Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation

In response to infection, CD8(+) T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127(low)KLRG1(high)) and memory precursor effector cells (CD127(high)KLRG1(low)) from an early effector cell that is CD127(low)KLRG1(low) in phenotype. CD8(+) T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12-dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8(+) T cell functionality and memory subset distribution, but in an IL-12-independent manner. Population dynamics were dramatically different during secondary CD8(+) T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127(high)KLRG1(high) memory cells, both of which were intrinsic to the memory CD8(+) T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8(+) T cell differentiation.     

Molecular and functional profiling of memory CD8 T cell differentiation

How and when memory T cells form during an immune response are long-standing questions. To better understand memory CD8 T cell development, a time course of gene expression and functional changes in antigen-specific T cells during viral infection was evaluated. The expression of many genes continued to change after viral clearance in accordance with changes in CD8 T cell functional properties. Even though memory cell precursors were present at the peak of the immune response, these cells did not display hallmark functional traits of memory T cells. However, these cells gradually acquired the memory cell qualities of self-renewal and rapid recall to antigen suggesting the model that antigen-specific CD8 T cells progressively differentiate into memory cells following viral infection.     

Regulation of effector and memory CD8+ T cell function by inflammatory cytokines

Cells communicate with each other through the production and secretion of cytokines, which are integral to the host response to infection. Once recognized by specific cytokine receptors expressed on the cell surface, these exogenous signals direct the biological function of a cell in order to adapt to their microenvironment. CD8⁺ T cells are critical immune cells that play an important role in the control and elimination of intracellular pathogens. Current findings have demonstrated that cytokines influence all aspects of the CD8⁺ T cell response to infection or immunization. The cytokine milieu induced at the time of activation impacts the overall magnitude and function of the effector CD8⁺ T cell response and the generation of functional memory CD8⁺ T cells. This review will focus on the impact of inflammatory cytokines on different aspects of CD8⁺ T cell biology.     

Selective T cell expansion during aging of CD8 memory repertoires to influenza revealed by modeling

The aging of T cell memory is often considered in terms of senescence, a process viewed as decay and loss of memory T cells. How senescence would affect memory is a function of the initial structure of the memory repertoire and whether the clonotypes that make up the repertoire decay at random. We examine this issue using the T cell memory generated to the conserved influenza A epitope M1(58-66), which induces a strong, focused, but polyclonal CD8 T cell response in HLA-A2 individuals. We analyzed the CD8 T cell memory repertoires in eight healthy middle-aged and eight healthy older blood donors representing an average age difference of ～ 27 y. Although the repertoires show broadly similar clonotype distributions, the number of observable clonotypes decreases significantly. This decrease disproportionally affects low-frequency clonotypes. Rank frequency analysis shows the same two-component clonotype distribution described earlier for these repertoires. The first component includes lower frequency clonotypes for which distribution can be described by a power law. The slope of this first component is significantly steeper in the older cohort. Generating a representative repertoire for each healthy cohort allowed agent-based modeling of the aging process. Interestingly, simple senescence of middle-aged repertoires is insufficient to describe the older clonotype distribution. Rather, a selective clonotype expansion must be included to achieve the best fit. We propose that responses to periodic virus exposure may drive such expansion, ensuring that the remaining clonotypes are optimized for continued protection.     

Distinct effects of saracatinib on memory CD8+ T cell differentiation

Immunologic memory involving CD8(+) T cells is a hallmark of an adaptive Ag-specific immune response and constitutes a critical component of protective immunity. Designing approaches that enhance long-term T cell memory would, for the most part, fortify vaccines and enhance host protection against infectious diseases and, perhaps, cancer immunotherapy. A better understanding of the cellular programs involved in the Ag-specific T cell response has led to new approaches that target the magnitude and quality of the memory T cell response. In this article, we show that T cells from TCR transgenic mice for the nucleoprotein of influenza virus NP68 exhibit the distinct phases--priming, expansion, contraction, and memory--of an Ag-specific T cell response when exposed in vitro to the cognate peptide. Saracatinib, a specific inhibitor of Src family kinases, administered at low doses during the expansion or contraction phases, increased CD62L(high)/CD44(high) central memory CD8(+) T cells and IFN-γ production but suppressed immunity when added during the priming phase. These effects by saracatinib were not accompanied by the expected decline of Src family kinases but were accompanied by Akt-mammalian target of rapamycin suppression and/or mediated via another pathway. Increased central memory cells by saracatinib were recapitulated in mice using a poxvirus-based influenza vaccine, thus underscoring the importance of dose and timing of the inhibitor in the context of memory T cell differentiation. Finally, vaccine plus saracatinib treatment showed better protection against tumor challenge. The immune-potentiating effects on CD8(+) T cells by a low dose of saracatinib might afford better protection from pathogens or cancer when combined with vaccine.     

Discriminating between different pathways of memory CD8+ T cell differentiation

Despite the rapid accumulation of quantitative data on the dynamics of CD8(+) T cell responses following acute viral or bacterial infections of mice, the pathways of differentiation of naive CD8(+) T cells into memory during an immune response remain controversial. Currently, three models have been proposed. In the "stem cell-associated differentiation" model, following activation, naive T cells differentiate into stem cell-like memory cells, which then convert into terminally differentiated short-lived effector cells. In the "linear differentiation" model, following activation, naive T cells first differentiate into effectors, and after Ag clearance, effectors convert into memory cells. Finally, in the "progressive differentiation" model, naive T cells differentiate into memory or effector cells depending on the amount of specific stimulation received, with weaker stimulation resulting in formation of memory cells. This study investigates whether the mathematical models formulated from these hypotheses are consistent with the data on the dynamics of the CD8(+) T cell response to lymphocytic choriomeningitis virus during acute infection of mice. Findings indicate that two models, the stem cell-associated differentiation model and the progressive differentiation model, in which differentiation of cells is strongly linked to the number of cell divisions, fail to describe the data at biologically reasonable parameter values. This work suggests additional experimental tests that may allow for further discrimination between different models of CD8(+) T cell differentiation in acute infections.     

Protective CD8 memory T cell responses to mouse melanoma are generated in the absence of CD4 T cell help

Background

We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma.

Methodology and Principal Findings

To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (T_reg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete T_reg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision.

Conclusions and Significance

This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen

Whether memory CD8 T cells can be reactivated in nonlymphoid tissues is unclear. Using mice lacking the spleen, lymph nodes, or both, we show that the secondary T cell response, but not homeostatic maintenance of memory cells, required lymphoid tissue. Whereas primary and secondary CD8 T cell responses to vesicular stomatitis virus infection were lymph node dependent, responses to Listeria monocytogenes infection were driven primarily in the spleen. Memory cell subset reactivation was also regulated by location of the responding population and the pathogen. Thus, CD62Llow effector memory T cells (TEM) cells responded nearly as well as CD62Lhigh central memory T cells (TCM) and TCM cells after L. monocytogenes infection, and both subsets generated equivalent populations of secondary memory cells. In contrast, TCM cells, but not TEM cells, mounted a robust response to vesicular stomatitis virus infection. TCM and TEM cells also required lymphoid tissue to mount recall responses, and the bone marrow did not contribute significantly to the response of either subset. Our findings indicated that characteristics of the infectious agent and the migratory preferences of memory cells dictated the secondary lymphoid tissue requirement for the recall response to infection.     

CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help dependent, it remains controversial when the "help" is needed. In this study, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to vaccinia virus infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to vaccinia virus infection and may help to design effective vaccine strategies.     

Activated iNKT cells promote memory CD8+ T cell differentiation during viral infection

α-Galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8(+) T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8(+) T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8(+) T cells, as a consequence of reduced inflammation.     

Effector CD8 T cell development: a balancing act between memory cell potential and terminal differentiation

Immune responses to infection are optimally designed to generate large numbers of effector T cells while simultaneously minimizing the collateral damage of their potentially lethal actions and generating memory T cells to protect against subsequent encounter with pathogens. Much remains to be discovered about how these equally essential processes are balanced to enhance health and longevity and, more specifically, what factors control effector T cell expansion, differentiation, and memory cell formation. The innate immune system plays a prominent role in the delicate balance of these decisions. Insights into these questions from recent work in the area of effector CD8 T cell differentiation will be discussed.     

Il-12 enhances CD8 T cell homeostatic expansion

The size of the T lymphocyte pool is maintained by regulation of T cell production, proliferation, and survival. Under the pressure of a T lymphopenic environment, mature naive T cells begin to proliferate in the absence of Ag, a process called homeostatic expansion. Homeostatic expansion involves TCR recognition of self peptide/MHC ligands, but less is known about the soluble factors that regulate this process. Here we show that IL-12 dramatically enhanced the homeostatic proliferation of CD8 T cells. In contrast, IL-2 had no beneficial effect on homeostatic expansion and, in fact, inhibited T cell expansion induced by IL-12. Using gene-targeted mice, we showed that IL-12 acted directly on the T cells to enhance homeostatic expansion, but that IL-12 cannot override the requirement for TCR interaction with self peptide/MHC ligands in homeostatic expansion. These data indicate that inflammatory cytokines may modulate T cell homeostasis after lymphopenia and have implications for regulation of the T cell repertoire and autoimmunity.     

TCR affinity promotes CD8+ T cell expansion by regulating survival

Ligation with high affinity ligands are known to induce T lymphocytes to become fully activated effector cells while ligation with low affinity ligands (or partial agonists) may result in a delayed or incomplete response. We have examined the quantitative features of CD8(+) T cell proliferation induced by peptides of different TCR affinities at a range of concentrations in the mouse OT-I model. Both the frequency of cells responding and the average time taken for cells to reach their first division are affected by peptide concentration and affinity. Consecutive division times, however, remained largely unaffected by these variables. Importantly, we identified affinity to be the sole regulator of cell death in subsequent division. These results suggest a mechanism whereby TCR affinity detection can modulate the subsequent rate of T cell growth and ensure the dominance of higher affinity clones over time.     

Strength of stimulus and clonal competition impact the rate of memory CD8 T cell differentiation

The developmental pathways of long-lived memory CD8 T cells and the lineage relationship between memory T cell subsets remain controversial. Although some studies indicate the two major memory T cell subsets, central memory T (T(CM)) and effector memory T (T(EM)), are related lineages, others suggest that these subsets arise and are maintained independently of one another. In this study, we have investigated this issue and examined the differentiation of memory CD8 T cell subsets by tracking the lineage relationships of both endogenous and TCR transgenic CD8 T cell responses after acute infection. Our data indicate that TCR transgenic as well as nontransgenic T(EM) differentiate into T(CM) in the absence of Ag. Moreover, the rate of memory CD8 T cell differentiation from T(EM) into the self-renewing and long-lived pool of T(CM) is influenced by signals received during priming, including Ag levels, clonal competition, and/or the duration of infection. Although some T(EM) appear to not progress to T(CM), the vast majority of T(CM) are derived from T(EM). Thus, long-lasting, Ag-independent CD8 T cell memory results from progressive differentiation of memory CD8 T cells, and the rate of memory T cell differentiation is governed by events occurring early during T cell priming.     

Cutting edge: immediate RANTES secretion by resting memory CD8 T cells following antigenic stimulation

The efficiency of CD8 memory response relies partially on the modification of cellular functional capacities. To identify effector functions that can be modified following priming, we have compared the chemokines produced by naive and memory CD8 T cells. Our results show that in contrast to naive cells, resting memory CD8 T cells contain high levels of RANTES mRNA. As a result, they have the capacity to rapidly secrete RANTES upon ex vivo antigenic stimulation. In contrast to that of IFN-gamma, RANTES secretion is mainly due to the translation of the pre-existing mRNA.     

Suppression of memory CD8 T cell generation and function by tryptophan catabolism

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of T(CM) cells or T(CM) cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, T(CM) cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of T(EM) cells or T(EM) cell-mediated allograft rejection, suggesting that T(EM) cells, unlike T(CM) cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.     

Generation of CD8 T cell memory is regulated by IL-12

Various signals during infection influence CD8 T cell memory generation, but these factors have yet to be fully defined. IL-12 is a proinflammatory cytokine that has been shown to enhance IFN-gamma-producing T cell responses and has been widely tested as a vaccine adjuvant. In this study, we show that IL-12-deficient mice generate a weaker primary CD8 T cell response and are more susceptible to Listeria monocytogenes infection, but have substantially more memory CD8 T cells and greater protective immunity against reinfection. Kinetic analyses show that in the absence of IL-12 there is a reduced contraction of Ag-specific CD8 T cells and a gradual increase in memory CD8 T cells as a result of increased homeostatic renewal. By signaling directly through its receptor on CD8 T cells, IL-12 influences their differentiation to favor the generation of fully activated effectors, but hinders the formation of CD8 T cell memory precursors and differentiation of long-term CD8 T cell memory(.) These results have implications for understanding memory T cell development and enhancing vaccine efficacy, and offer new insight into the role of IL-12 in coordinating the innate and adaptive immune response.