Constitutive expression of IL-7 receptor alpha does not support increased expansion or prevent contraction of antigen-specific CD4 or CD8 T cells following Listeria monocytogenes infection

Expression of IL-7Ralpha (CD127) has been suggested as a major determinant in the survival of memory T cell precursors. We investigated whether constitutive expression of IL-7Ralpha on T cells increased expansion and/or decreased contraction of endogenous Ag-specific CD4 and CD8 T cells following infection with Listeria monocytogenes. The results indicate that constitutive expression of IL-7Ralpha alone was not enough to impart an expansion or survival advantage to CD8 T cells responding to infection, and did not increase memory CD8 T cell numbers over those observed in wild-type controls. Constitutive expression of IL-7Ralpha did allow for slightly prolonged expansion of Ag-specific CD4 T cells; however, it did not alter the contraction phase or protect against the waning of memory T cell numbers at later times after infection. Memory CD4 and CD8 T cells generated in IL-7Ralpha transgenic mice expanded similarly to wild-type T cells after secondary infection, and immunized IL-7Ralpha transgenic mice were fully protected against lethal bacterial challenge demonstrating that constitutive expression of IL-7Ralpha does not impair, or markedly improve memory/secondary effector T cell function. These results indicate that expression of IL-7Ralpha alone does not support increased survival of effector Ag-specific CD4 or CD8 T cells into the memory phase following bacterial infection.     

Duration of infection and antigen display have minimal influence on the kinetics of the CD4+ T cell response to Listeria monocytogenes infection

The T cell response to infection consists of clonal expansion of effector cells, followed by contraction to memory levels. It was previously thought that the duration of infection determines the magnitude and kinetics of the T cell response. However, recent analysis revealed that transition between the expansion and contraction phases of the Ag-specific CD8+ T cell response is not affected by experimental manipulation in the duration of infection or Ag display. We studied whether the duration of infection and Ag display influenced the kinetics of the Ag-specific CD4+ T cell response to Listeria monocytogenes (LM) infection. We found that truncating infection and Ag display with antibiotic treatment as early as 24 h postinfection had minimal impact on the expansion or contraction of CD4+ T cells; however, the magnitudes of the Ag-specific CD4+ and CD8+ T cell responses were differentially affected by the timing of antibiotic treatment. Treatment of LM-infected mice with antibiotics at 24 h postinfection did not prevent generation of detectable CD4+ and CD8+ memory T cells at 28 days after infection, vigorous secondary expansion of these memory T cells, or protection against a subsequent LM challenge. These results demonstrate that events within the first few days of infection stimulate CD4+ and CD8+ T cell responses that are capable of carrying out the full program of expansion and contraction to functional memory, independently of prolonged infection or Ag display.     

Overexpression of IL-15 in vivo increases antigen-driven memory CD8+ T cells following a microbe exposure.

To elucidate potential roles of IL-15 in the maintenance of memory CD8+ T cells, we followed the fate of Ag-specific CD8+ T cells directly visualized with MHC class I tetramers coupled with listeriolysin O (LLO)(91-99) in IL-15 transgenic (Tg) mice after Listeria monocytogenes infection. The numbers of LLO(91-99)-positive memory CD8+ T cells were significantly higher at 3 and 6 wk after infection than those in non-Tg mice. The LLO(91-99)-positive CD8+ T cells produced IFN-gamma in response to LLO(91-99), and an adoptive transfer of CD8+ T cells from IL-15 Tg mice infected with L. monocytogenes conferred a higher level of resistance against L. monocytogenes in normal mice. The CD44+ CD8+ T cells from infected IL-15 Tg mice expressed the higher level of Bcl-2. Transferred CD44+ CD8+ T cells divided more vigorously in naive IL-15 Tg mice than in non-Tg mice. These results suggest that IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8+ T cells following microbial exposure via promotion of cell survival and homeostatic proliferation.     

Role of CD28 for the generation and expansion of antigen-specific CD8(+) T lymphocytes during infection with Listeria monocytogenes.

Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8(+) T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8(+) T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-gamma production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28(-/-) mice we observed significantly reduced numbers of Listeria-specific CD8(+) T cells and only marginal levels of specific IFN-gamma production and cytotoxicity. Although frequencies were also reduced in CD28(-/-) mice during secondary response, we detected a considerable population of Listeria-specific CD8(+) T cells in these mice. In parallel, IFN-gamma production and cytotoxicity were observed, revealing that Listeria-specific CD8(+) T cells in CD28(-/-) mice expressed normal effector functions. Consistent with their impaired CD8(+) T cell activation, CD28(-/-) mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8(+) T cells in listeriosis. However, Ag-specific CD8(+) T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.     

Induction of telomerase activity and maintenance of telomere length in virus-specific effector and memory CD8+ T cells

Acute viral infections induce extensive proliferation and differentiation of virus-specific CD8+ T cells. One mechanism reported to regulate the proliferative capacity of activated lymphocytes is mediated by the effect of telomerase in maintaining the length of telomeres in proliferating cells. We examined the regulation of telomerase activity and telomere length in naive CD8+ T cells and in virus-specific CD8+ T cells isolated from mice infected with lymphocytic choriomeningitis virus. These studies reveal that, compared with naive CD8+ T cells, which express little or no telomerase activity, Ag-specific effector and long-lived memory CD8+ T cells express high levels of telomerase activity. Despite the extensive clonal expansion that occurs during acute lymphocytic choriomeningitis virus infection, telomere length is maintained in both effector and memory CD8+ T cells. These results suggest that induction of telomerase activity in Ag-specific effector and memory CD8+ T cells is important for the extensive clonal expansion of both primary and secondary effector cells and for the maintenance and longevity of the memory CD8+ T cell population.     

Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers

Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8(+) T cell memory, but exhibit reduced levels of protection against virulent LM. In this study, Ag-specific CD8(+) T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM. Perforin deficiency resulted in an approximately 5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8(+) T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time. However, perforin-deficient CD8(+) T cells exhibited reduced in vivo cytotoxic function compared to WT CD8(+) T cells. Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice. Thus, increasing the number of memory CD8(+) T cells can overcome diminished per-cell protective immunity in the absence of perforin.     

Innate immune cells contribute to the IFN-gamma-dependent regulation of antigen-specific CD8+ T cell homeostasis

IFN-gamma has a dual function in the regulation of T cell homeostasis. It promotes the expansion of effector T cells and simultaneously programs their contraction. The cellular mechanisms leading to this functional dichotomy of IFN-gamma have not been identified to date. In this study we show: 1) that expansion of wild-type CD8+ T cells is defective in IFN-gamma-deficient mice but increased in IFN-gammaR-deficient mice; and 2) that contraction of the effector CD8+ T cell pool is impaired in both mouse strains. Furthermore, we show that CD11b+ cells responding to IFN-gamma are sufficient to limit CD8+ T cell expansion and promote contraction. The data presented here reveal that IFN-gamma directly promotes CD8+ T cell expansion and simultaneously induces suppressive functions in CD11b+ cells that counter-regulate CD8+ T cell expansion, promote contraction, and limit memory formation. Thus, innate immune cells contribute to the IFN-gamma-dependent regulation of Ag-specific CD8+ T cell homeostasis.     

Differing roles of inflammation and antigen in T cell proliferation and memory generation

Recent studies have demonstrated that viral and bacterial infections can induce dramatic in vivo expansion of Ag-specific T lymphocytes. Although presentation of Ag is critical for activation of naive T cells, it is less clear how dependent subsequent in vivo T cell proliferation and memory generation are upon Ag. We investigated T cell expansion and memory generation in mice infected alternately with strains of Listeria monocytogenes that contained or lacked an immunodominant, MHC class I-restricted T cell epitope. We found substantial differences in the responses of effector and memory T cells to inflammatory stimuli. Although effector T cells undergo in vivo expansion in response to bacterial infection in the absence of Ag, memory T cells show no evidence for such bystander activation. However, Ag-independent expansion of effector T cells does not result in increased memory T cell frequencies, indicating that Ag presentation is critical for effective memory T cell generation. Early reinfection of mice with L. monocytogenes before the maximal primary T cell response induces typical memory expansion, suggesting that the capacity for a memory T cell response exists within the primary effector population. Our findings demonstrate that T cell effector proliferation and memory generation are temporally overlapping processes with differing requirements for Ag.     

A specific role for B cells in the generation of CD8 T cell memory by recombinant Listeria monocytogenes

In this study, we investigated whether B cells play a role in the induction and maintenance of CD8 T cell memory after immunization with an intracellular bacterium, Listeria monocytogenes. Our results show that B cells play a minimal role in the initial activation and Ag-driven expansion of CD8 T lymphocytes. However, absence of B cells results in increased death of activated CD8 T cells during the contraction phase, leading to a lower level of Ag-specific CD8 T cell memory. Once memory is established, B cells are no longer required for the long-term maintenance and rapid recall response of memory CD8 T cells. Increased contraction of Ag-specific CD8 T cells in B cell-deficient mice is not due to impaired CD4 T cell responses since priming of epitope-specific CD4 T cell responses is normal in B cell-deficient mice following L. monocytogenes infection. Furthermore, no exaggerated contraction of Ag-specific CD8 T cells is evident in CD4 knockout mice. Thus, B cells play a specific role in modulating the contraction of CD8 T cell responses following immunization. Elucidation of factors that regulate the death phase may allow us to manipulate this process to increase the level of immunological memory and thus, vaccine efficacy.     

The role of p53 in regulating antiviral T cell responses

It is now well established that viral infections can induce large expansions of Ag-specific CD8(+) T cells. These cells divide very rapidly with an estimated doubling time of approximately 6 h. When virus is cleared, the vast majority of these effector CD8 T cells undergo apoptosis. The remaining memory cells persist at constant levels and provide the basis for the accelerated recall response upon rechallenge. The molecular mechanisms that control the rapid proliferation and death of Ag-specific T cells are poorly understood. Because of its important role in controlling cell proliferation and death, we examined antiviral immune responses in p53(-/-) mice using lymphocytic choriomeningitis virus. We found that effector CD8 and CD4 responses were comparable but that memory levels were slightly higher in -/- mice compared with +/+ mice. The lack of a major difference in virus-specific T cell responses between +/+ and -/- mice suggests that p53 only plays a minor role in regulating the proliferation, apoptosis, and maintenance of Ag-specific T cells. Thus, it appears that the primary function of p53 is in controlling "illegitimate" proliferation and tumor development and not in regulating Ag-specific T cell responses.     

Tyk2 signaling in host environment plays an important role in contraction of antigen-specific CD8+ T cells following a microbial infection

Tyrosine kinase 2 (Tyk2), a member of JAK signal transducer family contributes to the signals triggered by IL-12 for IFN-gamma production. To elucidate potential roles of Tyk2 in generation and maintenance of Ag-specific CD8+ T cells, we followed the fate of OVA-specific CD8+ T cells in Tyk2-deficient (-/-) mice after infection with recombinant Listeria monocytogenes expressing OVA (rLM-OVA). Results showed that the numbers of OVA(257-264)/K(b) tetramer-positive CD8+ T cells in Tyk2(-/-) mice were almost the same as those in Tyk2(+/+) mice at the expansion phase on day 7 but were significantly larger in Tyk2(-/-) mice than those in Tyk2(+/+) mice at the contraction phase on day 10 and at the memory phase on day 60 after infection. The intracellular expression level of active caspase-3 was significantly decreased in the OVA-specific CD8+ T cells of Tyk2(-/-) mice on day 7 compared with those of Tyk2(+/+) mice. Adaptive transfer experiments revealed that Tyk2 signaling in other factors rather than CD8+ T cells played a regulatory role in CD8+ T cell contraction following infection. Administration of exogenous IFN-gamma from day 6 to day 9 restored the CD8+ T cell contraction in Tyk2(-/-) mice after infection with rLM-OVA. These results suggest that Tyk2 signaling for IFN-gamma production in host environment plays an important role in contraction of effector CD8+ T cells following a microbial infection.     

IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival

Mice lacking CD137L (4-1BBL) show normal primary expansion and contraction of the CD8+ T cell response to influenza virus, but exhibit a defect in Ag-specific CD8+ T cell numbers at 3-6 wk postinfection. Previous results showed that the decrease in CD8+ T cell numbers in this model is not due to a programming defect during primary expansion. Thus, it appears that 4-1BB/4-1BBL interactions control the number of surviving CD8+ effector memory cells, late in the primary response. In this report, we asked how 4-1BB on T cells could play a role after Ag has apparently been cleared from the host. We show that IL-15, a cytokine involved in regulation of CD8+ memory T cell survival, induces the expression of 4-1BB on CD8+CD44(high) memory phenotype T cells, but not on CD4+ T cells. The Ag-independent induction of 4-1BB by IL-15 was dependent on MAPK p38 and ERK activation. Transfer of in vitro-generated OT-I CD8+ memory T cells into unimmunized wild-type or 4-1BBL-deficient hosts revealed a 2- to 3-fold survival advantage when 4-1BBL was present, recapitulating the effect seen in the endogenous response to influenza in mice. Decreases in the overall number of memory CD8+ T cells were also observed in the bone marrow of unmanipulated 4-1BBL-deficient mice. These data suggest a model whereby 4-1BB expression on memory CD8+ T cells, perhaps due to encounter with IL-15 in the bone marrow, allows 4-1BB/4-1BBL interactions to maintain memory CD8 T cell survival in the absence of Ag.     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.     

T cell conditioning explains early disappearance of the memory CD8 T cell response to infection

Memory CD8 T cells respond more rapidly to acute intracellular infections than naive CD8 T cells. An understanding of the biological processes involved in memory CD8 T cell recognition of Ag and up-regulation of effector mechanism necessitates analyzing memory CD8 T cells at early time points after infection. In the current study, we show that memory CD8 T cells ostensibly disappear from the spleens, blood, and peripheral organs of mice early after infection with Listeria monocytogenes. This disappearance is critically dependent on Ag, and cell-associated Ag alone can mediate this phenomenon. Further investigations, however, suggest that this disappearance is secondary to T cell-APC interactions, also known as T cell conditioning, and disruption of these putative interactions during splenic processing improves recovery of Ag-specific memory CD8 T cell populations after immunization. Conventional analyses of memory CD8 T cell populations early after infection and possibly in the presence of low levels of Ag (as during chronic infections) may exclude significant numbers of the responding CD8 T cell population.     

IL-15 regulates CD8+ T cell contraction during primary infection

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257-264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L-CD127- effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.     

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.     

A role for IFN-gamma from antigen-specific CD8+ T cells in protective immunity to Listeria monocytogenes

Whether IFN-gamma contributes to the per-cell protective capacity of memory CD8(+) T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8(+) T cells via immunization of wild-type (WT) and IFN-gamma-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8(+) T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8(+) T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8(+) T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8(+) T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8(+) T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8(+) T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8(+) T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-gamma production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-gamma-deficient LM-specific memory CD8(+) T cells.     

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.     

Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells

IL-15 and IL-15Ralpha are required for generation of memory-phenotype CD8 T cells in unimmunized mice. However, the role of IL-15 in primary expansion and generation of Ag-specific memory CD8 T cells in vivo has not been investigated. We characterized the CD8 T cell response against vesicular stomatitis virus (VSV) in IL-15(-/-) and IL-15Ralpha(-/-) mice. Surprisingly, IL-15 was required for primary expansion of VSV-specific CD8 T cells. The generation of VSV-specific memory CD8 T cells was also impaired without IL-15 signaling, and this defect correlated with a decrease in memory CD8 T cell turnover. Despite minimal proliferation without IL-15, a subset of memory cells survived long-term. IL-15Ralpha expression was low on naive CD8 T cells, up-regulated on Ag-specific effector cells, and sustained on memory cells. Thus, IL-15 was important for the generation and the subsequent maintenance of antiviral memory CD8 T cells.