MHC class Ia protein. Also, it was found that MHC class Ia protein and CD8 coimmunoprecipitated from T cells (Bushkin et al. 1988), and that cross-linking anti-MHC I (with antibody W6/32) and anti-CD8 (with antibody OKT8) in the presence of IL-2 enhances proliferation over cross-linked anti-MHC I alone (Bushkin et al. 1990).  Since that time, researchers have suspected that CD8 binds to MHC class Ia protein and functions to transduce a signal.

Cell-cell adhesion assays and crystallographic data of the structures of CD8aa and MHC class Ia suggested that CD8aa could possibly bind to MHC I molecules bivalently (Giblin et al. 1994).  Using surface plasmon resonance, Jelonek et al. (1998) found that  peptides derived from the CD8a chain could bind to the peptide binding cleft of the MHC class Ia protein H-2Ld, even when the carboxyl terminus of the peptide is unavailable, raising the specter of a third mode of how CD8 could bind MHC class Ia and function to transduce a signal.  Recently, it was shown that malfolding MHC I may cause CD8 to bind to it; antibodies to malfolded MHC I coimmunoprecipitate CD8 (Santos et al. 2004).

The results of the predictive protein binding of Qa-2 and CD8 using the docking program ClusPro suggests that Qa-2 and CD8aa may bind (Figure 31).  This binding is strikingly similar to the X-ray crystallographic data for CD8aa and H-2Kb (Kern et al. 1998), with the complementary-determining region (CDR) loops interacting with the a3 domain of Qa-2.  In comparison, the computed binding of Qa-2 and CD8ab protein suggest that Qa-2 and CD8ab do not bind, or that they bind poorly.  Studies suggest that the CDR loops of the CD8ab protein bind to the a3 domain of the MHC class I protein (Chang et al. 2005).  In this study, the CDR loops of the CD8ab protein interact with the a2 domain, in both Qa-2 and H-2Kb.  The possibility that CD8aa may bind Qa-2 in the embryo is supported by evidence in our laboratory that that CD8a is expressed in mouse embryos (unpublished data).

Evidence contradicting the hypothesis

The hypothesis is not supported by some of the results found in this study.  First, if Qa-2 signals via CD8, cross-linking CD8 itself should provide an equivalent signal.  However, when CD8a is cross-linked with secondary antibody in the presence of PMA, proliferation of T cells is not induced.  This experiment has been performed previously, and the results found in this thesis concur (e.g., Bushkin et al. 1990).  This result suggests that CD8aa is either not involved or is not the sole signaling partner in Qa-2 signaling.  Using T cells from CD8a-negative mice, this study found that cross-linking Qa-2 in the presence of PMA induces proliferation.  While not directly refuting the hypothesis, this data supports the existence of other molecules involved in the signaling of Qa-2.

            The hypothesis of the thesis is not supported by several studies by other researchers.  These studies suggest that Qa-2 and CD8aa do not bind at all.  Qa-2 encoded by the Q9 gene is known to bind poorly to CD8 in studies of CTL selection of Qa-2 target cells (Aldrich et al. 1991).  Aldrich et al.  found poor target lysis by primary H-2Ld CTLs of target cells constructed from Qa-2-negative L cells containing a chimeric protein of the a3 domain of Qa-2 and the a1 and a2 domains of H-2Ld in both transmembrane and GPI forms.  Similarily, Teitell et al. (1993) found poor target lysis by H-2Ld CTLs of target cells constructed from CIR B cells containing the a3 domain of Qa-2 and a1/a2 domains of H-2Ld.  Further, Teitell et al. (1993) found poor cell-cell adhesion between these CIR B cells and CHO cells containing hCD8aa.   Finally, the crystallographic data of He et al. (2001) suggests that CD8aa is likely to bind poorly to Qa-2 protein.  These studies suggest poor binding of Qa-2 and CD8aa.  However, it is important to note that many of these studies tested the binding of Qa-2 and CD8aa in a trans orientation, not a cis orientation as suggested in our model.  CD8aa may bind poorly to Qa-2 in the usual trans configuration, but it may bind in several different manners.  On the contrary, the predicted binding configuration of Qa-2 and CD8aa found in this thesis suggests that Qa-2 and CD8aa may, in fact, bind.

            Arcaro et al. (2000) found that CD8aa is excluded from rafts, whereas CD8ab is resident in rafts.  This distribution is the result of palmitoylations in the two CD8 chains.  In CD8b, two palmitoylations exist, whereas CD8a contains a single palmitoylation, causing CD8aa to have weak lipid raft localization (Fragoso et al. 2003).   Arcaro found that CD8aa is mostly unbound to Lck, because CD8aa and Lck are segregated to different areas of the membrane.  CD8aa and Qa-2 could bind, but that would require CD8aa to translocate from non-raft to raft.  Although problematic, it is not without precedence.  For example, IL-2 is composed of three chains, one of which is outside of lipid rafts, and the other two within lipid rafts (Marmor and Julius 2001).

Is Qa-2 behaving as a MHC I protein, or as a GPI-anchor protein during signal transduction?

Qa-2 is a both a MHC I protein, and a GPI-anchored protein.  Researchers have found that cross-linked MHC I proteins, and cross-linked GPI-anchored proteins, both induce proliferation in T and other cell types in the presence of PMA.  Consequently, researchers have proposed similar mechanisms for these observations.

Many researchers have focused on the proliferative effects of cross-linking MHC class Ia molecules (Table 3).  The effects of cross-linking antibodies for MHC I protein plus PMA on T cells was studied by Geppert et al. (1988) and Geppert and Lipsky (1988).  These researchers found synergistic effects using various combinations of anti-CD3e, anti-MHC I, anti-CD4, anti-CD8, and anti-CD2.  Also, it was found that anti-CD4 and anti-CD3e used in immobilized format had a synergistic effect on T cell proliferation, but an antagonistic effect when cells were exposed to soluble anti-CD4 plus immobilized anti-CD3e.  The results in this thesis concur with these observations.

In further studies, Gur et al. continued to analyze the mechanisms by which cross-linked anti-MHC class Ia transduces signals (Gur et al. 1990; Gur et al. 1997; Gur et al. 1999).  Gur et al. 1990 found that although the cytoplasmic tail of the MHC Ia protein HLA-A2 is phosphorylated after cross-linking, stimulation of Jurkat cells transgenecally expressing a form of HLA-A2 without the tail still proliferate in the presence of IL-2.  In later experiments, this group investigated the role of the extracellular, transmembrane, and cytoplasmic portions of HLA-A2 in signal transduction, and concluded that the extracellular portion of the MHC I molecule was responsible for signaling, and not the transmembrane domain or cytoplasmic tail (Gur et al. 1997; Gur et al. 1999).

Other researchers examined the role of anti-MHC I in signal transduction using SMS and EC cells (Bian et al. 1997; Harris et al. 1997; Bian et al. 1998; Bian and Reed 1999b; Bian and Reed 1999a; Nath et al. 1999; Bian and Reed 2001; Jin et al. 2002; Jin et al. 2004; Lepin et al. 2004; Jin et al. 2005).  These workers found that binding of anti-MHC class Ia caused SMS and EC cell proliferation.  Recently, Jin et al. 2004 studied activation of PI3K and Akt after MCH Ia cross-linking using western blotting detection of specific residues in Akt.  Of particular interest, these researchers found both the residues whose phosphorylation is required for activation, Ser473 and Thr308, of Akt were phosphorylated after cross-linking anti-MHC Ia.  In contrast, our laboratory has found that cross-linking of Qa-2 induces phosphorylation of Thr308 only (De Fazio and Warner, submitted for publication).  These findings suggest that Qa-2 and HLA-A2 do not share exactly the same mechanism for signal transduction.

A third group of researchers has demonstrated proliferation, Ca+2 mobilization, tyrosine phosphorylation of Lck, Zap70, and CD3z, and activation of the PI3K pathway following antibody binding to MHC class Ia (Skov et al. 1995; Bregenholt et al. 1996; Skov et al. 1997a; Skov et al. 1997b; Pedersen et al. 1998; Skov 1998; Skov et al. 1998; Pedersen et al. 1999a; Pedersen et al. 1999b).  However, these experiments employed anti-b2m, so it is not clear if a similar signal was generated.

For MHC class Ia molecules, one hypothesized mechanism for the signal transduction is via endocytosis (Bamezai et al. 1989).  This study found that immobilized anti-Qa-2 and anti-CD3e are synergistic on T cell proliferation.  Since immobilization of anti-Qa-2 prevents endocytosis of the Qa-2 protein, it is unlikely that endocytosis is involved in Qa-2 signal transduction.

A second body of research has focused on the activational effects of cross-linking GPI-anchored molecules (Table 4).  This set of research is very large, including at least 20 GPI-anchored proteins and an additional number of ligands.  For simplicity, the protein Thy-1 (CD90) will be considered here, because it is currently the most well investigated (Table 5).  Within this topic, there are over a dozen different groups of researchers.  Gunter et al. 1984 initially found that cross-linking Thy-1 stimulated IL-2 secretion, Ca+2 mobilization, and proliferation.  Kroczek et al. (1986) showed that proliferation of T cells is dependent on PMA.   In an attempt to find coreceptors for Thy-1, costimulation of Thy-1 and CD3e was studied and showed that the z chain of CD3 is phosphorylated without any costimulation, but to a greater extent and with the same pattern as with anti-CD3e (Klausner et al. 1987; Hsi et al. 1989).  Several studies followed to identify the state of tyrosine phosphorylation, kinase activity, and association via coimmunoprecipitation (