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.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.     

E2F1 and E2F2 are differentially required for homeostasis-driven and antigen-induced T cell proliferation in vivo

Homeostasis-driven T cell proliferation occurs in response to a lymphopenic environment and is mediated by TCR and IL-7 signaling. In this report, we demonstrate a defect in the proliferation of murine naive and memory T cells lacking both E2F1 and E2F2 in response to lymphopenic conditions, suggesting that E2F1 and E2F2 function redundantly downstream of TCR and/or IL-7 signaling during homeostasis-driven proliferation. In contrast, T cell proliferation in response to antigenic stimulation is either unaffected (in vivo) or potentiated (ex vivo) by loss of E2F1 and E2F2, indicating divergent requirements for these E2F factors in T cell proliferation mediated by distinct stimuli. E2F1/E2F2 double knockout (DKO) T cells enter S phase in response to homeostatic signaling, but fail to divide, suggesting that S phase progression is either incomplete or defective. In addition, E2F1/E2F2 DKO mice do not recover normal T cell numbers following exposure to a sublethal dose of radiation, indicating that this defect in homeostasis-driven proliferation is physiologically relevant. Consistent with their failure in cell cycle progression, the differentiation of DKO T cells into memory T cells in response to homeostatic signals is significantly reduced. These observations support the idea that proliferation is required for memory T cell formation and also have implications for the development of clinical strategies to minimize the occurrence of lymphopenia-induced autoimmunity.     

The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance

IL-7 is well known as a lymphopoietic cytokine, but recent studies have also identified a critical role for IL-7 in peripheral T cell homeostasis. IL-7 is well poised to serve as a homeostatic cytokine because it is produced by resting stromal cells, the IL-7R is present on most T cells, and IL-7 down-regulates its own receptor. These features allow IL-7 to signal large numbers of resting T cells and to be efficiently used when supplies are limiting. Consistent with this, in normal hosts, IL-7 is required for survival of naive T cell populations, and IL-7 contributes to homeostatic cycling of naive and memory cells. In addition, lymphopenic hosts accumulate increased levels of IL-7, and the supranormal levels are largely responsible for inducing homeostatic peripheral expansion in response to lymphopenia. Thus, IL-7 plays critical and nonredundant roles in both T cell lymphopoiesis and in maintaining and restoring peripheral T cell homeostasis.     

Expression of IL-17 in human memory CD45RO+ T lymphocytes and its regulation by protein kinase A pathway.

In the present study, the authors compared the interleukin 17 (IL-17 expression of human naive and phenotypically defined memory T cells as well as its regulation by cAMP pathway. Our data showed that IL-17 mRNA was highly expressed in memory human peripheral CD8(+)45RO+T cells and CD4(+)45RO+T cells when peripheral blood mononuclear cells were first stimulated with ionomycin/PMA. IL-17 expression in memory CD8(+)T cells required accessory signals since culture of ionomycin/PMA-activated CD8(+)45RO+T cells alone did not result to IL-17 expression. In contrast, memory CD4(+)T cell population seems to be more independent. IL-17 and interferon gamma(IFN-gamma) mRNA were both inhibited in the presence of PGE2 or the cAMP analogue (dibutyryl-cAMP), while the anti-inflammatory cytokine IL-10 was highly increased. In contrast, naive CD45RA+T cells were unable to express IL-17 whatever the culture conditions. Naive CD4(+)and CD8(+)T cells were sensitive to the PKA regulatory pathway since they represent a significant source of IL-10 when PBMC were first cultured with ionomycin/PMA in the presence of either PGE2 or db-cAMP. The authors showed that naive cells are highly dependent to their microenvironment, since culture of ionomycin/PMA-activated CD45RA+T cells alone did not result in detectable levels of cytokines even in the presence of PGE2. Results also showed that PGE2 induced quite the same levels of intracellular cAMP in naive and memory cells suggesting that these cell populations are equally sensitive to PGE2. However, we suggest that PGE2 may be more efficient in blocking both IL-17 and IFN-gamma expression in already primed memory T cells, rather than in suppressing naive T cells that could represent a significant source of IL-10. Data suggest that PKA activation pathway plays a critical role in the regulation of cytokine profiles and consequently the functional properties of both human naive and memory CD4(+) and CD8(+)T cells during the immune and inflammatory processes.     

CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells

CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

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.     

Differential effects of IL-27 on human B cell subsets

IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL-12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig-stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig-activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation.     

IL-7R alpha gene expression is inversely correlated with cell cycle progression in IL-7-stimulated T lymphocytes

IL-7 plays a major role in T lymphocyte homeostasis and has been proposed as an immune adjuvant for lymphopenic patients. This prospect is based, at least in part, on the short-term expansion of peripheral T cells in rIL7-treated mice and primates. Nevertheless, in vivo, following initial increases in T cell proliferation and numbers, lymphocytes return to a quiescent state. As the bases for this cell cycle exit have not yet been elucidated, it is important to assess the long-term biological effects of IL-7 on quiescent human T lymphocyte subsets. In this study, we find that IL-7-stimulated CD4+ naive lymphocytes enter into cell cycle with significantly delayed kinetics as compared with the memory population. Importantly though, these lymphocytes exit from the cell cycle despite the continuous replenishment of rIL-7. This response is distinct in memory and naive CD4+ lymphocytes with memory cells starting to exit from cycle by day 10 vs day 18 for naive cells. Return to quiescence is associated with a cessation in IL-7R signaling as demonstrated by an abrogation of STAT-5 phosphorylation, despite an up-regulation of surface IL-7Ralpha. Indeed, an initial 10-fold decrease in IL-7Ralpha mRNA levels is followed by increased IL-7Ralpha expression in naive as well as memory T cells, with kinetics paralleling cell cycle exit. Altogether, our data demonstrate that IL-7 promotes the extended survival of both naive and memory CD4+ T cells, whereas cycling of these two subsets is distinct and transient. Thus, IL-7 therapy should be designed to allow optimal responsiveness of naive and memory T cell subsets.     

Conditioning of Langerhans cells induced by a primary CD8 T cell response to self-antigen in vivo

Using a previously described model of autoimmune skin disease, we addressed the question of how CD8 T cell responsiveness to self-Ag is regulated during chronic inflammation. In this model, CD8 T cells expand and induce tissue pathology directed at an epidermal self-Ag. However, we show here that this primary CD8 T cell response prevented subsequent expansion of a second CD8 T cell population with the same specificity. This lack of T cell accumulation was not due to Ag elimination, nor was it due to competition between the two T cell populations. However, skin-specific dendritic cells that present Ag in this model--Langerhans cells--underwent significant phenotypic changes associated with a compromised ability to stimulate naive T cells. Our study suggests that conditioning of dendritic cells may play a role in maintaining unresponsiveness to self-Ag during chronic inflammation.     

Intrinsic differences in the proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune responses

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with IL-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division 20-30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38(+) blasts in a manner that increased with consecutive division number. These differentiated CD38(+) B cells divided faster than did CD38(-) memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38(+) and CD38(-) cells present in cultures of memory B cells was increased by IL-10. In contrast, IL-2 enhanced proliferation of CD38(-) and CD38(+) memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.     

Increased expression of SLAM receptors SLAMF3 and SLAMF6 in systemic lupus erythematosus T lymphocytes promotes Th17 differentiation

Altered T cell function in systemic lupus erythematosus (SLE) is determined by various molecular and cellular abnormalities, including increased IL-17 production. Recent evidence suggests a crucial role for signaling lymphocyte activation molecules (SLAMs) in the expression of autoimmunity. In this study, we demonstrate that SLAMF3 and SLAMF6 expression is increased on the surface of SLE T cells compared with normal cells. SLAM coengagement with CD3 under Th17 polarizing conditions results in increased IL-17 production. SLAMF3 and SLAMF6 T cell surface expression and IL-17 levels significantly correlate with disease activity in SLE patients. Both naive and memory CD4(+) T cells produce more IL-17 in response to SLAM costimulation as compared with CD28 costimulation. In naive CD4(+) cells, IL-17 production after CD28 costimulation peaks on day 3, whereas costimulation with anti-SLAMF3 and anti-SLAMF6 Abs results in a prolonged and yet increasing production during 6 d. Unlike costimulation with anti-CD28, SLAM costimulation requires the presence of the adaptor molecule SLAM-associated protein. Thus, engagement of SLAMF3 and SLAMF6 along with Ag-mediated CD3/TCR stimulation represents an important source of IL-17 production, and disruption of this interaction with decoy receptors or blocking Abs should mitigate disease expression in SLE and other autoimmune conditions.     

Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand black mice

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.     

Persistence and function of central and effector memory CD4+ T cells following infection with a gastrointestinal helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.     

TCR signal transduction in antigen-specific memory CD8 T cells

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.