CD8 T Cell Differentiation during Viral Infection Open Access

Gill, Amanda (Spring 2023)

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CD8 T cells play a crucial role in antiviral immunity. In order to develop better vaccines and T cell-based therapies, we need to understand how CD8 T cells differentiate in different viral contexts, and what mechanisms regulate their fate and functional capacity. We now have a better understanding of which processes favor effective memory responses during acute infection, as well as which factors lead to sustained antiviral responses during chronic infection.

We examined memory CD8 T cell differentiation using the acute lymphocytic choriomeningitis virus (LCMV) infection model. Through these studies, we were able to define a late-memory phenotype and use this to guide our analyses of the fate decisions occurring within the first few days after infection. We determined that long-lived memory CD8 T cells localized to the splenic white pulp and expressed TCF-1, IL-7Ra, CD62L, CXCR3, and CD28. This phenotype may be of interest as a target for functional immunity following vaccination or infection. We next sought to examine how these cells were generated, so we analyzed CD8 T cell differentiation in the earliest phase of the acute response—before the first division of an activated CD8 T cell, as well as in the first few days following infection. Remarkably, these studies showed heterogeneity emerging in the antigen-specific population within 24 hours. At this time, at least two distinct populations of cells were identified—one population that was more effector-like, and one that appeared to have a memory precursor “precursor” phenotype. This finding suggests that memory fate determining decisions may occur substantially earlier than previously thought, and that interventions to improve memory responses may need to occur concomitantly early.

One of the earliest proteins to be expressed on CD8 T cells following infection is CD69, which is now widely used as a marker of cellular activation and tissue-residency. We investigated which mechanisms might enable such rapid expression. We found that both TCR and type I interferon signaling could upregulate CD69 on antigen-specific CD8 T cells as early as 1 day after infection. Type I interferon also regulated the expression of CD69 on naïve, non-specific CD8 T cells within the same time frame. Surprisingly, we found that naïve CD8 T cells actually harbored high levels of Cd69 message and pre-formed protein. We hypothesize that these intracellular stores enable rapid recruitment of CD8 T cells following an immune insult.

Finally, we investigated the basic mechanisms of immune dysfunction during chronic infection, when antigen is never cleared and classical memory CD8 T cells do not develop. In this case, PD-1+TCF-1+ stem-like CD8 T cells act as critical resource cells that sustain the antiviral response. They also provide the proliferative burst of effector CD8 T cells after PD-1 directed immunotherapy. We sought to determine what happens to the number of these progenitor cells in the setting of increased effector differentiation. Not only were stem-like CD8 T cells maintained following PD-1 blockade, but they actually increased their self-renewal in an mTOR-dependent manner, indicating that the PD-1 inhibitory pathway also regulates this process. At the same time, the stem-cell like transcriptional signature and in vivo functionality remained intact. These findings have broad implications for the improvement of immunotherapy in humans.

Table of Contents

Chapter 1: Introduction. 16

I. CD8 T cell responses during viral infection. 16

II. CD8 T cell differentiation during acute infection. 16

1. Early activation and expansion. 16

2. Contraction. 18

3. Memory precursor differentiation. 18

4. Generation of long-lived memory. 20

5. Memory subsets. 22

5.1 Effector memory vs. central memory. 22

5.2 Tissue-resident memory cells. 23

6. Location and cellular fate. 25

7. Earliest activation and differentiation. 27

III. CD8 T cell differentiation during chronic infection. 29

1. CD8 T cell exhaustion. 29

2. Stem-like CD8 T cells. 31

3. Transitory effectors and terminal differentiation. 32

4. Role of the PD-1 pathway in CD8 T cell exhaustion. 33

5. PD-1 pathway blockade. 35

6. Mechanisms of reinvigoration following PD-1 pathway blockade. 36

Chapter 2: Longitudinal analysis of the phenotype, transcriptional profile, and anatomic location of memory CD8 T cell subsets after acute viral infection. 40

I. Summary. 40

II. Introduction. 41

III. Results. 43

IV. Discussion. 50

V. Materials and Methods. 52

Chapter 3: Early fate decisions and the generation of early memory precursor CD8 T cells. 63

I. Summary. 63

II. Introduction. 63

III. Results. 65

IV. Discussion. 69

V. Materials and Methods. 70

Chapter 4: Intracellular stores facilitate rapid expression of CD69 on antigen-specific and non-specific CD8 T cells following TCR or cytokine stimulation. 82

I. Summary. 82

II. Introduction. 82

III. Results. 85

IV. Discussion. 89

V. Materials and Methods. 91

Chapter 5: PD-1 blockade not only promotes effector cell differentiation, but also increases the self-renewal of stem-like CD8 T cells to maintain the number of progenitor cells. 107

I. Summary. 107

II. Introduction. 107

III. Results. 108

IV. Discussion. 113

V. Materials and Methods. 114

Chapter 6: Discussion. 132

I. Evidence of early memory precursor “precursors”. 132

II. Functional role of CD69 in orchestrating the early immune response. 133

III. PD-1 regulates stem-like CD8 T cells’ self-renewal 134

IV. PD-1 blockade does not functionally or transcriptionally alter stem-like CD8 T cells. 136

References. 137

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