IL-12 signaling drives CD8+ T cell IFN-gamma production and differentiation of KLRG1+ effector subpopulations during Toxoplasma gondii Infection

IFN-gamma-producing CD8(+) T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8(+) T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8(+) T cell-intrinsic IL-12 signaling was required for the generation of IFN-gamma-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1(+) effector subpopulations, but not the memory precursor-containing KLRG1(-) effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1(+) effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-gamma expression but also in the development of heterogeneous subpopulations of effector CD8(+) T cells generated in response to the intracellular parasite T. gondii.     

Bcl-2 allows effector and memory CD8+ T cells to tolerate higher expression of Bim

As acute infections resolve, most effector CD8(+) T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8(+) T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8(+) T cells reported to have a longer lifespan (i.e., KLRG1(low)CD127(high)) have increased levels of Bcl-2 compared with their shorter-lived KLRG1(high)CD127(low) counterparts. Surprisingly, we found that these effector KLRG1(low)CD127(high) CD8(+) T cells also had increased levels of Bim compared with KLRG1(high)CD127(low) cells. Similar effects were observed in memory cells, in which CD8(+) central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8(+) effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8(+) T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8(+) T cells. Finally, we found that Bim levels were significantly increased in effector CD8(+) T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.     

KLRG1+NKG2A+ CD8 T cells mediate protection and participate in memory responses during γ-herpesvirus infection

Functional CD8 T cell effector and memory responses are generated and maintained during murine γ-herpesvirus 68 (γHV68) persistent infection despite continuous presentation of viral lytic Ags. However, the identity of the CD8 T cell subpopulations that mediate effective recall responses and that can participate in the generation of protective memory to a γ-herpesvirus infection remains unknown. During γHV68 persistence, ～75% of γHV68-specific CD8 T cells coexpress the NK receptors killer cell lectin-like receptor G1 (KLRG1) and NKG2A. In this study, we take advantage of this unique phenotype to analyze the capacity of CD8 T cells expressing or not expressing KLRG1 and NKG2A to mediate effector and memory responses. Our results show that γHV68-specific KLRG1(+)NKG2A(+) CD8 T cells have an effector memory phenotype as well as characteristics of polyfunctional effector cells such us IFN-γ and TNF-α production, killing capacity, and are more efficient at protecting against a γHV68 challenge than their NKG2A(-)KLRG1(-) counterparts. Nevertheless, γHV68-specific NKG2A(+)KLRG1(+) CD8 T cells express IL-7 and IL-15 receptors, can survive long-term without cognate Ag, and subsequently mount a protective response during antigenic recall. These results highlight the plasticity of the immune system to generate protective effector and proliferative memory responses during virus persistence from a pool of KLRG1(+)NKG2A(+) effector memory CD8 T cells.     

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.     

Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.     

Expression of killer cell lectin-like receptor G1 on antigen-specific human CD8+ T lymphocytes during active, latent, and resolved infection and its relation with CD57

Killer cell lectin-like receptor G1 (KLRG1) is one of several inhibitory killer cell lectin-like receptors expressed by NK cells and T lymphocytes, mainly CD8(+) effector/memory cells that can secrete cytokines but have poor proliferative capacity. Using multiparameter flow cytometry, we studied KLRG1 expression on CD8(+) T cells specific for epitopes of CMV, EBV, influenza, and HIV. Over 92% of CD8(+) cells specific for CMV or EBV expressed KLRG1 during the latent stage of these chronic infections. CD8(+) T cell cells specific for HIV epitopes were mostly (72-89%) KLRG1(+), even though not quite at the level of predominance noted with CMV or EBV. Lower frequency of KLRG1 expression was observed among CD8(+) cells specific for influenza (40-73%), a resolved infection without a latent stage. We further observed that CD8(+) cells expressing CD57, a marker of replicative senescence, also expressed KLRG1; however, a population of CD57(-)KLRG1(+) cells was also identified. This population may represent a "memory" phenotype, because they also expressed CD27, CD28, CCR7, and CD127. In contrast, CD57(+)KLRG1(+) cells did not express CD27, CD28, and CCR7, and expressed CD127 at a much lower frequency, indicating that they represent effector cells that are truly terminally differentiated. The combination of KLRG1 and CD57 expression might thus aid in refining functional characterization of CD8(+) T cell subsets.     

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.     

Functional CD8 T cell memory responding to persistent latent infection is maintained for life

Aging is associated with depressed naive T cell responses, but it is less clear whether T cell memory established early in life also becomes impaired with age. This is particularly important for T cells responding to latent persistent infection, which need to remain functional and capable of controlling the infection over the lifetime; however, repeated stimulation over the lifetime may dysregulate their maintenance or function, potentially contributing to impaired immunity in the elderly. Systemic infection with HSV-1, a persistent latent virus, is associated with memory inflation of virus-specific CD8 T cells. We tested how these inflated memory cells are maintained from adulthood into old age. We found no significant differences in the numbers (i.e., blood, spleen), ex vivo Ag-specific IFN-γ production, and in vivo recall response to HSV-1 (i.e., proliferation, IFN-γ production, cytolysis) between adult and old memory T cells. There was a discrete shift from dominantly effector memory phenotype in the adults to a central memory-like phenotype in the old mice, with fewer old cells expressing the killer cell lectin-like receptor G1 (KLRG1). Adult and old KLRG1(+) memory CD8 T cells were functionally identical: both produced IFN-γ but could minimally proliferate in response to viral challenge. Interestingly, regardless of age, KLRG1(+) cells retained the ability to proliferate and survive in response to homeostatic signals, both in vitro (culture with IL-7 and IL-15) and in vivo (expansion following transfer into lymphopenic recipients). This finding demonstrates that functional effector memory T cells, including those expressing KLRG-1, are maintained and are functional for life, despite the presence of persistent viral infection.     

Differential microenvironment localization of effector and memory CD8 T cells

CD8 T cells are critical for the clearance of intracellular pathogens. Upon infection, naive CD8 T cells differentiate into effector cells that target and eliminate infected cells. Following clearance of the pathogen, most effector cells die, although a small fraction survives to establish a memory population. Subsequent exposure to the same pathogen induces a rapid response of memory T cells and efficient elimination of the pathogen. Although much is known about the CD8 T cell response, the precise microenvironment location of effector and memory CD8 T cells in secondary lymphoid organs is not well characterized. In this study, we present an in situ analysis of the localization of effector and memory CD8 T cells during the murine immune response to lymphocytic choriomenginits virus. We identified the location of these cells using a transgenic mouse model system in which CD8 T cells are irreversibly tagged with yellow fluorescent protein (YFP) after activation. After infection, YFP+ CD8 T cells were initially observed within T cell zones. Later, these cells were found in the red pulp and a disruption of all CD8 T cell zones was observed. After resolution of the immune response, YFP+ memory CD8 T cells were observed primarily in T cells zones. Thus, in the spleens of mice, effector CD8 T cells localize to the red pulp and memory CD8 T cells localize to the T cell zones. Upon rechallenge, memory CD8 T cells rapidly proliferate and the secondary effector CD8 T cells are found in the red pulp.     

OX40 costimulatory signals potentiate the memory commitment of effector CD8+ T cells

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8(+) T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8(+) T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8(+) T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1(low) MPECs, and, subsequently, markedly impaired the generation of memory CD8(+) T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1(low) MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8(+) T cells.     

Progressive loss of memory T cell potential and commitment to exhaustion during chronic viral infection

T cell exhaustion and loss of memory potential occur during many chronic viral infections and cancer. We investigated when during chronic viral infection virus-specific CD8 T cells lose the potential to form memory. Virus-specific CD8 T cells from established chronic infection were unable to become memory CD8 T cells if removed from infection. However, at earlier stages of chronic infection, these virus-specific CD8 T cells retained the potential to partially or fully revert to a memory differentiation program after transfer to infection-free mice. Conversely, effector CD8 T cells primed during acute infection were not protected from exhaustion if transferred to a chronic infection. We also tested whether memory and exhausted CD8 T cells arose from different subpopulations of effector CD8 T cells and found that only the KLRG1(lo) memory precursor subset gave rise to exhausted CD8 T cells. Together, these studies demonstrate that CD8 T cell exhaustion is a progressive developmental process. Early during chronic infection, the fate of virus-specific CD8 T cells remains plastic, while later, exhausted CD8 T cells become fixed in their differentiation state. Moreover, exhausted CD8 T cells arise from the memory precursor and not the terminally differentiated subset of effector CD8 T cells. These studies have implications for our understanding of senescence versus exhaustion and for therapeutic interventions during chronic infection.     

Viral infections induce abundant numbers of senescent CD8 T cells.

Viral infections are often accompanied by extensive proliferation of reactive CD8 T cells. After a defined number of divisions, normal somatic cells enter a nonreplicative stage termed senescence. In the present study we have identified the inhibitory killer cell lectin-like receptor G1 (KLRG1) as a unique marker for replicative senescence of murine CD8 T cells. KLRG1 expression was induced in a substantial portion (30-60%) of CD8 T cells in C57BL/6 mice infected with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus, or vaccinia virus. Similarly, KLRG1 was found on a large fraction of LCMV gp33 peptide-specific TCR-transgenic (tg) effector and memory cells activated in vivo using an adoptive transfer model. Transfer experiments with CFSE-labeled TCR-tg cells into LCMV-infected hosts further indicated that induction of KLRG1 expression required an extensive number of cell divisions. Most importantly, KLRG1(+) TCR-tg effector/memory cells could efficiently lyse target cells and secrete cytokines, but were severely impaired in their ability to proliferate after Ag stimulation. Thus, this study demonstrates that senescent CD8 T cells are induced in abundant numbers during viral infections in vivo.     

Differential Regulation of Effector- and Central-Memory Responses to Toxoplasma gondii Infection by IL-12 Revealed by Tracking of Tgd057-Specific CD8+ T Cells

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-γ-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K^b-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-γ production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-γ recall responses. Importantly, isolated CD62L^hi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-γ-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.