A role for the thermal environment in defining co-stimulation requirements for CD4(+) T cell activation.

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever.

Elevation in body temperature to fever range enhances and prolongs subsequent responsiveness of macrophages to endotoxin challenge.

Macrophages are often considered the sentries in innate immunity, sounding early immunological alarms, a function which speeds the response to infection. Compared to the large volume of studies on regulation of macrophage function by pathogens or cytokines, relatively little attention has been devoted to the role of physical parameters such as temperature. Given that temperature is elevated during fever, a long-recognized cardinal feature of inflammation, it is possible that macrophage function is responsive to thermal signals. To explore this idea, we used LPS to model an aseptic endotoxin-induced inflammatory response in BALB/c mice and found that raising mouse body temperature by mild external heat treatment significantly enhances subsequent LPS-induced release of TNF-α into the peritoneal fluid. It also reprograms macrophages, resulting in sustained subsequent responsiveness to LPS, i.e., this treatment reduces "endotoxin tolerance" in vitro and in vivo. At the molecular level, elevating body temperature of mice results in a increase in LPS-induced downstream signaling including enhanced phosphorylation of IKK and IκB, NF-κB nuclear translocation and binding to the TNF-α promoter in macrophages upon secondary stimulation. Mild heat treatment also induces expression of HSP70 and use of HSP70 inhibitors (KNK437 or Pifithrin-µ) largely abrogates the ability of the thermal treatment to enhance TNF-α, suggesting that the induction of HSP70 is important for mediation of thermal effects on macrophage function. Collectively, these results support the idea that there has been integration between the evolution of body temperature regulation and macrophage function that could help to explain the known survival benefits of fever in organisms following infection.

Effector CD8+ T cell IFN-γ production and cytotoxicity are enhanced by mild hyperthermia.

PURPOSE: Clinical trials combining hyperthermia with radiation and/or chemotherapy for cancer treatment have resulted in improved overall survival and control of local recurrences. The contribution of thermally enhanced anti-immune function in these effects is of considerable interest, but not understood; studies on the fundamental effects of elevated temperature on immune effector cells are needed. The goal of this study is to investigate the potential of mild hyperthermia to impact tumour antigen-specific (Ag) effector CD8+ T cell functions. METHOD: Pmel-1 Ag-specific CD8+ T cells were exposed to mild hyperthermia and tested for changes in IFN-γ production and cytotoxicity. Additionally, overall plasma membrane organisation and the phosphorylation of signalling proteins were also investigated following heat treatment. RESULTS: Exposing effector Pmel-1-specific CD8+ T cells to mild hyperthermia (39.5°C) resulted in significantly enhanced Ag-specific IFN-γ production and tumour target cell killing compared to that seen using lower temperatures (33° and 37°C). Further, inhibition of protein synthesis during hyperthermia did not reduce subsequent Ag-induced IFN-γ production by CD8+ T cells. Correlated with these effects, we observed a distinct clustering of GM1(+) lipid microdomains at the plasma membrane and enhanced phosphorylation of LAT and PKCθ which may be related to an observed enhancement of Ag-specific effector CD8+ T cell IFN-γ gene transcription following mild hyperthermia. However, mitogen-mediated production of IFN-γ, which bypasses T cell receptor activation with antigen, was not enhanced. CONCLUSIONS: Antigen-dependent effector T cell activity is enhanced following mild hyperthermia. These effects could potentially occur in patients being treated with thermal therapies. These data also provide support for the use of thermal therapy as an adjuvant for immunotherapies to improve CD8+ effector cell function.

Differentiation of CD8+ T cells into effector cells is enhanced by physiological range hyperthermia.

In this study, we asked whether exposure to different physiologically relevant temperatures (33°C, 37°C, and 39.5°C) could affect subsequent antigen-specific, activation-related events of naive CD8(+) T cells. We observed that temporary exposure of CD62L(hi)CD44(lo) Pmel-1 CD8(+) cells to 39.5°C prior to their antigen-dependent activation with gp100(25-33) peptide-pulsed C57BL/6 splenocytes resulted in a greater percentage of cells, which eventually differentiated into CD62L(lo)CD44(hi) effector cells compared with cells incubated at 33°C and 37°C. However, the proliferation rate of naive CD8(+) T cells was not affected by mild heating. While exploring these effects further, we observed that mild heating of CD8(+) T cells resulted in the reversible clustering of GM1(+) CD-microdomains in the plasma membrane. This could be attributable to a decrease in line tension in the plasma membrane, as we also observed an increase in membrane fluidity at higher temperatures. Importantly, this same clustering phenomenon was observed in CD8(+) T cells isolated from spleen, LNs, and peripheral blood following mild whole-body heating of mice. Further, we observed that mild heating also resulted in the clustering of TCRß and the CD8 coreceptor but not CD71R. Finally, we observed an enhanced rate of antigen-specific conjugate formation with APCs following mild heating, which could account for the difference in the extent of differentiation. Overall, these novel findings may help us to further understand the impact of physiologically relevant temperature shifts on the regulation of antigen-specific CD8(+) T cell activation and the subsequent generation of effector cells.

Essential role of Skp2-mediated p27 degradation in growth and adaptive expansion of pancreatic beta cells.

Diabetes results from an inadequate mass of functional beta cells, due to either beta cell loss caused by immune assault or the lack of compensation to overcome insulin resistance. Elucidating the mechanisms that regulate beta cell mass has important ramifications for fostering beta cell regeneration and the treatment of diabetes. We report here that Skp2, a substrate recognition component of Skp1-Cul1-F-box (SCF) ubiquitin ligase, played an essential and specific role in regulating the cellular abundance of p27 and was a critical determinant of beta cell proliferation. In Skp2(-/-) mice, accumulation of p27 resulted in enlarged polyploid beta cells as a result of endoreduplication replacing proliferation. Despite beta cell hypertrophy, Skp2(-/-) mice exhibited diminished beta cell mass, hypoinsulinemia, and glucose intolerance. Increased insulin resistance resulting from diet-induced obesity caused Skp2(-/-) mice to become overtly diabetic, because beta cell growth in the absence of cell division was insufficient to compensate for increased metabolic demand. These results indicate that the Skp2-mediated degradation pathway regulating the cellular degradation of p27 is essential for establishing beta cell mass and to respond to increased metabolic demand associated with insulin resistance.

Regulatory T cells can mediate their function through the stimulation of APCs to produce immunosuppressive nitric oxide.

Regulatory T cells (Tr cells) play a critical role in inducing immune tolerance. It remains largely unclear how various types of Tr cells perform their regulatory function. We have studied the underlying regulatory mechanism of a population of autoantigen-specific CD4+ Tr cells. These T cells are specific for the glutamic acid decarboxylase p206-220 peptide and are isolated from the diabetes-resistant nonobese-resistant mice. Although these T cells express T-bet and display a Th1 phenotype, they are able to inhibit diabetes. Their regulatory function is dependent on both IFN-gamma and cell contact with target cells. These Tr cells can mediate their cell contact-dependent regulatory function by secreting IFN-gamma which stimulates APCs to produce NO. NO is necessary for the Tr cells to inhibit the proliferation of pathogenic T cells and the development of diabetes. Therefore, we have identified a novel mechanism by which these Tr cells can exert their regulatory function. These results also provide an explanation as to why IFN-gamma may play both pathogenic and immunomodulatory roles in autoimmune diseases.

Maintenance of CD8+ T cells during acute viral infection of the central nervous system requires CD4+ T cells but not interleukin-2.

The JHM strain of mouse hepatitis virus (JHMV) is rapidly cleared from the central nervous system (CNS) by CD8(+) T cells. In the absence of CD4(+) T cells, fewer CD8(+) T cells are found within the CNS in association with a coordinate increase in apoptotic lymphocytes. Previous data suggested that CD4(+) T cells may support CD8(+) T cells through secretion of interleukin-2 (IL-2). To determine the in vivo role of IL-2 during CNS infection, IL-2 signaling was inhibited via administration of a neutralizing IL-2-specific monoclonal antibody (mAb). In contrast to depletion of CD4(+) T cells, inhibition of IL-2 signaling did not influence CD8(+) T cell infiltration, effector cell function or survival within the CNS. These data suggest that the cellular immune response to acute neurotropic JHMV infection requires a distinct CD4(+) T cell component, but is independent of a requirement for IL-2 for induction, activation, recruitment, and/or maintenance of CD8(+) T cells within the CNS during acute infection.

Polarized development of memory cell-like IFN-gamma-producing cells in the absence of TCR zeta-chain.

TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.

Zeta-associated protein of 70 kDa (ZAP-70), but not Syk, tyrosine kinase can mediate apoptosis of T cells through the Fas/Fas ligand, caspase-8 and caspase-3 pathways.

The TCR zeta-chain-associated protein of 70 kDA (ZAP-70) and Syk tyrosine kinases play critical roles in regulating TCR-mediated signal transduction. They not only share some overlapped functions but also may play unique roles in regulating the function and development of T cells. However, it is not known whether they have different effects on the activation and activation-induced cell death of T cells. To address this question, we generated cDNAs encoding chimeric molecules that a tailless TCR zeta-chain was directly linked to truncated ZAP-70 (Z/ZAP) or Syk (Z/Syk) molecules lacking the two Src homology 2 domains. Transfection of these molecules into zeta-chain-deficient cells restored their TCR expression. In addition, Z/ZAP and Z/Syk transfectants but not control cells demonstrated kinase activities in phosphorylating an exogenous substrate specific for ZAP-70 and Syk kinases. Z/ZAP transfectants activated through TCRs underwent a faster time course of apoptosis and had a greater percentage of apoptotic cells than that of Z/Syk and control cells. Activated Z/ZAP transfectants increased Fas and Fas ligand (FasL) expression 3- and 40-fold, respectively. Blocking of the Fas/FasL interaction could inhibit the apoptosis of Z/ZAP transfectants. In contrast, although activated Z/Syk transfectants could increase FasL expression, their Fas expression actually decreased and the percentage of apoptotic cells did not increase. Further studies of the mechanisms revealed that activation of Z/ZAP but not Z/Syk transfectants resulted in rapid activation of caspase-3 and caspase-8 that could also be inhibited by blocking Fas/FasL interaction. These results demonstrated that ZAP-70 and Syk play distinct roles in T cell activation and activation-induced cell death.