CD8 T cell memory development is influenced by direct interactions with CD4 T cells and tissue localization during T cell activation and clonal expansion Open Access

Romagnoli, Pablo (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/p2676w07r?locale=en
Published

Abstract

Immunological memory against intracellular pathogens is highly dependant on the
number, quality and location of memory CD8 T cells, all features defined during CD8 T
cell memory formation. In terms of quality formation, CD8 T cells need CD4 T cells to
develop into effective memory CD8 T cells. Since murine CD8 T cells do not transcribe
MHC class II genes, several models have proposed antigen-presenting cells (APCs) as
intermediaries that allow CD8 T cells to receive CD4 T cell help. Here, we demonstrate
that activated murine CD8 T cells possess MHC class II molecules acquired from APCs
via a process called trogocytosis. Transferred MHC class II molecules are functionally
competent and enable activated CD8 T cells to receive CD4 T cell help enhancing their
recall responses upon challenge. These observations indicate that direct CD8:CD4 T cell
interactions may significantly contribute to the development of memory CD8 T cells.
The location in which CD8 T cells get activated can also influence generation of
CD8 T cell memory. Naive CD8 T cells responding to infection in different tissues
integrate signals that determine their phenotype and functional properties as effector or
memory CD8 T cells. Here, we show that CD8 T cells responding to acute LCMV
infection leave blood to populate several lymphoid tissues (spleen, PLN, MLN and bone
marrow). We find that while the phenotype of LCMV-specific CD8 T cells varies with
the tissue, CD8 T cells from bone marrow display a memory precursor phenotype,
CD62LloCD25loLy6Chi different to the mixed phenotype observed in CD8 T cells from
spleen or PLN 3 days post LCMV infection. Interestingly, T-bet is absent in LCMV-
specific CD8 T cells from bone marrow, in contrast to the high T-bet expression found on
LCMV-specific CD8 T cells in PLN and spleen. Differences in phenotype also correlate
with the cytokine profile of CD8 T cells, detecting less IFNγ-producers in bone marrow
than in spleen upon ex vivo stimulation. These findings suggest that memory precursors
arise in bone marrow early during LCMV infection. Taken together, we show two
unappreciated contributions to CD8 T cell memory development.

Table of Contents

Chapter 1: Defining moments in the life of CD8 T cells……………...1
1.1 Immunological memory.…………………………………………………..2
1.2 CD8 T cell activation……………………………………….……………..3
1.3 CD8 T cell memory……………………...………………………………...4
1.4 CD4 T cell help……………………………………….………….………..5
1.5 T:T interactions………………………….…......….………….…………...10
1.5.1 MHC class II on T cells…...……………….………..….………......11
1.5.2 Immunological synapse…...………………….…………...…...…...13
1.5.3 Intercellular transfer of proteins and Trogocytosis…...…………...15

1.6 CD8 T cell migration………………………………….…….………..…...18
1.7 References…...……………….………………..….………….……….…...22
Chapter 2: CD8 T cell memory recall is enhanced by novel direct
interactions with CD4 T cells enabled by MHC class II
transferred from APCs …………............................................44

2.1 Abstract………………..…………………………………………………...45
2.2 Introduction………………………………………………………………...46
2.3 Materials and Methods……………...……………………………………...48
2.3.1 Mouse Strains……………...…………………………………...…...48
2.3.2 Pathogens and infections……………...…………………...…...…...48
2.3.3 Cell suspensions and flow cytometry……………….…...……...…...49
2.3.4 Peptides…...…………………..……………………...…………......49
2.3.5 Cell culture…..………………..……………………...…………......50
2.3.6 In vitro CD4 T cell differentiation…………………....…………......50
2.3.7 Adoptive Transfer…………………………………….…………......51
2.3.8 Statistical Analysis……...…………………………….…………......51

2.4 Results…….………………..……………………………....……………....52
2.4.1 MHC class II is present on recently activated murine CD8 T cells
in vitro as well as in vivo……….....................................................52
2.4.2 MHC class II is transiently present on responding transgenic
CD8 T cells after infection…..........................................................53
2.4.3 MHC class II is transferred onto CD8 T cells from APCs….............54
2.4.4 MHC Class II on activated CD8 T cells mediate direct stimulation
of experienced CD4 T cells..….......................................................56
2.4.5 In vitro interactions between MHC Class II+ activated CD8 T cells
and experienced CD4 T cells improve the in vivo recall response
of CD8 T cells..……………...........................................................57

2.5 Discussion………………..………………………………………………...59
2.6 Figures………………..…………………..………………………....……...65
2.7 Acknowledgements………………..………………..……………....……...80
2.8 Footnotes………………..………………..……………………........……...80
2.9 References………………..………………..……………....…………….....81
Chapter 3: Memory precursor phenotype is detected early on
CD8 T cells responding to acute LCMV infection

in bone marrow…………….……………………………..…....89
3.1 Abstract………………..…………………………………………………..90
3.2 Introduction………………..…………………..………………………......91
3.3 Materials and Methods………….……………..…………………………..94
3.3.1 Mouse Strains………………..……...…..…………………...…......94
3.3.2 Pathogens and Infections………...……..……………………...…...94
3.3.3 Cell suspensions and flow cytometry…….…………..……...……...94
3.3.4 Peptides………………..………………..………………...………...95
3.3.5 Adoptive Transfer………………..………………..…...…………....95
3.3.6 Statistical Analysis…….………………..…………...……………...95

3.4 Results…….………………..……………………………....……………...96
3.4.1 Responder CD8 T cell locate to different lymphoid tissues than
bystanders CD8 T cells and divide up to 10 times
during the first 3 days of LCMV infection……………….…….....96
3.4.2 Responder CD8 T cells from bone marrow display a memory
precursor phenotype early during LCMV infection……….....…..97
3.4.3 Endogenous CD8 T cells from bone marrow responding to
LCMV infection also display lower CD25 expressio..……….…..98
3.4.4 Lower percentage of IFNg-producer responder CD8 T cells
are found in bone marrow than in spleen.……………...……......99

3.5 Discussion…….………………..…………………………….……...…....100
3.6 Figures…….………………..………….…………………….……….…..105
3.7 Acknowledgements….……..………….…………………….…………...115
3.8 Footnotes.……..………….…………………….………………………...115
3.9 References.………………....………….…………………….…………...116
Chapter 4: Conclusions………………………………………...……………123
4.1 Why research on Immunological Memory is important…...……..…...…124
4.2 Memory CD8 T cells and our research findings……………...…..…...…124
4.3 How direct CD8:CD4 T cell interactions impact
the quality of CD8 T cell memory...................................................125
4.4 How location impacts on CD8 T cell memory formation……..…………129
4.5 Conclusions………………………………………………...…..……...…132
4.6 References………………………………………………...……....…...…134
List of Figures
Chapter 2
Figure 2.1: MHC class II is present on recently activated murine
CD8 T cells in vitro as well as in vivo………………………..65
Figure 2.2: MHC class II is transiently present on responding
transgenic CD8 T cells after infection………………………..67
Figure 2.3: MHC class II is transferred onto CD8 T cells from APCs…...69
Figure 2.4: MHC class II on activated CD8 T cells mediate direct
stimulation of experienced CD4 T cells……………….……..71
Figure 2.5: In vitro interactions between MHC class II+ activated
CD8 T cells and experienced CD4 T cells improve
the in vivo recall response of CD8 T cells…………….……..72
Figure 2.6: MHC class II and CD54 is present around Day 2, 3 and 4
on gp33-specific CD8 T cells in MLN, spleen and PLN
respectively after LCMV infection…………….…………….73
Figure 2.7: MHC class II is present only on specific CD8 T cells
after activation in vitro and in vivo…………….…………….74
Figure 2.8: MHC class II is not present on endogenous CD8 T cells
that do not respond to LCMV infection in vivo……………...76
Figure 2.9: CD4 T cells stimulation with activated CD8 T cells is not due
to DC contamination...…………………………………….....77
Figure 2.10: No difference in absolute numbers between adoptively
transferred helped or unhelped CD8 T cells before
delayed challenge………………………………………….....79
Chapter 3
Figure 3.1: Responder CD8 T cell locate to different lymphoid tissues
than bystanders CD8 T cells and divide up to 10 times
during the first 3 days of LCMV infection……….……..…..105
Figure 3.2: Responder CD8 T cells from bone marrow display a memory
precursor phenotype early during LCMV infection………...107
Figure 3.3: Endogenous CD8 T cells from bone marrow responding
to LCMV infection also display lower CD25 expression…...108
Figure 3.4: Lower percentage of IFNg-producer responder CD8 T cells
are found in bone marrow than in spleen ……….…………..109
Figure 3.5: Phenotype of bystander CD8 T cells remains unchanged
after 3 days of LCMV infection...…………………………...110
Figure 3.6: Responder CD8 T cells show similar activation phenotype
in PLN, MLN and Liver after 3 days of LCMV infection.….111
Figure 3.7: CD8 T cells responding to LCMV show similar differentiation
markers in spleen, PLN and bone marrow …………….....…112
Figure 3.8: Phenotype of endogenous bystander CD8 T cells remains
unchanged after 3 days of LCMV infection regardless of
tissue location ……………………………………………....113
Figure 3.9: Responder CD8 T cells display similar Eomesodermin in
different lymphoid tissues after 3 days of LCMV infection..114

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