Constitutive expression of a chimeric receptor that delivers IL-2/IL-15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells

We have prepared transgenic mice whose T cells constitutively express a chimeric receptor combining extracellular human IL-4R and intracellular IL-2Rbeta segments. This receptor can transmit IL-2/IL-15-like signals in response to human, but not mouse, IL-4. We used these animals to explore to what extent functional IL-2R/IL-15R expression controls the capacity of T cells to proliferate in response to IL-2/IL-15-like signals. After activation with Con A, naive transgenic CD8+ and CD4+ T cells respond to human IL-4 as well as to IL-2. Without prior activation, they failed to proliferate in response to human IL-4, although human IL-4 did prolong their survival. Thus, IL-2-induced proliferation of activated T cells requires at least one other Ag-induced change apart from the induction of a functional IL-2R. However, a fraction of CD8+CD44high T cells proliferate in human IL-4 without antigenic stimulation or syngeneic feeder cells. In contrast, CD4+CD44high T cells are not constitutively responsive to human IL-4. We conclude that although all transgenic T cells express a functional chimeric receptor, only some CD8+CD44high T cells contain all molecules required for entry into the cell cycle in response to human IL-4 or IL-15.     

IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

GSTM1 null allele is a risk factor for gastric cancer development in Asians

IL-1 strikingly enhances antigen-driven responses of CD4 and CD8 T cells. It is substantially more effective than LPS and when added to a priming regime of antigen plus LPS, it strikingly enhances cell expansion. The effect is mediated by direct action on CD4 and CD8 T cells; the response occurs when OT-I or OT-II cells are transferred to B6 IL-1R1-/- recipients and only cells that express IL-1 receptors can respond. The major mechanism through which IL-1 enhances responses is by increasing survival of responding cells. IL-1 enhances the proportion of responding CD4 T cells that differentiate into Th17 cells and increases the proportion of responding CD8 cells that express granzyme B. Of a wide range of cytokines tested, only IL-1α and IL-1β mediate this function. The potency of IL-1 as an enhancer of T cell responses suggests that it could act to enhance responses to weak vaccines and that the pathway utilized by IL-1 might be considered in the design of new generations of adjuvants.     

Loss of IL-7 receptor alpha on CD4+ T cells defines terminally differentiated B cell-helping effector T cells in a B cell-rich lymphoid tissue

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Ralpha (CD127) and the common gamma-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4(+) T cells uniformly express CD127, the memory/effector (non-FOXP3(+)) CD4(+) T cells are divided into CD127(+) and CD127(-) cells. The CD127(-) T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127(+) T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127(-) T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127(+) memory T cells, CD127(-) T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127(-) T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.     

IL-2 and IL-4 stimulate different subpopulations of double-negative thymocytes

Double-negative (CD4-/CD8-) thymocytes from young adult mice can be separated into two distinct subpopulations on the basis of the binding of mAb 7D4 directed against the receptor for IL-2. The 7D4+ cells have predominantly nonrearranged TCR beta-chain genes and express incomplete 1.0-kb beta-messages, whereas the 7D4- cells have rearranged beta-genes and express complete 1.3-kb as well as incomplete 1.0-kb beta-messages. These two populations of double-negative thymocytes also differ in their responses to IL-2 and IL-4. The 7D4+ cells are nonresponsive to IL-2 alone or IL-2 plus PMA but they are stimulated to proliferate by the combination of IL-4 and PMA. In contrast, the 7D4- cells vigorously proliferate in response to IL-2 alone or IL-2 plus PMA but they respond poorly to IL-4 alone or IL-4 plus PMA. These results suggest that IL-2 and IL-4 may be involved in the stimulation of immature thymocytes at distinct steps of their differentiation. IL-4 together with PMA stimulate immature thymocytes which seem to express the IL-2R but do not respond to IL-2.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Autocrine regulation of IL-21 production in human T lymphocytes

IL-21 has pathologic function in immune-inflammatory diseases. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21R, and the common gamma-chain. IL-21 is mostly produced by CD4(+) T cells, but molecular mechanisms that regulate IL-21 synthesis are not fully understood. The fact that CD4(+) T cells express high levels of IL-21R and are capable of functionally responding to IL-21 raises the possibility that IL-21 may regulate its own production. We here show that IL-21 enhances IL-21 RNA and protein expression in human peripheral blood CD3(+) T cells in a dose- and time-dependent fashion. Additionally, both IL-7 and IL-15, but not IL-4, induce IL-21, thus suggesting that common gamma-chain signals are not sufficient to promote IL-21 synthesis. Analysis of molecular mechanisms underlying IL-21 induction reveals that IL-21 activates Stat3 and enhances its recruitment to IL-21 gene promoter. Pharmacologic inhibition and knockdown of Stat3 by small interference RNA largely prevent IL-21 induction in IL-21-treated cells. Consistently, IL-21 is inducible in T cells by IL-6, another cytokine that activates Stat3. Finally, we show that IL-21 positively regulates its own expression in human intestinal CD3(+) lamina propria lymphocytes, and blockade of endogenous IL-21 in cultures of CD3(+) lamina propria lymphocytes isolated from patients with Crohn's disease, a chronic inflammatory bowel disease characterized by high IL-21, down-regulates Stat3 activation and IL-21 expression. These data suggest the existence of a positive autocrine loop that could help to amplify and stabilize IL-21-driven, T cell-mediated responses.     

Upregulated IL-7 receptor α expression on colitogenic memory CD4+ T cells may participate in the development and persistence of chronic colitis

We have previously demonstrated that IL-7 is essential for the persistence of colitis as a survival factor of colitogenic IL-7Rα-expressing memory CD4(+) T cells. Because IL-7Rα is broadly expressed on various immune cells, it is possible that the persistence of colitogenic CD4(+) T cells is affected by other IL-7Rα-expressing non-T cells. To test this hypothesis, we conducted two adoptive transfer colitis experiments using IL-7Rα(-/-) CD4(+)CD25(-) donor cells and IL-7Rα(-/-) × RAG-2(-/-) recipient mice, respectively. First, IL-7Rα expression on colitic lamina propria (LP) CD4(+) T cells was significantly higher than on normal LP CD4(+) T cells, whereas expression on other colitic LP immune cells, (e.g., NK cells, macrophages, myeloid dendritic cells) was conversely lower than that of paired LP cells in normal mice, resulting in predominantly higher expression of IL-7Rα on colitogenic LP CD4(+) cells, which allows them to exclusively use IL-7. Furthermore, RAG-2(-/-) mice transferred with IL-7Rα(-/-) CD4(+)CD25(-) T cells did not develop colitis, although LP CD4(+) T cells from mice transferred with IL-7Rα(-/-) CD4(+)CD25(-) T cells were differentiated to CD4(+)CD44(high)CD62L(-) effector-memory T cells. Finally, IL-7Rα(-/-) × RAG-2(-/-) mice transferred with CD4(+)CD25(-) T cells developed colitis similar to RAG-2(-/-) mice transferred with CD4(+)CD25(-) T cells. These results suggest that IL-7Rα expression on colitogenic CD4(+) T cells, but not on other cells, is essential for the development of chronic colitis. Therefore, therapeutic approaches targeting the IL-7/IL-7R signaling pathway in colitogenic CD4(+) T cells may be feasible for the treatment of inflammatory bowel diseases.     

IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype

IL-21, the most recently described member of the common gamma-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-alpha have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-gamma or IL-4 production and can partially block IL-12 induction of IFN-gamma production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44(high), PD-1(low), CD25(low), CD134(low), and CD137(low). Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-gamma.     

Human IL-7: a novel T cell growth factor

IL-7 is a hemopoietic growth factor that induces the proliferation of early B lineage cells. In the course of studies to determine its effect on human bone marrow cells, we noted a marked outgrowth of mature T cells. When T cells from the circulation were cultured with IL-7, a dose-dependent proliferative response was observed. The target cells included both the CD4+ and CD8+ subpopulations of T cells, but the memory T cells (CD45R-) were better responders than unprimed T cells (CD45R+). IL-7 induced the expression of receptors for IL-2 and transferrin and higher levels of the 4F2 activation Ag. Although T cell responses to suboptimal concentrations of IL-7 were enhanced by the addition of IL-2, the proliferative response to IL-7 was not inhibited by neutralizing antibody to the IL-2R (Tac), nor was IL-2 secretion detected in this response. This response pattern of mature T cells suggests an important role for IL-7 in normal T cell physiology in humans.     

The relationship of cytomegalovirus (CMV) infection with circulatory IFN-α levels and IL-7 receptor α expression on CD8+ T cells in human aging

The IL-7 receptor alpha (IL-7Rα) is the high affinity receptor for IL-7 which is essential for T cell homeostasis. We recently reported an age-associated expansion of human effector memory (EM) CD8(+) T cells expressing IL-7Rα low (IL-7Rα(low)), which could be detrimental to hosts by occupying "immunological space". We investigated the potential mechanisms for this phenomenon, focusing on cytomegalovirus (CMV) infection and INF-α. In the elderly (age ≥ 65), CMV infection was associated with a decreased frequency of na?ve CD8(+) T cells as well as with an increased frequency of total EM and IL-7Rα(low) EM CD8(+) T cells. However, in the young (age ≤ 40), this viral infection was associated only with an increased frequency of IL-7Rα(low) EM CD8(+) T cells. There was no association found between CMV immune status and plasma levels of IFN-α. In CMV-infected young and elderly people, INF-α levels had no correlation with the frequency of IL-7Rα(low) EM CD8(+) T cells although this cytokine levels correlated with the frequency of IL-7Rα(low) CD45RA(+) EM CD8(+) T cells in CMV-uninfected elderly people. Our findings suggest that the effect of CMV infection on the frequency of CD8(+) T cell subsets may begin with IL-7Rα(low) EM CD8(+) T cells and spread to other subsets with aging. Also, IFN-α could be associated with the expansion of IL-7Rα(low) CD45RA(+) EM CD8(+) T cells in the CMV-uninfected elderly.     

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.     

Hodgkin's disease and anaplastic large cell lymphoma revisited

In cultures, and in tissues as well, Hodgkin's and Reed-Sternberg (H-RS) cells and anaplastic large cell lymphoma (ALCL) cells are known to express a variety of cytokines, including IL-1, -5, -6, -8, -9, TNF-, GM-CSF, M-CSF, TGF-, CD70, CD80, and CD86. Various numbers of H-RS/ALCL cells may express cytokine receptors (R), such as CD30, CD40, IL-2R (CD25/CD122), IL-6R (CD126), IL-7R (CD127), TNF-R (CD120), TGF--R (CD105/endoglin), M-CSF-R (CD115), and SCF-R (CD117/c-kit receptor). All of these cytokines and cytokine receptors are implicated in the growth regulation of H-RS/ALCL cells, the histopathologic alterations in tissues, and the clinical manifestations in patients with Hodgkin's disease (HD) or ALCL. Many of these cytokines or cytokine receptors also play an important role in the pathogenesis of other types of lymphomas. In this review, we describe the cytokine or cytokine-receptor expression that is diacritic for H-RS/ALCL cells. The identification of such unique cytokine-cytokine receptor interactions is likely to explain the biologic property that distinguishes HD/ALCL from other types of lymphomas. These interactions include those of CD30L-CD30, CD40L-CD40, CD70-CD27, CD80/CD86-CD28, SCF-CD117, IL-9-IL9R, and IL-7-IL-7R. The H-RS/ALCL cells express IL-9 and two cytokine receptors, CD30 and CD117, which are observed infrequently in NHLs. Although IL-7 expression is not restricted to H-RS/ALCL cells, the expression of IL-7 in conjunction with IL-9 and/or CD117 may be regarded as unique for HD/ALCL because of an unusual combination and a synergistic activity among these cytokines. The expression of CD70 and CD80/CD86 (as cytokines) may exert a unique effect in HD because of intimate contact between H-RS cells and CD27/CD28-positive T cells. The expression of these costimulators (CD70 and CD80/CD86) and other adhesion/constimulator molecules such as CD54 and CD58, along with the secretion of soluble cytokines such as Il-1, IL-6, IL-7, or TNFs by H-RS/ALCL cells, could result in the profound T-cell proliferation often seen in lymph nodes involved by HD and some ALCL. On the other hand, the expression of CD30L and CD40L by surrounding T cells may affect the proliferation of H-RS/ALCL cells. The cytokine-cytokine receptor interaction between H-RS cells and T cells via direct cell-cell contact is bidirectional, a situation not commonly seen in NHLs.     

Role of the intracellular domain of IL-7 receptor in T cell development

Signals from the IL-7R are uniquely required for T cell development and maintenance, despite the resemblance of IL-7R to other cytokine receptors and the apparent sharing of common signaling pathways. This unique requirement could either reflect unique expression of IL-7R or IL-7, or it could indicate that the IL-7R delivers unique signals. To determine whether the IL-7R provided unique signals, we exchanged its intracellular domain with that of other cytokine receptors: IL-4R, IL-9R, and prolactin receptor (PRLR). Chimeric receptors were used to reconstitute development of IL-7R(-/-) hemopoietic progenitors by transducing the receptors in retroviral vectors. Whereas IL-7R(-/-) thymocytes are arrested at the double-negative stage, IL-4R, IL-9R, or PRLR all imparted some progression to the double-positive stage. IL-4R and PRLR gave only small numbers of thymocytes, whereas IL-9R gave robust alphabeta T cell development and reconstitution of peripheral CD4 and CD8 cells, indicating that it can duplicate many of the functions of IL-7R. However, IL-9R failed to reconstitute rearrangement of the TCRgamma locus or development of gammadelta T cells. Thus, the IL-7R signals required in the alphabeta T cell lineage (such as survival and proliferation) are not unique to this receptor, whereas rearrangement of the TCRgamma locus may require a signal that is not shared by other receptors.     

Cutting edge: soluble IL-6R is produced by IL-6R ectodomain shedding in activated CD4 T cells

IL-6 trans-signaling via the soluble IL-6R (sIL-6R) plays an important role in the progression of several autoimmune diseases and cancer by providing IL-6-responsiveness to cells lacking IL-6R. However, the potential sources of sIL-6R are less understood. In this study we show that sIL-6R is produced by both naive and memory CD4 T cells upon TCR activation. The production of sIL-6R by activated CD4 T cells is mediated by shedding of the membrane-bound IL-6R, and this process correlates with the expression of the metalloproteinase ADAM17 in these cells. In contrast to CD4 T cells, CD8 T cells do not express ADAM17 and their production of sIL-6R is negligible. Thus, during an immune response CD4 T cells are an important source of sIL-6R. Production of sIL-6R by autoreactive CD4 T cells may contribute to their role in the development of autoimmune disease by conferring IL-6-responsiveness to cells lacking IL-6R such as synoviocytes.     

Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS,IL-4, and IL-21, but not CD28, IL-7, and IL-15 responses

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4(+) T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Ralpha expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.     

Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor

Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R(+)B220(+)CD19(-)NK1.1(-)Ly-6C(-) cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment.