The emerging role of T cell Ig mucin 1 in alloimmune responses in an experimental mouse transplant model

T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1-specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1-specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1-specific antibody treatment was more effective in Th1-type cytokine-deficient Stat4(-/-) recipients as compared with Th2-type cytokine-deficient Stat6(-/-) recipients. Subtherapeutic doses of rapamycin plus TIM-1-specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1-specific antibody. Importantly, CD4(+)CD25(+) Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive CD4(+)CD25(+) Tregs. Consistent with this, TIM-1-specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance.     

Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones

Interleukin 12 (IL-12) facilitates the generation of a T helper type 1 (Th1) response, with high interferon gamma (IFN-gamma) production, while inhibiting the generation of IL-4-producing Th2 cells in polyclonal cultures of both human and murine T cells and in vivo in the mouse. In this study, we analyzed the effect of IL-12, present during cloning of human T cells, on the cytokine profile of the clones. The culture system used allows growth of clones from virtually every T cell, and thus excludes the possibility that selection of precommitted Th cell precursors plays a role in determining characteristics of the clones. IL-12 present during the cloning procedures endowed both CD4+ and CD8+ clones with the ability to produce IFN-gamma at levels severalfold higher than those observed in clones generated in the absence of IL-12. This priming was stable because the high levels of IFN-gamma production were maintained when the clones were cultured in the absence of IL-12 for 11 d. The CD4+ and some of the CD8+ clones produced variable amounts of IL-4. Unlike IFN-gamma, IL-4 production was not significantly different in clones generated in the presence or absence of IL-12. These data suggest that IL-12 primes the clone progenitors, inducing their differentiation to high IFN-gamma-producing clones. The suppression of IL-4-producing cells observed in polyclonally generated T cells in vivo and in vitro in the presence of IL-12 is not observed in this clonal model, suggesting that the suppression depends more on positive selection of non-IL-4-producing cells than on differentiation of individual clones. However, antigen- specific established Th2 clones that were unable to produce IFN-gamma with any other inducer did produce IFN-gamma at low but significant levels when stimulated with IL-12 in combination with specific antigen or insoluble anti-CD3 antibodies. This induction of IFN-gamma gene expression was transient, because culture of the established clones with IL-12 for up to 1 wk did not convert them into IFN-gamma producers when stimulated in the absence of IL-12. These results suggest that Th clones respond to IL-12 treatment either with a stable priming for IFN- gamma production or with only a transient low level expression of the IFN-gamma gene, depending on their stage of differentiation.     

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-gamma production in mice

Histamine receptor H1 (H1R) is a susceptibility gene in both experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune orchitis (EAO), 2 classical T cell-mediated models of organ-specific autoimmune disease. Here we showed that expression of H1R in naive CD4+ T cells was required for maximal IFN-gamma production but was dispensable for proliferation. Moreover, H1R signaling at the time of TCR ligation was required for activation of p38 MAPK, a known regulator of IFN-gamma expression. Importantly, selective reexpression of H1R in CD4+ T cells fully complemented both the IFN-gamma production and the EAE susceptibility of H1R-deficient mice. These data suggest that the presence of H1R in CD4+ T cells and its interaction with histamine regulates early TCR signals that lead to Th1 differentiation and autoimmune disease.     

Specific regulation of T helper cell 1-mediated murine colitis by CEACAM1

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell surface molecule that has been proposed to negatively regulate T cell function. We have shown that CEACAM1 is associated with specific regulation of T helper cell (Th)1 pathways, T-bet-mediated Th1 cytokine signaling, and Th1-mediated immunopathology in vivo. Mice treated with anti-mouse CEACAM1-specific monoclonal antibody (mAb) CC1 during the effector phase exhibited a reduced severity of trinitrobenzene sulfonic acid colitis in association with decreased interferon (IFN)-gamma production. Although oxazolone colitis has been reported as Th2 mediated, mice treated with the CC1 mAb or a CEACAM1-Fc chimeric protein exhibited a reduced severity of colitis in association with a significant reduction of IFN-gamma and T-bet activation, whereas signal transducer and activator of antigen 4 activation was unaffected. Both interleukin-4 and IFN-gamma gene-deficient mice exhibited less severe colitis induction by oxazolone. Direct ligation of T cells in vitro with the murine hepatitis virus spike protein, a natural ligand for the N-domain of CEACAM1, inhibited the differentiation of naive cells into Th1 but not Th2 cells and activation of Th1 but not Th2 cytokine production. These results indicate that CEACAM1 isoforms are a novel class of activation-induced cell surface molecules on T cells that function in the specific regulation of Th1-mediated inflammation such as that associated with inflammatory bowel disease.     

In vivo priming of CD4 T cells that produce interleukin (IL)-2 but not IL-4 or interferon (IFN)-gamma, and can subsequently differentiate into IL-4- or IFN-gamma-secreting cells

The differentiation of antigen-stimulated naive CD4 T cells into T helper (Th)1 or Th2 effector cells can be prevented in vitro by transforming growth factor (TGF)-beta and anti-interferon (IFN)-gamma. These cells proliferate and synthesize interleukin (IL)-2 but not IFN-gamma or IL-4, and can differentiate into either Th1 or Th2 cells. We have now used two-color Elispots to reveal substantial numbers of primed cells producing IL-2 but not IL-4 or IFN-gamma during the Th1- or Th2-biased immune responses induced by soluble proteins or with adjuvants. These cells were CD4(+)CD44(high) and were present during immediate and long-term immune responses of normal mice. Naive T cell receptor for antigen (TCR) transgenic (DO11.10) T cells were primed in vivo after adoptive transfer into normal hosts and FACS((R)) cloned under conditions that did not allow further differentiation. After clonal proliferation, aliquots of each clone were cultured in Th1- or Th2-inducing conditions. Many in vivo-primed cells were uncommitted, secreting IL-2 but not IL-4 or IFN-gamma at the first cloning step, but secreting either IL-4 or IFN-gamma after differentiation in the appropriate conditions. These in vivo-primed, uncommitted, IL-2-producing cells may constitute an expanded pool of antigen-specific cells that provide extra flexibility for immune responses by differentiating into Th1 or Th2 phenotypes later during the same or subsequent immune responses.     

Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells

The effects exerted on the in vitro development of antigen-specific T cell lines and T cell clones by addition or neutralization of interleukin 12 (IL-12) in lymphocyte bulk culture were examined. T cell lines specific for Dermatophagoides pteronyssinus group I (Der p I) derived in the presence of IL-12 exhibited reduced ability to produce IL-4 and increased ability to produce interferon gamma (IFN-gamma), and developed into Der p I-specific CD4+ T cell clones showing a T helper type 0 (Th0)- or Th1-, instead of Th2-, like cytokine profile. In contrast, purified protein derivative (PPD)-specific T cell lines derived in the presence of anti-IL-12 antibody exhibited an increased ability to produce IL-4 and developed into PPD-specific CD4+ T cell clones showing a Th0-, instead of Th1-, like profile. The influence of IL-12 on the cytokine secretion profile of Der p I-specific T cell lines was not prevented by addition to lymphocyte bulk cultures of anti- IFN-gamma antibody, but could be at least partially inhibited by the removal from bulk cultures of CD16+ cells. Thus, IL-12 and CD16+ cells appear to have inhibitory effects on the development of IL-4-producing cells and to play an inductive role in promoting Th1-like responses.     

EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) maintains the viral episome in all host cells infected with EBV. Recently, EBNA1 was found to be the main EBV latency antigen for CD4+ T cells and could be recognized in cultures from all donors tested. We now identify a polarized Th1 phenotype and obtain evidence for its presence in vivo. When T cells were stimulated with dendritic cells infected with vaccinia vectors expressing EBNA1, 18 of 19 donors secreted IFN-gamma, whereas only two of 19 secreted IL-4. Magnetic selection was then used to isolate cells from fresh blood based on EBNA1-induced cytokine production. Specific IFN-gamma CD4+ cell lines were established from six of six donors and IL-4 lines from three of six. Only the Th1 lines specifically lysed targets expressing three different sources of EBNA1 protein. When the IgG isotype of EBNA1 plasma Ab's was tested, most specific Ab's were IgG1 and of a high titer, confirming a Th1 response to EBNA1 in vivo. Ab's to other microbial antigens generally were not skewed toward IgG1. Given emerging evidence that Th1 CD4+ T cells have several critical roles in host defense to viral infection and tumors, we propose that EBNA1-specific CD4+ Th1 cells contribute to resistance to EBV and EBV-associated malignancies.     

Recombinant interleukin 12 cures mice infected with Leishmania major

Resistant C57BL/6 mice infected with Leishmania major are self-healing, whereas susceptible BALB/c mice fail to contain cutaneous infection and subsequently undergo fatal visceral dissemination. These disparate outcomes are mediated by dissimilar expansions of T helper type 1 (Th1) and Th2 CD4+ T lymphocyte subsets in vivo during cure and progression of disease. Because interleukin 12 (IL-12) has potent T cell growth and interferon gamma (IFN-gamma) stimulatory effects, we studied its effect on CD4+ T cell differentiation during murine leishmaniasis. Treatment with recombinant murine (rMu)IL-12 during the first week of infection cured 89% of normally susceptible BALB/c mice, as defined by decreased size of infected footpads and 1,000-10,000-fold reduced parasite burdens, and provided durable resistance against reinfection. Cure was associated with markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, but preserved or increased production of IFN-gamma relative to untreated mice. IL-4 and IFN-gamma mRNA associated with CD4+ T lymphocytes isolated from infected lymph nodes showed similar reciprocal changes in response to rMuIL-12 therapy. A single injection of anti-IFN-gamma monoclonal antibody abrogated the protective effect of rMuIL-12 therapy and restored Th2 cytokine responses. We conclude that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma- dependent fashion during murine leishmaniasis. Since BALB/c leishmaniasis cannot be cured with rMuIFN-gamma alone, additional direct effects of IL-12 during T cell subset selection are suggested. Because rMuIL-12 is uniquely protective in this well-characterized model of chronic parasitism, differences in IL-12 production may underlie heterogenous host responses to L. major and other intracellular pathogens.     

Interferon gamma signaling alters the function of T helper type 1 cells

One mechanism regulating the ability of different subsets of T helper (Th) cells to respond to cytokines is the differential expression of cytokine receptors. For example, Th2 cells express both chains of the interferon gamma receptor (IFN-gammaR), whereas Th1 cells do not express the second chain of the IFN-gammaR (IFN-gammaR2) and are therefore unresponsive to IFN-gamma. To determine whether the regulation of IFN-gammaR2 expression, and therefore IFN-gamma responsiveness, is important for the differentiation of naive CD4(+) T cells into Th1 cells or for Th1 effector function, we generated mice in which transgenic (TG) expression of IFN-gammaR2 is controlled by the CD2 promoter and enhancer. CD4(+) T cells from IFN-gammaR2 TG mice exhibit impaired Th1 polarization potential in vitro. TG mice also display several defects in Th1-dependent immunity in vivo, including attenuated delayed-type hypersensitivity responses and decreased antigen-specific IFN-gamma production. In addition, TG mice mount impaired Th1 responses against Leishmania major, as manifested by increased parasitemia and more severe lesions than their wild-type littermates. Together, these data suggest that the sustained expression of IFN-gammaR2 inhibits Th1 differentiation and function. Therefore, the acquisition of an IFN-gamma-unresponsive phenotype in Th1 cells plays a crucial role in the development and function of these cells.     

Redirecting human CD4+ T lymphocytes to the MHC class I-restricted melanoma antigen MAGE-A1 by TCR alphabeta gene transfer requires CD8alpha

Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes has demonstrated its effectiveness in the eradication of cancer and virally infected cells. Clinical trails and in vitro studies have focused on CD8+ cytotoxic T-cell receptor (TCR) alphabeta lymphocytes since these cells directly kill virally infected- and tumor cells after antigen-specific recognition via their TCR alphabeta. However, increasing evidence suggests that induction of sustained immunity against cancer and viral infections depends on the presence of tumor- or virus-specific CD4+ T lymphocytes, which are restricted by MHC class II. Here, we show that these MHC class II-restricted CD4+ T lymphocytes can efficiently be redirected to MHC class I-restricted tumor cells by retroviral introduction of an HLA-A1/MAGE-A1-specific chimeric two-chain TCR ValphaCalphazeta/VbetaCbetazeta (tcTCR/zeta). However, TCR-transduced CD4+ T lymphocytes were only able to specifically bind to HLA-A1/MAGE-A1 complexes and respond to HLA-A1+/MAGE-A1+ melanoma cells when the CD8alpha gene was cointroduced. These CD4+/CD8alpha+/TCR(POS) T lymphocytes produce IFN-gamma, TNFalpha and IL-2 when specifically stimulated via the introduced TCR with immobilized HLA-A1/MAGE-A1 complexes or HLA-A1+/MAGE-A1+ melanoma cells. Furthermore, introduction of the CD8alpha gene into TCR(POS) T lymphocytes rendered these T lymphocytes cytotoxic for HLA-A1+/MAGE-A1+ melanoma cells. These results demonstrate that human CD4+ T lymphocytes when genetically grafted with an HLA-A1/MAGE-A1-specific TCR and CD8alpha are induced to kill and produce cytokines upon specific interaction with the relevant melanoma cells. Hence, CD4+ T lymphocytes, in addition to CD8+ T lymphocytes, may be critical effector cells for adoptive immuno-gene therapy to generate a sustained tumor-specific immune response in cancer patients.     

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.     

Dectin-1 diversifies Aspergillus fumigatus-specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation

Pulmonary infection of mice with Aspergillus fumigatus induces concurrent T helper type 1 (Th1) and Th17 responses that depend on Toll-like receptor/MyD88 and Dectin-1, respectively. However, the mechanisms balancing Th1 and Th17 CD4 T cell populations during infection remain incompletely defined. In this study, we show that Dectin-1 deficiency disproportionally increases Th1 responses and decreases Th17 differentiation after A. fumigatus infection. Dectin-1 signaling in A. fumigatus-infected wild-type mice reduces IFN-γ and IL-12p40 expression in the lung, thereby decreasing T-bet expression in responding CD4 T cells and enhancing Th17 responses. Absence of IFN-γ or IL-12p35 in infected mice or T-bet in responding CD4 T cells enhances Th17 differentiation, independent of Dectin-1 expression, in A. fumigatus-infected mice. Transient deletion of monocyte-derived dendritic cells also reduces Th1 and boosts Th17 differentiation of A. fumigatus-specific CD4 T cells. Our findings indicate that Dectin-1-mediated signals alter CD4 T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells, thereby decreasing T-bet expression in A. fumigatus-specific CD4 T cells and enabling Th17 differentiation.     

Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and beta(2)- microglobulin-deficient BALB/c mice

Continuous administration of soluble proteins, delivered over a 10-d period by a mini-osmotic pump implanted subcutaneously, induces a long- lasting inhibition of antigen-specific T cell proliferation in lymph node cells from BALB/c mice subsequently primed with antigen in adjuvant. The decreased T cell proliferative response is associated with a down-regulation of the T helper cell (Th)1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma and with a strong increase in the secretion of the Th2 cytokines IL-4 and IL-5 by antigen specific CD4+ T cells. This is accompanied by predominant inhibition of antigen- specific antibody production of IgG2a and IgG2b, rather than IgG1 isotype. Interestingly, inhibition of Th1 and priming of Th2 cells is also induced in beta(2) microglobulin-deficient BALB/c mice, indicating that neither CD8+ nor CD4+ NK1.1+ T cells, respectively, are required. The polarization in Th2 cells is stably maintained by T cell lines, all composed of CD4+/CD8- cells expressing T cell receptor for antigen (TCR) alpha/beta chains, derived from BALB/c mice treated with continuous antigen administration, indicating that they originate from Th2 cells fully differentiated in vivo. This polarization is induced in BALB/c mice by continuous administration of any protein antigen tested, including soluble extracts from pathogenic microorganisms. Priming of Th2 cells is dose dependent and it is optimal for low rather than high doses of protein. Blocking endogenous IL-4 in vivo inhibits expansion of antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen, indicating the involvement of two independent mechanisms. Consistent with this, Th2 cell development, but not inhibition of Th1 cells, depends on non-major histocompatibility complex genetic predisposition, since the Th2 response is amplified in BALB/c as compared to DBA/2, C3H, or C57BL/6 mice whereas tested. These findings support the hypothesis that continuous release of low amounts of protein antigens from pathogenic microorganisms may polarize the immune response toward a Th2 phenotype in susceptible mouse strains.