Antigen processing and T cell priming by mouse dendritic cells

Antigen processing and T cell priming

by mouse dendritic cells

Alexander Hal Drakesmith

A thesis submitted for the degree of

Doctor of Philosophy

March 1998

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Abstract

For a vertebrate to survive and reproduce, it must be able to fight infection efficiently

throughout its life-span. The vertebrate immune system performs this function by

recognising, responding to, killing and remembering pathogens. A crucial component of

immunity is the dendritic cell, which responds to inflammatory signals such as

cytokines which are produced because of infection. The invading pathogen is captured

and partially degraded by dendritic cells via a mechanism termed antigen processing.

Dendritic cells are specialised to prime specific T cells against small regions (called

determinants) derived from the pathogen by antigen processing. The activated T cells

then control the immune response which fights the infection.

This thesis is a study of how antigen processing by dendritic cells can be affected by

cytokines. The determinants processed and presented from a model antigen are shown

to vary considerably depending upon which cytokines the dendritic cells are exposed to.

In particular, determinants that are normally not presented (cryptic determinants) can

become very immunogenic: the cytokine exposed dendritic cells activate T cells against

these cryptic deteminants both in vitro and in vivo.

How one particular cytokine, interleukin-6, can cause these effects is further

investigated. Although the exact mechanism is not elucidated, interleukin-6 is shown to

differentiate dendritic cells in novel ways, including acidifying certain intracellular

compartments which are involved in antigen processing. Because pH influences many

aspects of antigen processing, the display of cryptic determinants by interleukin-6

Acknowledgements

I have had an extremely enjoyable time in the Tumour Immunology Unit. This is

entirely due to the people I have been lucky enough to work with.

Dr. Hans Stauss was my first supervisor, has been ever ready with support and advice,

and shows that comprehensive knowledge of cytotoxic T lymphocytes and European

football are not mutually exclusive. Professor Peter Beverley has been my main

supervisor and even in complicated times has always been inspiring, great fun, a

reasonable off-spinner, and an estimable supplier of fine Chardonnay. Professor Benny

Chain at UCL also supervised me, and his energy, enthusiasm and interesting ideas for

experiments contributed much to this thesis.

In the lab, Maria Dahl, Christine Hughes, Marcos Timon, Diana Wallace, Anne Marit-

Sponaas, Ray Hicks and Lindsey Goff helped me when I didn't have a clue. Peter Noble

always had a question. Pip O'Brien was a fantastic friend who made the lab a more

unpredictable place. The four who were with me at the end, like in 'Zulu' or (perhaps

more accurately) the Alamo, lovely but mad Mala Maini, Christian Zilch and my main

men Harry White and Sorena Kiani-Alikhan, were understanding in the extreme as I

hogged the computers and went slowly insane.

At UCL Immunology, Debbie O'Neil, Patrick Medd, Mike Binks, Ness Woodhead, Tim

Stonehouse, Torben Lund, Tom MacDougal, Peter Bunyard and the wondrous Lucienne

Lopes all helped me out technically, told me something important that I didn't know or

bought beer. Upstairs, Maureen Cohen, Simon McAdam, Steve Furrow, John Bingham,

Caroline O'Hare, Inusha De Silva, Victoria Spanswick, Lucia Christodoulides, John

Hartley and Peter McHugh also helped me and generally added to the camaraderie.

At the La Jolla Institute for Allergy and Immunology, California, Susanne Schneider

was extremely generous and hospitable, and Eli Sercarz was fun and gave good advice.

Contents

Title page 1

Abstract 2

Acknowledgements 4

Contents 5

List of Figures and Tables 11

Abbreviations 14

Chapter 1

General Introduction: dendritic cells, antigen processing

and hierarchy of T cell determinants 18

Introduction 19

Origins of dendritic cells 23

First sightings 23

A novel cell type in lymphoid organs 25 Dendritic cells prime naive T cells and T-dependent inunune reponses 25 Dendritic cell maturation and migration 26 Differentiation of dendritic cells from precursors 29 Molecular events of dendritic cell maturation and T cell priming 31 Generation of the T cell repertoire 33 MHC class II restricted processing of exogenously acquired antigen 36 Accessory proteins in MHC class II restricted processing 37 Endocytic compartments involved in antigen processing 38 Dominant and cryptic T cell determinants 40 Determinant capture and protection 42 Cryptic determinants, tolerance and autoimmunity 47 Antigen processing by different cell types 48

Chapter 2

Materials and Methods 50

Plastics 51

Mice 51

Reagents 51

Cytokines 51

Radioactive Isotopes 51

Peptides 52

Media 54

Sera 54

Buffers 54

Antibodies 55

Cell lines 56

Cell counting 58

Cry op reservation and retrieval of cells 58

Immunofluorescence staining of cell surface markers 58

Enriching dendritic cells from spleen 59

Grovying dendritic cells from bone marrow precursors 59

Allogeneic mixed lymphocyte proliferation assays 60

Induction of cytotoxic T lymphocytes 61

Using dendritic cells to generate allogeneic CTL in vitro 61

Using dendritic cells to generate syngeneic CTL in vitro 61

CTL assay 61

Target cells 61

Effector cells 62

MHC Class I peptide binding assay 62

Antigen processing assays 63

Immunisation of mice with dendritic cells 63

Antigen presenting cell separation 64

Endocytosis assay 65

Intracellular staining and confocal microscopy 65

Mounting cells onto slides 66

Confocal microscopy 66

Enzyme Linked Immunosorbent Assay (ELISA) 66 Pulsing dendritic cells with radioactive antigen 67

Messenger RNA differential display 68

RNA extraction 68

Cleaning extracted RNA 69

Reverse transcription of RNA 69

Polymerase chain reaction 69

6% denaturing polyacrylamide gel electrophoresis 70

Chapter 3

Characterisation of mouse dendritic cells 71

Introduction 72

Results 74

50-fold enrichment of dendritic cells from mouse spleen 74

Spleen dendritic cells are strong stimulators of allo-responses 76

Culturing bone marrow to grow dendritic cells in vitro 78

Mature bone marrow derived dendritic cells induce strong responses 80

Phenotype of day 8 dendritic cells 82

Day 8 dendritic cells can stimulate allo-specific CTL 84

Relative stabilisation abilities of three H-2Kb binding peptides 85

Comparison of splenic and bone marrow derived dendritic cells

in inducing syngeneic peptide specific CTL 86

Discussion 89

Comparison of splenic and bone marrow derived dendritic cells 89

Using dendritic cells to prime anti-cancer responses 90

Summary 92

Chapter 4

Effects of cytokines on antigen processing and

T cell priming by dendritic cells 93

Introduction 94

Results 98

Surface markers of T-T cell hybridomas 98

Effects of cytokines on the processing of HEL in vitro 98

Effects of cytokines on the differentiation/maturation of dendritic cells 104

Interleukin-6 treated dendritic cells prime T cells in vivo 108

Injection of EL-6 with HEL in adjuvant alters the anti-HEL T cell response 110

Dendritic cells draining HEL plus IL-6 immunisations present the cryptic

determinant HEL 2-16 112

Discussion 114

IL-4 114

TNF-a 115

IFN-y 116

Effects of IL-6 on dendritic cells 118

Mechanisms of escape from self tolerance 120

Molecular mimicry 120

Superantigen effects 121

CD2 ligation 121

Direct priming 121

Chapter 5

Action of IL-6 on antigen processing pathways

of dendritic cells 124

Introduction 125

Results 128

Endocytic progress of dextran through a dendritic cell 128

Characteristics of fluorochromes at different pH 130

IL-6 treated dendritic cells quench the fluorescence of endocytosed pH

sensitive markers 132

Visualisation of acidic vesicles in dendritic cells and colocalisation with

endocytosed mated al 136

Lysosome associated membrane protein-1 is peripherally located

in IL-6 treated dendritic cells 139

Levels and distribution of other molecules associated with

antigen processing in IL-6 treated and control dendritic cells 141

Antigen proteolysis by IL-6 treated and control dendritic cells 142

Altered gene expression in dendritic cells after IL-6 treatment 144

Discussion 145

IL-6 regulated acidification of peripheral vesicles 145

Role of pH in antigen processing 148

Protein unfolding 148

Proteolytic degradation 149

Peptide loading 150

Additional factors which may affect determinant selection by

dendritic cells 151

Chapter 6

Conclusions and perspectives:

multiple pathways of dendritic cell differentiation 156

Introduction 157

Many types of receptor molecules on dendritic cells 157

Antigen receptors (pattern recognition receptors) 157

Chemokine receptors 158

The diverse roles of cytokine receptors on dendritic cells 159

Induction of IL-12 secretion and TH-1 -type responses 159

Th-2-type responses or tolerance 160

Direct effects of pathogen on dendritic cells 161

Abnormal dendritic cell function and disease 163

Summary 165

Conclusions 168

Appendix I

Plastic ware 169

Appendix II

Chemical and biological reagents 170

Recombinant cytokines 172

Appendix III

Buffers and solutions 173

Elisa buffers 173

Molecular biology buffers 174

List of Figures

Figure 1.1 A dendritic cell 22

Figure 1.2 Migration patterns of dendritic cells in vivo 28

Figure 1.3 Scheme for determinant capture and protection 46

Figure 3.1 Constituents of the dendritic cell enriched cell fraction 74

Figure 3.2 FACS analysis of the dendritic cell enriched cell fraction 75

Figure 3.3 Splenic dendritic cells stimulate an allogeneic mixed

lymphocyte reaction 76

Figure 3.4 Splenic dendritic cells prime allo-specific CTL 77

Figure 3.5 Development of bone marrow cultures over time 78

Figure 3.6 Number of viable cells in developing bone marrow cultures 79

Figure 3.7 Day 7 bone marrow derived dendritic cells in culture 80

Figure 3.8 Time course of allogeneic mixed lymphocyte reaction 81

Figure 3.9 Time course of syngeneic mixed lymphocyte reaction 82

Figure 3.10 FACS analysis of day 8 bone marrow derived dendritic cells 83

Figure 3.11 Bone marrow derived dendritic cells prime allo-specific CTL 84

Figure 3.12 MHC class I stabilisation by various H-2 binding peptides 85

Figure 3.13 Comparison of the abilities of splenic and bone marrow derived

dendritic cells in inducing peptide specific syngeneic CTL 87

Figure 3.14 Blocking B7 costimulatory molecules on dendritic cells inhibits

CTL priming 88

Figure 4.1 MHC class II binding determinants of Hen Eggwhite Lysozyme 95

Figure 4.2 Comparison of responses to cryptic and dominant determinants 96

Figure 4.3 Effect of IL-4 on the processing of HEL by dendritic cells 99

Figure 4.4 Effect of TNF-a on the processing of HEL by dendritic cells 100

Figure 4.5 Effect of IFN-y on the processing of HEL by dendritic cells 101

Figure 4.7 Effect of cytokines on fluid-phase endocytosis by dendritic cells 105

Figure 4.8 Surface levels of MHC class II on cytokine treated dendritic cells 106

Figure 4.9 Effect of cytokines on dendritic cell viability 107

Figure 4.10 Immune response to injected IL-6 and HEL treated dendritic cells 109

Figure 4.11 Immune response to injected IL-6 plus HEL in adjuvant 111

Figure 4.12 Dendritic cells from lymph nodes draining IL-6 plus HEL

immunisation sites present cryptic determinants 113

Figure 5.1 Confocal analysis of dendritic cells endocytosing dextran 129

Figure 5.2 Behaviour of FTTC at different pH 131

Figure 5.3 Fluid-phase endocytosis of FITC conjugated markers by

IL-6 treated dendritic ells 132

Figure 5.4 Effect of fixation on the fluorescence of IL-6 treated

dendritic cells 133

Figure 5.5 Receptor-mediated endocytosis of FITC conjugated markers 134

Figure 5.6 Pulse-chase endocytosis of IL-6 treated dendritic cells 135

Figure 5.7 Structure of DAMP 136

Figure 5.8 Location of acidic vesicles in dendritic cells 137

Figure 5.9 Colocalisation of DAMP and endocytosed dextran 138

Figure 5.10 Positions of lysosome associated membrane protein-1 and

the transferrin receptor in dendritic cells 140

Figure 5.11 Degradation of iodinated bovine serum albumin by dendritic cells 143

Figure 5.12 Differential display of mRNA from dendritic cells 145

Figure 5.13 Effects of chloroquine on dendritic cell viability 153

List of Tables

Table 2.1 Storage procedures for certain reagents

Table 2.2 Details of peptides used

Table 2.3 First layer antibodies

Table 2.4 Second layer antibodies and streptavidin cychrome C

Table 2.5 Cell lines used as targets in CTL assays

Table 2.6 Summary of T cell hybridomas

52

53

55

56

57

57

Table 5.1 Characteristics of markers used in endocytosis assays

Table 5.2 Intracellular staining of dendritic cells for levels of

proteolytic enzymes and MHC class II

130

Abbreviations

adenine or adenosine

amino acid

antibody

antigen presenting cell

adenosine triphosphate

The American Type Culture Collection

basepairs

becquerel (1 disintegration/sec)

bovine serum albumin

cytosine

cluster of differentiation

complementary DNA

complete Freund's adjuvant

curie (3.7 x lO^® Bq)

MHC class II containing vesicle

MHC class II associated invariant chain peptide

counts per minute

chloroquine

chromium

cytotoxic T lymphocyte

Dalton

N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3- aminopropyl)methylamine dihydrochloride

DC/IL-6 DEPC DMSO DNA DNAse DNP dNTP dpi DTT EBV ECACC EDTA ELISA E:T ratio EtBr EtOH FACS PCS FITC g G [3H]TdR HCl HEL HIV HLA

as for DC, except IL-6 added for the last 18 hours of culture diethyl pyrocarbonate dimethylsulfoxide deoxyribonucleic acid deoxyribonuclease dinitrophenol deoxyribonucleoside triphosphate

dots per inch

dithiothreitol

Epstein-Barr virus

The European Collection of Animal Cell Cultures

ethylenediamine tetraacetic acid (disodium salt)

enzyme linked immunosorbent assay

effector to target cell ratio

ethidiumbromide

ethanol

fluorescence activated cell sorter

foetal calf serum

fluorescein isothiocyanate

gram or gravitational force

guanidine

tritiated thymidine

hydrochloric acid

hen eggwhite lysozyme

human immunodeficiency virus

HPLC high-performance liquid chromatography

HPV human papillomavirus

ICRF Imperial Cancer Research Fund

IFA incomplete Fruend's adjuvant

IFN interferon

Ig immunoglobulin

li invariant chain

IL interleukin

IMDM Iscove's modified Dulbecco's medium

kb kilobase

KCl potassium chloride

kDa kilodalton

LAMP lysosome associated membrane protein

LCMV lymphocytic choriomeningitis virus

M molar

mAh monoclonal antibody

2-ME p-Mercaptoethanol

MEM minimal essential medium

MHC major histocompatibility complex

MIIC MHC class II containing compartment

MMLV Moloney murine leukemia virus

mRNA messenger RNA

M-tropic macrophage infecting HIV strain

Mw molecular weight

NaAc sodium acetate

NOD non-obese diabetic

OD260 optical absorbance at 260 nm

OD450 optical absorbance at 450 nm

ova ovalbumin

PAGE polyacrylamide gel electrophoresis

PBSA phosphate buffered saline

PCR polymerase chain reaction

PHA phyto haemagglutinin

RNA ribonucleic acid

RNAse ribonuclease

rpm revolutions per minutes

SDS sodium dodecyl sulphate

T thymidine

TCR T cell receptor

TE tris-EDTA buffer

TEMED N, N, N', N', tetramethylethylenediamine

Tf-R Transferrin receptor

TH-1 T helper type 1 cell

TH-2 T helper type 2 cell

TNF tumour necrosis factor

TR-Dx Texas Red conjugated dextran

TRITC tetramethylrhodmamine isothiocyanate

T-tropic T cell infecting HIV strain

V volt

vol volume

Chapter 1

General introduction:

dendritic cells,

antigen processing

Introduction

Vertebrate immunity, consisting of the innate and adaptive immune systems, has

evolved under selection from both infection and the needs of the host. Innate immunity

is based on the recognition of a limited number of common features of pathogens, is

fast acting, but does not improve over time in its ability to fight pathogen. Acquired

immunity is slower acting, more specific to particular antigens, and improves on

repeated exposure to the same infection(Austyn and Wood, 1993; Roitt et al, 1985).

Innate immunity consists of several basic biochemical and physical defences against

pathogens, including the skin, the acidic environment of the gut and the activity of

lysozyme, and some more advanced molecular and cellular mechanisms. This latter

category includes phagocytes, which are cells such as eosinophils, basophils,

neutrophils, Kupffer cells and macrophages which recognise, internalise and destroy

infectious material. Natural killer cells are able to lyse infected cells (and some tumour

cells), providing another mechanism of innate defense. Finally the levels of some serum

proteins increase in response to infection, for instance C-reactive protein, which binds

the C protein of pneumococci bacteria, and promotes subsequent attatchment of

complement(Austyn and Wood, 1993; Roitt et al, 1985).

The complement system is made up from a number of soluble protein factors which are

involved in several arms of both innate and adaptive immunity. Some complement

factors can directly bind polysaccharides found on a number of microbes and aid their

capture by phagocytes possessing complement receptors, in a process called

opsonisation. Another complement factor, C5a, attracts neutrophils to sites of infection.

Complement can also lyse bacteria via the formation of the membrane attack complex

which punches a hole in bacterial cell walls. This complex is formed during two

multistep enzymatic cascades, termed the classical and the alternative pathways. The

pivotal event in both these sequences is the proteolytic cleavage of factor C3 to yield

activated C3 and C3a. Activated C3 generates other complement components, building

Intrinsic to many of the mechanisms of innate immunity is the ability to recognise

pathogen as being foreign. This can occur because most invading microorganisms have

clear structural differences compared to higher organisms, such as the components of

their cell walls. Receptors encoded by higher organisms can recognise these differences

and alert both innate and adaptive immunity(Medzhitov and Janeway, 1997).

Adaptive immunity, unlike innate immunity, is not 'ready to go': it requires activation

signals which often originate from the innate immune system. The adaptive immune

system's main components are lymphocytes, particularly thymus derived lymphocytes

(T cells) and bone marrow derived lymphocytes (B cells). These two cell types encode

receptors which unlike innate receptors are clonal and can rearrange, giving a greater

diversity and specificity to pathogen recognition. T cells expressing T cell receptors

(TCR) consisting of one a and one p chain can be restricted by Major

Histocompatability (MHC) class I or class II molecules presenting pathogenic antigen

in the form of short peptides, termed determinants (Townsend et al, 1986; Zinkemagel

and Doherty, 1974). B cells secrete immunoglobulin (Ig) in many different forms,

which can bind specific three-dimensional conformations of pathogenic antigen. Ig also

binds and activates complement, which results in the destruction of the bound pathogen

by phagocytosis or lysis(Austyn and Wood, 1993; Roitt et al, 1985).

Clearly, it is important that innate immunity and adaptive immunity cooperate. This

occurs by many mechanisms, including complement and Ig, chemotactic signals

released after pathogen recognition, and importantly, the recruitment and maturation of

dendritic cells. The first cell type of the adaptive immune system to be activated is

usually the CD4“^ helper T cell. These cells are the most important components of

adaptive immunity as they control Ig release by B cells, cell killing by CD8+ cytotoxic

T cells (CTL), and immunological memory. Together, these features of the adaptive

immune system are crucial for the longer life-span of vertebrates(Medzhitov and

This introductory chapter will deal with the discovery, lineage and properties of

dendritic cells, in particular their special ability to prime naive T cells in lymph nodes

against antigen found in the periphery. How antigen is processed so that determinants

are presented by MHC class II molecules to helper T cells is outlined. Although an

antigen can contain many potential determinants, T cell immune responses tend to be

focussed on just one or two of these, termed the dominant deteminants. Additionally, T

cell self tolerance is also targeted to dominant self determinants. This phenomenon is

called immunodominance(Sercarz et al, 1993); how immunodominance arises and its

É ^ '

Figure 1.1

A dendritic cell, viewed in suspension under phase contrast using a confocal

Origins of dendritic cells

"Really, if the lower orders don't set us a good example, what on earth is the use of

them?"

-Oscar Wilde (The Importance of Being Earnest)

The invertebrate immune system, which is the forerunner of the innate immune system

of vertebrates, does not have T or B cells(Roitt et al, 1985). In invertebrates, pathogen

recognition results in host defence mechanisms such as the secretion of anti-microbial

peptides appropriate to the particular type of invading pathogen(Medzhitov and

Janeway, 1998). In vertebrates, pathogen recognition leads to an innate response, but

the adaptive immune system including pathogen-specific lymphocytes can also be

activated(Medzhitov and Janeway, 1997; Roitt et al, 1985); dendritic cells play a major

role in this process. Dendritic cells are able to recognise and capture infectious

pathogens, but are also specialised to prime T cells using MHC and other molecules of

the adaptive immune system(Steinman, 1991). Thus dendritic cells have characteristics

of both invertebrate and vertebrate immunity: they are a lower order cell made good. As

described later, dendritic cells are seeded in peripheral tissues of the body where they

act as 'sentinels' ready to be activated at the point of entry of infection. The first

peripheral dendritic cells to be identified were those of the skin, called Langerhans

cells.

First sightings

In 1868, Paul Langerhans saw cells with striking dendritic processes in the suprabasal

layer of human skin while staining epidermal sections with gold salts(Langerhans,

1868; Wolff, 1991). He was convinced that they were a type of nerve cell. However his

procedure was difficult to reproduce and gold staining was not specific for nerve cells.

Silver chromate staining was much more precise, and revealed to Santiago Ramon y

Prize for Medicine in 1906, along with Camillo Golgi). Silver chromate does not stain

Langerhans cells: for this reason and others Billingham and Medawar proposed in 1953

that Langerhans cells were not nerve cells, but were effete or dying melanocytes’ en

route to being shed at the skin surface(Wolff, 1991). This theory was discounted in

1961 by the findings of Birbeck et al, showing that Langerhans cells had a distinctive

marker lacked by melanocytes (the Birbeck granule) and were present in vitiligous

tissue where melanocytes were absent(Birbeck et al, 1961). However a (more distant)

relationship between melanocytes and Langerhans cells was still believed to exist until

limb bud transplantation experiments showed different lineages for these two cell types:

melanocytes did not reappear after transplantation of limb buds (as they are derived

from the neural crest), but Langerhans cells did, indicating a mesenchymal

origin (Wolff, 1991).

The first circumstantial evidence for a role in immune responses came from an

observation that Langerhans cells were found in the dermis and dermal lymphatics after

epidermal challenge of animals passively sensitised to dinitrophenol(Wolff, 1991). If

Langerhans cells were involved in immunity, they might possess known immunological

molecules. Three papers published contemporaneously in 1977 found this to be the

case, showing MHC class II, complement receptors and Fc receptors to be

expressed(Klareskog e ta l, 1977; Rowden e ta l, 1977; Stingl e ta l, 1977). The

capacity of Langerhans cells to stimulate proliferative and cytotoxic T cell responses

was demonstrated shortly afterwards(Stingl et al, 1978; Wolff, 1991). Because of their

surface Fc and complement receptors, and their ATPase activity, Langerhans cells were

thought to be of the macrophage system, even though they lacked other macrophage

markers such as CD 14, and expressed some non-macrophage markers such as NLDC-

145. The Langerhans cell lineage problem was finally resolved in 1985, when Schuler

and Steinman found that after an in vitro culture of 2-3 days, Langerhans cells

differentiated to become almost indistinguishable from lymphoid dendritic cells, which

A novel cell type in lymphoid organs

In January 1973 the first of five papers describing a novel cell type in peripheral

lymphoid organs was published(Steinman and Cohn, 1973). These papers established

the term, dendritic cell, as applying to a rare adherent cell type in mouse spleen, of low

buoyant density, possessing a distinctive morphology including very mobile processes,

few lysosomes, and lacking both lymphocytic markers and the endocytic activity of

macrophages(Steinman and Cohn, 1973; Steinman and Cohn, 1974). Dendritic cells

were shown to have a bone marrow origin and had a low rate of proliferation but turned

over rapidly in the spleen, at the rate of about 10% of the dendritic cell population per

day(Steinman et al, 1974). They were found in situ in the white pulp of spleen and

were cytologically very similar to previously identified interdigitating cells (Steinman et

al, 1975). The adherence of dendritic cells was found to be transient, allowing greater

purification which enabled further investigations into both phenotype (revealing surface

topography and expression of MHC class II) and immune function(Steinman et al,

1979).

Dendritic cells prime naive T cells and T-dependent immune reponses

The first direct evidence of an immunostimulatory property came when lymphoid

dendritic cells were found to be extremely potent inducers of the mixed allogeneic

lymphocyte reaction(Steinman and Witmer, 1978). Later, dendritic cells were shown to

be critical accessory cells in forming primary antibody responses in vitro{lnaba. et al,

1983); dendritic cells, T cells and B cells all cluster together during this process(Inaba

et al, 1984). In the case of antibody responses against hapten-conjugated peptides,

dendritic cells primed naive helper T cells from resting lymphocytes, which then

stimulated B cell growth and differentiation(Inaba and Steinman, 1985). As well as

activating helper CD4"^ T cells, dendritic cells were also directly primed allogeneic

CD8+ CTL in vitro, in both mouse and human systems(Inaba et al, 1987; Young and

Steinman, 1990). Additionally, dendritic cells primed syngeneic influenza virus-specific

Dendritic cells were also found to prime T cells in vivo. Splenic dendritic cells were

pulsed in vitro with protein antigens such as conalbumin or ovalbumin and injected into

the hind footpads of mice. T cells isolated from the draining popliteal lymph nodes

proliferated ex vivo to the protein in an MHC restricted manner(Inaba et ai, 1990).

Reciprocally, after protein antigen was administered intravenously or intraperitoneally,

dendritic cells in the mouse spleen were the major source of immunogenic

protein(Crowley et al., 1990). These findings have been extended by Guery et at, who

found that after injection of protein emulsified in adjuvant, dendritic cells but not B

cells in draining lymph nodes were able to present processed protein to T cells ex

v/vo(Guery et at., 1996). In parallel, other investigations aimed to elucidate the

properties of dendritic cells that could account for this ability to prime T cells in

lymphoid tissues against peripherally administered antigens.

Dendritic cell maturation and migration

Although splenic dendritic cells had been shown to turn over rapidly, the first clue that

dendritic cells could have a multi-stage life-cycle came from Schuler and Steinman who

showed that Langerhans cells differentiate during three days of in vitro culture to

become as immunostimulatory as lymphoid dendritic cells (Schuler and Steinman,

1985). This immediately suggested a relationship between the two cell types which was

further investigated. Freshly isolated Langerhans cells were found to synthesise and

express MHC class U(Pure et ai, 1990), phagocytose bacterial and yeast products (Reis

e Sousa et al, 1993) and to process antigen without efficiently priming naive T

cells(Schuler and Steinman, 1985). Upon ex vivo culture, these features altered so that

little new MHC class U was synthesised, phagocytosis and antigen processing were

reduced, Birbeck granules were lost and the capacity to stimulate naive T cells was now

very high: thirty of these cells stimulated three hundred thousand T cells (Schuler and

Steinman, 1985). As dendritic cells isolated from spleen had previously been shown to

be non-phagocytic, it was proposed that Langerhans cells might differentiate into

lymphoid dendritic cells in vivo. This process was termed the maturation of dendritic

Migration of Langerhans cells to lymph nodes via afferent lymph was directly shown by

skin explant experiments(Larsen et al, 1990a), and found to be induced by

inflammatory stimuli such as LPS, TNF-a and IL-lp(Cumberbatch and Kimber, 1992;

Roake et al, 1995). Similar observations were reported for the migration of rat gut

epithelial dendritic cells into afferent lymph(Liu and MacPherson, 1993; MacPherson et

al, 1995). These migrating cells had sheet-like membrane processes, and were named

veiled cells. They were non-phagocytic, but retained previously phagocytosed material.

Other routes for dendritic cell migration have been defined. In solid organs such as

heart, kidney and spleen, dendritic cells are found in interstitial spaces in connective

tissue. Austyn et al showed that when dendritic cells isolated from spleen were labelled

and injected intravenously into mice they homed back to specific regions of the

spleen (Austyn et al, 1988; Kupiec et al, 1988). In a murine cardiac allograft model,

dendritic cells were shown to migrate from the heart via the blood to the spleen (Larsen

et al, 1990b). This effect was increased by administration of LPS (Roake et al, 1995).

A third route for dendritic cell migration was suggested after intravenous administration

of particles into mice resulted in particle laden dendritic cells being recruited to liver

lymph nodes(Matsuno et al, 1996). The pathway was further investigated using

dendritic cells isolated from hepatic lymph: they were injected intravenously and

subsequently found in the paracortical regions of recipient mouse liver lymph nodes.

The translocation from blood to lymph did not occur at high endothelial venules, but at

liver sinusoids, with the probable involvement of Kupffer cells (Kudo et al, 1997). This

migratory route enables immunological sampling of antigen in hepatic blood. Dendritic

cells in the liver may also migrate to the spleen via the blood(Austyn, 1996).

Dendritic cells are found in different regions of the spleen: in the white pulp (both T cell

and B cell areas), in marginal zones and red pulp. These may represent stages of

migration through the spleen. Additionally, dendritic cells may also be able to go

directly into lymphoid tissue from blood without first going to peripheral sites(Austyn,

Bone marrow (CD34+ MHC c la s s II )

Interstitial spaces Spleen? Epithélia Liver

of solid organs

XFcR+, MHC c la s s 11+ endocytosis+ , B7"K

via liver sinusoids

Spleen Lymph node

(MHC c la s s ||bright g7+ endocytosis")

T

Liver lymph node

traffic through blood

— —► traffic through afferent lymph

Figure 1.2

Migration patterns of dendritic cells in vivo. CD34+ progenitor cells differentiate and

seed themselves as sentinel dendritic cells in various peripheral sites and possibly

marginal zones of the spleen. Inflammatory stimuli cause or increase migration out of

these sites into lymphoid organs where dendritic cells prime T cells.

A recurring theme in studying dendritic cells was that cells isolated from peripheral

sites were unable to prime naive T cells, but acquired this ability on migration to lymph

nodes or during in vitro culture. Elucidating the differentiation events that must occur

during this maturation process was tricky until methods became available to generate

large numbers of dendritic cells in vitro from their in vivo precursors.

Differentiation of dendritic ceils from precursors

The maturation of Langerhans cells into potent immunostimulatory cells in vitro could

be enhanced using the cytokine granulocyte-macrophage colony-stimulating-factor

(GM-CSF)(Witmer et al, 1987). As kératinocytes in epithelial tissue are a major source

of GM-CSF, this made sense. In the search for factors that could differentiate dendritic

cells in vitro from pre-Langerhans cell precursors, GM-CSF was the obvious choice.

Inaba et al showed that GM-CSF could differentiate mature dendritic cells from

precursors found in mouse blood and bone marrow, that the bone marrow precursor was

MHC class II" and that it could give rise to dendritic cells, granulocytes and

macrophages(Inaba et at., 1993; Inaba et ai, 1993; Inaba et al, 1992). In general, GM-

CSF is sufficient to generate dendritic cells from murine precursor cells, although

additional cytokines can improve the yield.

In the human, differentiation of mature T cell stimulatory dendritic cells from

precursors is evidently more complicated. Although GM-CSF and TNF-a can cooperate

to form dendritic cells from CD34+ MHC class II" cord blood precursors(Caux et ai,

1992), factors such as c-kit ligand(Szabolcs e ta l, 1995), IL-4(Sallusto and

Lanzavecchia, 1994), flt-3 ligand(Maraskovsky et aL, 1996) and other as yet

unidentified moieties in monocyte-conditioned medium can play other roles in dendritic

cell development. These roles include general enhancement of proliferation, inhibition

of macrophage differentiation, and stabilisation of the mature dendritic cell phenotype.

The requirements for these factors may also vary depending upon the starting precursor

cells: bone marrow cells, cord blood cells, peripheral blood CD34+ and CD 14"*" cells

have all been used.

Other differentiation pathways of dendritic cells have been shown to occur in vivo,

cells is important because of the potential use of dendritic cells in treating diseases: it is

vital to use dendritic cells which will have the appropriate migratory, antigen retaining

and T cell stimulating properties. Subsets of dendritic cells have been studied most

extensivley in the mouse, and have been defined phenotypically and functionally.

Differences between dendritic cells types were immediately obvious when it was found

that thymic dendritic cells, which delete thymocytes during negative selection, can

actually arise from the same precursor as thymocytes(Ardavin et al, 1993), and that a

subpopulation of both splenic and thymic dendritic cells expressed CD8a(Vremec et

al, 1992). It later became clear that CD8a-chain expression was a marker for thymic

and splenic dendritic cells arising from this early T cell/dendritic cell progenitor, which

seems expresses low levels of CD4(Wu et al, 1996). This lineage has been termed the

lymphoid lineage of dendritic cells and is separate from the myeloid lineage: lymphoid

dendritic cells can be differentiated in vitro from the precursor cells in the abscence of

GM-CSF(Saunders et al, 1996). CD8a expressing splenic dendritic cells do not

activate naive T cells; instead they kill CD4+ T cells by Fas/Fas ligand induced

apoptosis(Suss and Shortman, 1996), and limit production of IL-2 by CD8+ T cells,

reducing their proliferation(Kronin et al, 1996). In attempting to prime anti-cancer

immune responses, it would probably be a good idea to avoid using this lineage of

dendritic cells, as they could actually remove from the available repertoire T cells

specific for tumour antigen determinants. However, if one wanted to limit immune

responses for example in autoimmunity by inducing T cell tolerance to an autoantigen,

these cells could be useful.

The intracellular signalling and gene transcription events induced during the

differentiation of dendritic cells from their precursors are incompletely characterised.

The relB subunit of the NF-kB complex is likely candidate to be involved in controlling

maturation of dendritic cells from the myeloid lineage. RelB is expressed in splenic

dendritic cells, and transgenic mice with a disrupted relB gene had normal numbers of

Langerhans cells, but had impaired splenic antigen presenting function, indicating

The in vitro generation of dendritic cells (although not identical to dendritic cells

differentiating in vivo) enabled the phenotypical changes that accompany maturation

and T cell priming to be studied in greater detail.

Molecular events of dendritic cell maturation and T cell priming

The activation requirements of naive T cells have been studied independently of

dendritic cells. In particular, work in Jonathan Sprent's laboratory has used Drosophila

melanogaster cells transfected with various molecules to determine the minimum

equipment needed to prime a MHC class I restricted naive T cell of known specificity.

Three molecules were sufficient: the correct MHC class I-peptide complex, the

adhesion molecule ICAM-1, and the costimulatory molecule B7.1(Cai et al, 1996). As

noted below, dendritic cells acquire these molecules (and others) during their

maturation.

Sallusto et al used an in vitro model to investigate the maturation of dendritic

cells(Sallusto et al, 1995; Sallusto and Lanzavecchia, 1994). Their method involved

culturing human peripheral blood adherent cells in the presence of GM-CSF and IL-4.

After 8 days in these conditions, most cells were loosely adherent or non-adherent and

possessed motile dendritic processes. Phenotypically, these cells expressed high levels

of MHC class I, MHC class II, CD la, CD lb, CDlc, CDl lb, C D llc, CD40, CD44, B7,

ICAM-1, F c '^ n , lower levels of LFA-1, LFA-3 and invariant chain, but lacked CD 14

and T- or B cell markers. Functionally, they could stimulate a mixed allogeneic

leukocyte reaction (MLR), present tetanus toxoid to T cell clones, and could capture

antigen by means of macropinocytosis or mannose and Fc receptors, Intracellularly,

they possessed a large compartment accessible by endocytosis which contained MHC

class II, the proteolytic enzyme cathepsin D and the lysosome associated membrane

protein-1.

Because of their ability to internalise and process antigen and their surface phenotype,

these cells were likened to immature dendritic cells. When they were cultured with

some analagous to the maturation of Langerhans cells. Surface levels of MHC class I,

MHC class n, CD la, CD lb, CDlc, B7, CD40, ICAM-1, CD44 all increased as did the

capacity to stimulate an allogeneic MLR, while surface invariant chain, FcyRII,

macropinocytosis and the ability to process tetanus toxoid for presentation to T cells all

decreased. The intracellular MHC class II containing compartment disappeared. More

recent studies have delved further into the intracellular events that occur during

maturation, and are discussed in chapter 5. However the findings of the two Sallusto

papers summarised above show that mature dendritic cells possess characteristics

required for the activation of T cells, and that maturation is driven by inflammatory

stimuli present at sites of infection. The sensitivity of immature 'sentinel' dendritic cells

to environmental signals is further discussed in chapter 6.

T cell priming by mature dendritic cells is a multi-stage process. Initially, chemokines

secreted by dendritic cells induce migration of T cells, for example the C-C chemokine

DC-CKl preferentially attracts naive Tcells(Adema etal., 1997). Adhesion molecules

then allow antigen independent dendritic cell-T cell clustering(Inaba et al, 1989),

before specific MHC/TCR interactions with the help of B7/CD28 ligation act to trigger

the T cell. CD40 ligand on T cells binds CD40 on dendritic cells resulting in IL-12

release by the dendritic cell, which can influence the primed CD4'*' T cell to become a

Th-1 helper cell(Kelsall et al, 1996; Koch et al, 1996). CD40 ligation also protects

dendritic cells from apoptosis(Bjorck et al, 1997; Ludewig et al, 1995). The lifespan

of dendritic cells in lymphoid tissue and the mechanisms of dendritic cell death are not

yet fully understood.

The nature of the responding CD4+ T cells can sometimes determine whether or not an

infection is controlled. The pattern of cytokines produced by the responding cells in

addition to IL-2 is an important factor in mobilising other components of the immune

response. TH-1 cells secrete IFN-yand TNF-P while TH-2 cells make IL-4, IL-5, IL-6,

IL-10 and IL-13. Generally, but by no means always, TH-1 type immune responses are

associated with cell mediated immunity for example CTL killing virally infected cells,

antibodies and neutrophils. Although this dichotomy of T cell responses is more

obvious in the mouse than in the human, and the relationship between TH-1/TH-2 and a

particular type of infection is often blurred, the efficacy of the T cell response is largely

determined by the cytokines that T cells make(Abbas et al, 1996; Seder and Paul,

1994). The molecular events in which dendritic cells may be involved that could control

the TH-I/Th-2 decision are further discussed in chapter 6.

As well as priming T cells in the periphery, dendritic cells can also delete developing T

cells during negative selection in the thymus(Matzinger and Guerder, 1989). This and

other related issues are outlined below.

Generation of the T cell repertoire

Despite the fact that T cell immune reponses can be primed by dendritic cells presenting

determinants derived from foreign antigen, the majority of determinants displayed by

MHC molecules are in fact self in origin(Rudensky et al, 1991). As clinically

recognisable autoimmunity is not an everyday occurrence, there must be mechanisms

which avoid T cell self reactivity and invoke a state of T cell self tolerance: developing

self reactive T cells in the thymus are in fact deleted by negative selection. In order to

combat infection, the T cells that remain available in the repertoire must have TCR that

adequately recognise foreign determinants in the context of self MHC molecules: this

can be accounted for by thymic positive selection.

The earliest T cell progenitors are the multipotent and self renewing CD34+ (in

humans) haematopoeitic stem cells of adult bone marrow. In an incompletely

understood process, these stem cells can differentiate, change phenotype and lose the

ability to generate cells of other lineages (for example the myeloid lineage) while

becoming more committed to the lymphoid lineage. The identification of a self

renewing but lymphoid committed stem cell has not yet been forthcoming(Shortman

and Wu, 1996). Additionally, the T progenitors which traffic to and seed the thymus are

unknown, as are the factors which regulate their movement. In the mouse thymus, the

Shortman's group. This cell can also form thymic dendritic cells, natural killer cells and

B cells(Shortman and Wu, 1996). Committment to the T lineage probably occurs before

the functional rearrangement of the P or y TCR chains. The pre TCR alpha chain, in

association with a successfully rearanged p chain, rescues thymocytes from apoptosis.

This pre TCR dimer then induces differentiation, so that a functionally rearranged alpha

chain may be expressed(von Boehmer and Fehling, 1997). This cell expresses low

levels of mature TCR and is CD4 and CDS double positive. The vast majority (95-97%)

of double positive cells die in the thymus: much of this is due to neglect, in other words,

a lack of interaction with MHC molecules(von Boehmer and Fehling, 1997).

Early indications that positive selection of thymocytes recognising self MHC molecules

must take place came from bone marrow chimaera experiments (Jameson et al, 1995).

Inbred homozygous mice (A) were lethally irradiated and then rescued with bone

marrow from mice of mixed parentage, of which one parent was genetically identical to

the irradiated mouse (AxB Fi mice). The T cells that developed in the irradiated mice

after bone marrow transfer were almost entirely restricted to the MHC molecules of the

recipient and of the shared parent (A) and not to the MHC haplotype of mouse B. This

showed that non-bone marrow derived cells regulated host MHC positive selection of

bone marrow derived thymocytes(Jameson et al, 1995). More information about the

process of positive selection has been elucidated, such as the cell types responsible

(thymic epithelial cells but almost certainly other cell types too) and its location (the

thymic cortex)(Anderson et al, 1996). Experiments using TCR transgenic mice showed

that the same peptide/MHC complex could induce positive selection or negative

selection depending on the concentration of the peptide present: low concentrations of

peptide caused positive selection(Sebzda et al, 1994). Additionally, peptides which

were unable to activate mature T cells could mediate positive selection of developing

thymocytes(Hogquist et al, 1994a), while agonist peptides for T cell activation could

not induce positive selection(Hogquist et al, 1994b). It appears that thymocytes are

positively selected on the basis of having low avidity for self peptide/self MHC

peptide/self MHC complexes or allogeneic MHC. During positive selection, the TCR

on double positive thymocytes will come into contact with MHC class I or MHC class

II molecules: this will determine whether a CDS or a CD4 single positive thymocyte

will result(Jameson et al, 1995).

Thymocytes need to avoid negative selection in the thymus; this process has also been

investigated using TCR transgenic mice(Kisielow and von Boehmer, 1995). Negative

selection is the induction of apoptosis in thymocytes with high avidity for self peptides.

Avidity is the sum of the affinity of the T cell receptors, the density of peptide/MHC

complexes and the density of other costimulatory molecules. Perhaps because of this,

the cells that mediate negative selection, for instance thymic dendritic cells, tend to

have high levels of costimulatory molecules(Matzinger and Guerder, 1989). Generally,

negative selection deletes thymocytes recognising abundant self peptides with

intermediate or high affinity and thymocytes with TCR of high affinity for other self

peptides(Kisielow and von Boehmer, 1995). In turn, this implies that some thymocytes

may escape negative selection. If the TCR of these thymocytes have a low affinity for

self this should not be a problem, as the activation requirements for mature naive T cells

are more stringent than the threshold to induce negative selection in

thymocytes(Pirchner et al, 1991, Sebzda et al, 1994). However some T cells

recognising self peptide with high affinity may escape negative selection because the

self peptide is not expressed in the thymus. There could be two reasons for this: either

the peptide is not adequately processed from the protein it derives from by thymic

antigen presenting cells, or the protein is not expressed in the thymus. In the first case T

cells could be activated against the self peptide if it is processed efficiently elsewhere

by a different antigen presenting cell. In the second case, other mechanisms probably

exist to tolerise T cells against the large numbers of proteins whose expression is either

tissue or developmentally regulated to preclude thymic expression. This is called

peripheral tolerance, and although there is evidence to suggest that peripheral lymphoid

The development of a self-MHC recognising, but non-self reactive T cell repertoire is

dependent upon generating self peptides which are presented by MHC molecules.

Likewise, the ability of dendritic cells to prime T cells against MHC class II restricted

pathogenic antigen not only requires migration and costimulatory molecules, but also

involves the processing of protein antigen into a form capable of binding MHC class II,

and regulating the binding event itself. Antigen processing for MHC class II

presentation is discussed below, although it must be noted that only recently has antigen

processing by dendritic cells been directly studied.

MHC class II restricted processing of exogenously acquired antigen

In 1981 Ziegler and Unanue found that in order for Listeria monocytogenes antigens to

be recognised in the context of MHC class II molecules (la) on macrophages by CD4+

T cells. Listeria had to be phagocytosed and partially catabolised(Ziegler and Unanue,

1981). This catabolism was termed antigen processing and was temperature and energy

dependent. Further experiments showed antigen processing could be inhibited by prior

fixing of the macrophages, by lysosomo- and acido-tropic compounds such as

chloroquine and ammonia, or by the protease inhibitor leupeptin(Streicher et al, 1984).

Importantly, the degree of catabolism inhibition was reflected in similar blocking of

antigen presentation to T cells. Other studies showed that in vitro enzymatic or

chemical fragmentation of antigen could substitute for cellular

processing(Shimonkevitz et al, 1983), and that MHC class II molecules could form

stable complexes with synthetic peptides(Babbitt et al, 1985). This latter finding was

also shown to be the case for MHC class I molecules(Townsend et al, 1986), although

the normal origin of peptides for the two types of MHC molecules appeared to be

different: MHC class I presented endogenously derived peptide(Townsend and Bodmer,

1989) while peptides presented on MHC class II tended to be from exogenous

antigen(Braciale et al, 1987). Although there are important exceptions to both these

MHC class n was shown to be accessible to internalised antigen (Cress well, 1985)and

intracellular transport of MHC class II intersected the endocytic route(Neefjes et al,

1990). Other work showed that interection of MHC class II dimers with peptide led to

enhanced MHC class II stability^ vitro and in vivo , and that as a result(Germain and

Hendrix, 1991; Sadegh Nasseri and Germain, 1991), peptides can affect the lifespan of

MHC class II molecules within cells(Nelson et al, 1994), A major question raised by

these findings was how peptide binding to MHC class II was prevented in early

compartments such as the endoplasmic reticulum (ER) or the golgi body (where many

peptides would be present), but allowed in later compartments which contained peptides

derived from captured exogenous antigen. In fact MHC class II molecules do present

many self peptides(Rudensky et al, 1991), but that some MHC class II does remain

accessible to endocytosed and processed foreign antigen is due to the functions of

invariant chain, HLA-DM, HLA-DO and associated proteases.

Accessory proteins in MHC class II restricted processing

The stabilisation of MHC class II by peptides leads to the conclusion that newly

synthesised MHC class II was probably incompletely folded(Germain et al, 1996);

usually mechanisms in the ER would be expected to degrade such molecules. In 1979

Invariant chain (li) was found to coprecipitate with MHC class II from cell

lysates(Jones et al, 1979): li is a non-MHC encoded protein whose expression is

nevertheless co-regulated with MHC class II(Ceman and Sant, 1995). li exists in two

forms in the mouse, p31 and p41, and four forms in humans, p22, p35, p41 and

p43(Ceman and Sant, 1995). After translation in the ER, li trimerises and then binds

three MHC class II dimers: a nonanmer is formed(Roche et al, 1991). In the absence of

li, MHC class II was found in the ER in large misfolded aggregates or with ER proteins

complexed in the MHC class II binding site: this implied that li facilitated MHC class II

folding, and prevented premature access to the binding site(Bonnerot et al, 1994;

Marks et al, 1995). It also appears that trimérisation of li controls the intracellular

Mutations in the MHC class Il-like heterodimer HLA-DM were found to abolish

presentation of certain determinants from exogenously acquired antigen even though

MHC class II was successfully synthesised(Riberdy et al, 1992; Sette et al, 1992).

Peptides eluted from MHC class II molecules in these cells were shown to correspond

to amino acids 82-107 of li; this region was termed CLIP (Class II associated invariant

chain peptide). Further work showed that the function of normal HLA-DM was to

facilitate the exchange of CLIP from MHC class II for other more tightly binding

peptides(Denzin and Cress well, 1995; Sherman e ta l, 1995; Sloan e ta l, 1995). It

should be noted however that CLIP and HLA-DM have different binding affinities for

and regulatory activities on different alleles of MHC class II. HLA-DO has recently

been shown to counteract the function of HLA-DM(Denzin et al, 1997); the ratio of

DM/DO in a cell may therfore be crucial in determining how available MHC class II

molecules are for peptide loading. Additionally, proteases such as cathepsin S are

responsible for degrading non CLIP regions of li prior to CLIP removal(Riese et al,

1996), enabling DM/DO to gain access the MHC class II/CLIP complex.

The exact location of peptide loading onto MHC class II is still not clear, for one main

reason, that different determinants have different processing requirements, and so could

be loaded under different conditions. Moreover, present knowledge of the intracellular

location of antigen processing and peptide loading comes largely from studies with B

cell lines, not with dendritic cells, which are arguably the most important antigen

processing cell type. A summary of compartments thought to play a role in antigen

processing is given below.

Endocytic compartments involved in antigen processing

Generating MHC class II determinants from an antigen is a degradative process, while

transport of MHC class II involves membrane transport. Normally these two processes

are kept separate to protect surface receptors from being degraded, and to ensure that

degradative enzymes remain within the cell. The integration of these processes which

The first intracellular structure to be identified as containing both MHC class II and

molecules needed for antigen degradation was the MIIC, found by Peters et a l MÜCs

were multilamellar, close to the trans-Golgi network and although not directly accessed

by transferrin receptors (found in early endosomes), could be reached by

endocytosis (Peters eta l, 1991). They contained lysosome associated glycoproteins and

acid hydrolases. Whether MHC class II in MIIC was associated with li was unclear, but

generally only small amounts of li were present in these compartments. Attempts to

purify MIICs using density gradient centrifugation and free flow electrophoresis of

murine A20 B cell lysates identified an anodally deflected set of vesicles which were

termed CIIV(Amigorena et al, 1994). CHV and MIIC share many characteristics,

including accessibilty by endocytosis and having the ability to form stable MHC class

Il-peptide complexes. However CITV contain transferrin receptors, and are of lower

density than MIIC, suggesting that CIIV may be more endosomal than lysosomal.

The route of MHC class Il/Ii into the endocytic pathway has been studied in A20 cells.

This pathway is normally rapid, so in order to slow it down and reveal transport

intermediates, leupeptin was used, which inhibits li degradation. These experiments

showed that MHC class Il/Ii first travelled to early endosomes, and from there to

CIIV(Amigorena et al, 1995). During this process li was degraded so that MHC class II

was available for loading at the CIIV stage. This finding and the morphological

relationship with MIIC suggest that MHC class Il/Ii leave the golgi body, go to early

endosomes (and possibly even the surface) and then 'bounce back' via CIIV, then MIIC,

and then ultimately to perinuclear lysosomes for degradation if peptide loading has not

occurred(Castellino and Germain, 1995). If MHC class II has been stabilised by

peptide, the complex is transported to the surface, via a route which is at present very

uncertain.

The experiments outlined above, which show that both low and high density vesicles

contain stable MHC class Il/peptide complexes are biochemical in nature. If specific T

cells or antibodies against particular MHC class Il-peptide complexes are used to

appear, with either high or low density vesicles being identified (but not both),

depending on the determinant being investigated (Watts, 1997). This reflects the fact

that different determinants have different processing requirements and so may be

revealed in one compartment type but not in others, even though many compartments

can in principle load peptide. As discussed in chapter 5 some determinants can be

processed and presented independently of invariant chain, by loading onto recycling

MHC class II in early endosomes(Pinet et al, 1995). Overall, the intracellular sorting of

MHC class II (in B cells at least) seems to be regulated to allow maximum possible

exposure to the determinants generated in various different compartments as an antigen

is processed.

Given the ability of MHC class II to scan intracellularly most of the endocytic route for

determinants to bind to, the phenomenon of immunological dominance in T cell

reponses is perhaps surprising. This is the empirical observation that an in vivo T cell

response to an injected foreign antigen is usually narrowly focussed on one or two

determinants derived from that antigen, even though many more determinants may have

the potential to stimulate T cells(Sercarz et al, 1993). How this phenomenon may arise,

and its consequences for immunity are discussed below.

Dominant and cryptic T cell determinants

Immunodominance was first used in the context of B cells, referring to determinants to

which most of the immunoglobulins in a response were directed(Sercarz et al, 1993).

The term was later employed to describe a murine proliferative response to injected Hen

Eggwhite Lysozyme (HEL): T cells from mice immunised with whole HEL were found

to respond ex vivo to one cyanobromide fragment of HEL, despite the the fact that other

fragments were immunogenic when injected into mice(Maizels et al, 1980). Similar

observations were made for responses to other antigens including cytochrome c, lambda

repressor protein, insulin, myoglobin, ovalbumin and staphylococcal nuclease(Sercarz

Determinants are strictly defined as being dominant, subdominant or cryptic in the

following manner: a native foreign protein antigen is emulsified in adjuvant and

injected into mice. Ten days later, T cells isolated from draining lymph nodes are

cultured with synthetic peptides derived from the primary sequence of the antigen. The

peptide determinant which elicits the largest proliferative response is the dominant

determinant. Subdominant peptide determinants induce weak responses from

lymphocytes primed against whole antigen, while cryptic determinants are not

responded to. All three types of determinant can induce determinant specific responses

when injected into mice in the form of synthetic peptide, but, as a corollary to the

above, only T cells primed against dominant peptides in vivo will proliferate strongly to

whole antigen in in vitro recall assays(Maizels et al, 1980). An important point is that

the positions of the determinants (dominant, subdominant and cryptic) in an antigen are

usually different for different MHC class II alleles, for instance HEL 106-116 is

dominant in BALB/c mice (H-2^), but HEL 46-61 is dominant in CBA mice (H-2^).

These differences are mainly due to the properties of the peptide binding grooves of the

two MHC class II dimers, but other factors such as CLIP/MHC class II affinity and

levels of proteases may also have an influence. Once the pattern of determinant

hierarchy is established after the injection of a foreign native antigen, it is stable and

does not usually change over time(Sercarz et al, 1993).

A major reservation for these definitions is that proliferative assays detect primarily TH-

1-type responses. Therefore determinants which are dominant in TH-2 responses will

not be identified. Another problem is that MHC class Il/peptide complexes formed by

loading of exogenously added peptide onto recycling MHC class II molecules (as in in

vitro in recall assays) will not necessarily have the same conformation as complexes

formed in later compartments following processing of the whole antigen. This seems to

be the explanation behind findings of Viner et al who showed that some T cell

hybridomas isolated following immunisation of mice with a dominant peptide could not

recognise naturally processed whole antigen in vitro(VinQr et al, 1996). Another study

on the basis of poor proliferative responses was in fact well displayed by B cells after

processing the whole antigen in vitro : the reason behind the crypticity could have been

at the level of the T cell response, not in the processing of the determinant(Viner et al,

1995). There is the possibility however that while this determinant was well processed

and presented by B cells, it may not have been efficiently displayed by dendritic cells in

vivo , the crucial antigen presenting cell type in forming T cell responses. Differences

between antigen presenting cell types are discussed later.

Usually, dominant determinants are well presented and less immunogenic determinants

are poorly presented. The mechanisms by which this occurs are thought to be as a

consequence of how MHC class II binds partially degraded protein during antigen

processing.

Determinant capture and protection

Buus et al found that peptides with high binding affinity for MHC class II molecules

were also well presented(Buus et al, 1987). Schaeffer et al also observed this

phenomenon using peptides covering the entire sequence of staphylococcal nuclease,

but went further by analysing the immunigenicity of the peptides(Schaeffer et al,

1989). They found that strongly binding peptides were very stimulatory for T cells,

whereas peptides of lower affinity showed decreased immunogenicity. Adorini et al

examined whether peptide determinants could compete with each other for MHC class

II binding: they showed that a strongly binding peptide from mouse lysozyme could

prevent in vivo priming against HEL peptides if the self peptide was injected into mice

simultaneously with, but in excess to the HEL peptide(Adorini et al, 1988). Further

experiments demonstrated that this in vivo prevention effect was limited to situations

where one of the peptides involved was clearly a weaker binder(Sercarz et al, 1993).

These findings and observations showing that changes in regions of an antigen distant

from a determinant could affect that determinants' immunogenicity led to the hypothesis

of determinant capture and MHC protection to explain immunodominance. This has