A novel role of IL-15 in early activation of memory CD8+ CTL after reinfection

A rapid induction of effector functions in memory T cells provides rapid and intensified protection against reinfection. To determine potential roles of IL-15 in early expansion and activation of memory CD8+ T cells in secondary immune response, we examined the cell division and cytotoxicity of memory CD8+ T cells expressing OVA(257-264)/Kb-specific TCR that were transferred into IL-15-transgenic (Tg) mice, IL-15 knockout (KO) mice, or control C57BL/6 mice followed by challenge with recombinant Listeria monocytogenes expressing OVA (rLM-OVA). In vivo CTL activities and expression of granzyme B of the transferred CD8+ T cells were significantly higher in the IL-15 Tg mice but lower in the IL-15 KO mice than those in control mice at the early stage after challenge with rLM-OVA. In contrast, there was no difference in the cell division in IL-15 Tg mice and IL-15 KO mice compared with those in control mice. In vivo administration of rIL-15 conferred robust protection against reinfection via induction of granzyme B in the memory CD8+ T cells. These results suggest that IL-15 plays an important role in early activation of memory CD8+ T cells.     

IL-15 serves as a costimulator in determining the activity of autoreactive CD8 T cells in an experimental mouse model of graft-versus-host-like disease

To elucidate the mechanisms controlling peripheral tolerance, we established two transgenic (Tg) mouse strains expressing different levels of membrane-bound OVA (mOVA) as a skin-associated self-Ag. When we transferred autoreactive TCR-Tg CD8 T cells (OT-I cells), keratin 14 (K14)-mOVA(high) Tg mice developed autoreactive skin disease (graft-vs-host disease (GVHD)-like skin lesions) while K14-mOVA(low) Tg mice did not. OT-I cells in K14-mOVA(high) Tg mice were fully activated with full development of effector function. In contrast, OT-I cells in K14-mOVA(low) Tg mice proliferated but did not gain effector function. Exogenous IL-15 altered the functional status of OT-I cells and concomitantly induced disease in K14-mOVA(low) Tg mice. Conversely, neutralization of endogenous IL-15 activity in K14-mOVA(high) Tg mice attenuated GVHD-like skin lesions induced by OT-I cell transfer. Futhermore, K14-mOVA(high) Tg mice on IL-15 knockout or IL-15Ralpha knockout backgrounds did not develop skin lesions after adoptive transfer of OT-I cells. These results identify IL-15 as an indispensable costimulator that can determine the functional fate of autoreactive CD8 T cells and whether immunity or tolerance ensues, and they suggest that inhibition of IL-15 function may be efficacious in blocking expression of autoimmunity where a breach in peripheral tolerance is suspected.     

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.     

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.     

Cutting Edge: Limiting amounts of IL-7 do not control contraction of CD4+ T cell responses

During the acute T cell response most effector T cells die while some survive and become memory T cells. Selective expression of CD127 (IL-7Ralpha) on effector T cells has been proposed to engender their survival into the memory pool. We assessed the role of IL-7 in effector T cell survival using MHC class II tetramers to track a CD4+ T cell response following infection with a recombinant vaccinia virus (rVV-2W1S). Exogenous IL-7 prevented the contraction of the 2W1S-specific CD4+ T cell response after rVV-2W1S infection. IL-7 increased proliferation of, and Bcl-2 expression within, 2W1S-specific T cells; the latter was required for IL-7-driven prevention of contraction. Conversely, in vivo neutralization of IL-7 or Bcl-2 did not exacerbate the contraction of 2W1S-specific CD4+ T cells. These data suggest that IL-7 administration may enhance the survival of effector T cells but that IL-7 is not the limiting factor during normal contraction of the response.     

Cutting Edge: Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens

Constitutive presentation of tissue Ags by dendritic cells results in tolerance of autoreactive CD8+ T cells; however, the underlying molecular mechanisms are not well understood. In this study we show that programmed death (PD)-1, an inhibitory receptor of the CD28 family, is required for tolerance induction of autoreactive CD8+ T cells. An antagonistic Ab against PD-1 provoked destructive autoimmune diabetes in RIP-mOVA mice expressing chicken OVA in the pancreatic islet cells, which received naive OVA-specific CD8+ OT-I cells. This effect was mediated by the PD ligand (PD-L) PD-L1 but not by PD-L2. An increased number of effector OT-I cells recovered from the pancreatic lymph nodes of anti-PD-L1-treated mice showed down-regulation of PD-1. Furthermore, the blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect OT-I cell division but enhanced its granzyme B, IFN-gamma, and IL-2 production. Thus, during the presentation of tissue Ags to CD8+ T cells, PD-1/PD-L1 interaction crucially controls the effector differentiation of autoreactive T cells to maintain self-tolerance.     

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.     

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.     

Urinary bladder epithelium antigen induces CD8+ T cell tolerance, activation, and autoimmune response

The effort to explore the specific autoimmune mechanisms of urinary bladder has long been hindered due to a lack of proper animal models. To better elucidate this issue, we developed a novel line of transgenic (Tg) mice, designated as URO-OVA mice, that express the model Ag OVA as a "self"-Ag on the bladder epithelium. URO-OVA mice are naturally tolerant to OVA and show no response to OVA stimulation. Adoptive transfer of naive OVA-specific T cells showed cell proliferation, activation, and infiltration but no bladder histopathology. In contrast, adoptive transfer of activated OVA-specific T cells induced OVA-mediated histological bladder inflammation. Increased mast cells and up-regulated mRNA expressions of TNF-alpha, nerve growth factor, and substance P precursor were also observed in the inflamed bladder. To further facilitate bladder autoimmunity study, we crossbred URO-OVA mice with OVA-specific CD8(+) TCR Tg mice (OT-I mice) to generate a dual Tg line URO-OVA/OT-I mice. The latter mice naturally acquire clonal deletion for autoreactive OT-I CD8(+) T cells (partial deletion in the thymus and severe deletion in the periphery). Despite this clonal deletion, URO-OVA/OT-I mice spontaneously develop autoimmune cystitis at 10 wk of age. Further studies demonstrated that the inflamed bladder contained infiltrating OT-I CD8(+) T cells that had escaped clonal deletion and gained effector functions before developing histological bladder inflammation. Taken together, we demonstrate for the first time that the bladder epithelium actively presents self-Ag to the immune system and induces CD8(+) T cell tolerance, activation, and autoimmune response.     

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.     

Interleukin-4-mediated downregulation of cytotoxic T lymphocyte activity is associated with reduced proliferation of antigen-specific CD8+ T cells

During virus infection, exogenous IL-4 strongly downregulates expression of antiviral cytokines and cytotoxic T lymphocyte (CTL) responses. In this study, we have employed a T cell receptor (TCR) transgenic system to more closely investigate the effect of IL-4 on CTL activity. This system involves mice transgenic for an H2-Kb restricted TCR recognising an ovalbumin (OVA)-specific peptide (OT-I mice), and recombinant vaccinia viruses expressing the gene for OVA (VV-OVA), or OVA together with IL-4 (VV-OVA-IL-4). Spleen cells from OT-I mice were adoptively transferred to irradiated C57BL/6 mice infected with VV-OVA or VV-OVA-IL-4. Five days following transfer, markedly stronger CTL activity was detected in VV-OVA- than in VV-OVA-IL-4-infected recipients. The reduction in CTL activity was associated with a reduction in the number of OVA-specific CD8+ T cells. Proliferation of cells from VV-OVA-IL-4-infected recipients was dramatically reduced, and this is a likely explanation for the IL-4-mediated reduction in the total number of OVA-specific cells and the reduced cytotoxic activity. On a per cell basis, the production of IFNgamma and cytotoxic activity of OVA-specific CD8+ cells was not influenced by IL-4. Taken together, our results indicate that the reduction in CTL activity by exogenous IL-4 is due to a reduced number of antigen-specific effectors, and does not involve a downregulation of effector function of these cells.     

Overexpression of IL-15 in vivo enhances Tc1 response, which inhibits allergic inflammation in a murine model of asthma

IL-15, a pleiotropic cytokine, is involved in the inflammatory responses in various infectious and autoimmune diseases. We have recently constructed IL-15-transgenic (Tg) mice, which have an increased number of memory-type CD8+ T cells in the peripheral lymphoid tissues. In the present study, we found that eosinophilia and Th2-type cytokine production in the airway were severely attenuated in OVA-sensitized IL-15-Tg mice following OVA inhalation. IL-15-Tg mice preferentially developed Tc1 responses mediated by CD8+ T cells after OVA sensitization, and in vivo depletion of CD8+ T cells by anti-CD8 mAb aggravated the allergic airway inflammation in IL-15-Tg mice following OVA inhalation. Adoptive transfer of CD8+ T cells from OVA-sensitized IL-15-Tg mice into normal mice before OVA sensitization suppressed Th2 response to OVA in the normal mice. These results suggest that overexpression of IL-15 in vivo suppresses Th2-mediated-allergic airway response via induction of CD8+ T cell-mediated Tc1 response.     

Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells

Bcl-2 plays a critical role in regulating cell survival and apoptosis. We examined Bcl-2 expression in virus-specific CD8 T cells during the expansion, death, and memory phases of the T cell response following infection of mice with lymphocytic choriomeningitis virus (LCMV). Naive CD8 T cells expressed a basal level of Bcl-2 that was down-regulated in effector CD8 T cells just before the death phase. Bcl-2 levels remained low during the death phase but surviving memory CD8 T cells expressed higher levels of Bcl-2 than naive cells. These changes were shown to occur in LCMV TCR transgenic cells as well as virus-specific CD8 T cells in C57BL/6 and BALB/c mice identified by MHC class I tetramers. In all instances, memory CD8 T cells expressed higher levels of Bcl-2, suggesting that increased Bcl-2 expression plays a role in the long-term maintenance of memory CD8 T cells in vivo.     

The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia.

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2(+)CD8(+) OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1(+) recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVA(I)) containing the K(b) restricted OVA(257-264) epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant K(b) epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8(+) T cells reactive to a murine gamma-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.     

Evidence of a novel IL-2/15R beta-targeted cytokine involved in homeostatic proliferation of memory CD8+ T cells

The homeostasis of memory CD8+ T cells is regulated by cytokines. IL-15 is shown to promote the proliferation of memory CD8+ T cells, while IL-2 suppresses their division in vivo. This inhibitory effect of IL-2 appears to occur indirectly, through other cell populations including CD25+CD4+ T cells; however, the details of this mechanism remain unclear. In this study, we show that 1) both Ag-experienced and memory phenotype CD8+ T cells divided after the depletion of IL-2 in vivo; 2) this division occurred normally and CD44(high)IL-2/15Rbeta(high) CD8+ T cells generated after IL-2 depletion in IL-15 knockout (KO) and in IL-7-depleted IL-15 KO mice; 3) surprisingly, the blockade of IL-2/15Rbeta signaling in IL-2-depleted IL-15 KO mice completely abolished the division of memory CD8+ T cells, although the only cytokines known to act through IL-2/15Rbeta are IL-2 and IL-15; and 4) the expression of IL-2/15Rbeta molecules on memory CD8+ T cells was required for their division induced by IL-2 depletion. These results demonstrate that the depletion of IL-2 in vivo induced memory CD8+ T cell division by an IL-15-independent but by an IL-2/15Rbeta-dependent mechanism, suggesting the existence of a novel IL-2/15Rbeta-utilizing cytokine that acts directly on memory CD8+ T cells to promote cell division.     

Manipulating the rate of memory CD8+ T cell generation after acute infection

Infection with Listeria monocytogenes elicits expansion in numbers of Ag-specific CD8+ T cells, which then undergo programmed contraction. The remaining cells undergo further phenotypic and functional changes with time, eventually attaining the qualities of memory CD8+ T cells. In this study, we show that L. monocytogenes-specific CD8+ T cell populations primed in antibiotic-pretreated mice undergo brief effector phase, but rapidly develop phenotypic (CD127(high), CD43(low)) and functional (granzyme B(low), IL-2-producing) characteristics of memory CD8+ T cells. These early memory CD8+ T cells were capable of substantial secondary expansion in response to booster challenge at day 7 postinfection, resulting in significantly elevated numbers of secondary effector and memory CD8+ T cells and enhanced protective immunity compared with control-infected mice. Although early expansion in numbers is similar after L. monocytogenes infection of antibiotic-pretreated and control mice, the absence of sustained proliferation coupled with decreased killer cell lectin-like receptor G-1 up-regulation on responding CD8+ T cells may explain the rapid effector to memory CD8+ T cell transition. In addition, antibiotic treatment 2 days post-L. monocytogenes challenge accelerated the generation of CD8+ T cells with memory phenotype and function, and this accelerated memory generation was reversed in the presence of CpG-induced inflammation. Together, these data show that the rate at which Ag-specific CD8+ T cell populations acquire memory characteristics after infection is not fixed, but rather can be manipulated by limiting inflammation that will in turn modulate the timing and extent to which CD8+ T cells proliferate and up-regulate killer cell lectin-like receptor G-1 expression.     

Augmented IL-7 signaling during viral infection drives greater expansion of effector T cells but does not enhance memory

IL-7 signals are crucial for the survival of naive and memory T cells, and the IL-7R is expressed on the surface of these cells. Following viral infection, the IL-7R is expressed on only a subset of effector CD8 T cells, and has been demonstrated to be important for the survival of these memory precursors. IL-7 message levels remain relatively constant during the T cell response to lymphocytic choriomeningitis virus, but a short-lived burst of GM-CSF is observed soon after infection. Retroviral expression of a chimeric GM-CSF/IL-7R, in which binding of GM-CSF by T cells leads to IL-7 signaling, allows for the delivery of an IL-7 signal in all effector T cells expressing the receptor. In mice infected with lymphocytic choriomeningitis virus, CD8 and CD4 T cells transduced with this chimeric receptor underwent an enhanced proliferative response compared with untransduced populations in the same host. Similarly, TCR transgenic CD8 cells expressing the chimeric receptor produced higher effector numbers during the peak of the T cell response to infection. Surprisingly, the enhanced proliferation did not lead to higher memory numbers, as the subsequent contraction phase was more pronounced in the transduced cell populations. These findings demonstrate that artificial IL-7 signaling during an infection leads to significantly increased Ag-specific effector T cell numbers, but does not result in increased numbers of memory progeny. The extent of contraction may be dictated by intrinsic factors related to the number of prior cell divisions.     

Increased CD8+ T cell memory to concurrent infection at the expense of increased erosion of pre-existing memory: the paradoxical role of IL-15

The use of cytokines during vaccination, particularly IL-15, is being considered due to the unique ability of IL-15 to enhance the proliferation of memory CD8(+) T cells. However, as homeostatic mechanisms limit excessive lymphocyte expansion, we addressed the consequences of this enhancement of T cell memory by IL-15. Infection of mice with either recombinant Mycobacterium bovis (BCG) expressing IL-15 (BCG-IL-15) or BCG and purified IL-15 resulted in an increased CD44, IL-2Rbeta expression and increased frequency of IFN-gamma-secreting CD8(+) T cells. Surprisingly, the enhancement of memory to concurrent infection by IL-15 exacerbated the attrition of pre-existing memory. Infection of mice with Listeria monocytogenes expressing OVA resulted in potent OVA(257-264)-specific CD8(+) T cell memory, and a challenge of these mice with either BCG-IL-15 or BCG and purified IL-15 resulted in an increased erosion of OVA(257-264)-specific CD8(+) T cell memory, relative to BCG. Enhancement in the erosion of OVA-specific CD8(+) T cell memory by BCG-IL-15 resulted in a consequently greater impairment in protection against a challenge with OVA-expressing tumor cells. We thus raise important questions regarding vaccinations that are aimed at maximizing T cell memory without considering the impact on pre-existing T cell memory.