'Subthreshold stimulation' of allospecific delayed hypersensitivity by corneal allografts.

Corneal allografts contain few or no Ia+ Langerhans' cells and consistently fail to elicit allospecific delayed-type hypersensitivity (DTH). The present study examined the hypothesis that the inability of corneal allografts to induce allospecific DTH was the result of active suppression. However, the results indicated that an initial exposure to corneal allografts provided a subthreshold stimulus for the induction of DTH because hosts that received a second pair of corneal grafts 7 days later developed DTH. By contrast, hosts that received all four corneal allografts simultaneously, failed to display DTH to donor alloantigens. Hosts that received a single set of corneal allografts did not display DTH to donor alloantigens, yet their spleen cells were capable of transferring DTH to hosts subsequently stimulated with a single set of corneal grafts. The capacity of Langerhans' cell-free corneal grafts to prime the host so that a second set of corneal grafts stimulated DTH to donor alloantigens was termed 'subthreshold stimulation of DTH'. The ability of sequential sets of corneal grafts to induce DTH was abolished by fixation with 0.2% paraformaldehyde. Surprisingly, the single cell-layered corneal endothelium was found to be the crucial corneal component necessary for the induction of allospecific DTH. This unique spectrum of corneal alloimmunogenicity may contribute to the immunological privilege and high success rate of corneal allografts.     

UVB irradiation renders corneal allografts tolerogenic for allospecific delayed hypersensitivity responses.

Heterotopic corneal allografts treated with ultraviolet (UV) radiation not only failed to elicit allospecific delayed-type hypersensitivity (DTH) responses in mice but rendered the hosts tolerant to subsequent immunization with normally immunogenic corneal allografts. The immunological tolerance induced by UV-treated grafts was cyclophosphamide sensitive and antigen specific. Adoptive transfer studies revealed that the tolerance to donor alloantigens could be transferred with CD4+, CD8- T cells which suppressed alloimmune DTH responses at the afferent but not the efferent limb. The capacity of UV-treated corneal allografts to induce allospecific tolerance was related to UV irradiation and not simply a result of loss of corneal viability. Formalin-fixed corneal allografts could not produce similar tolerization for DTH responses. Selective debridement of either the corneal epithelium or endothelium revealed that the corneal endothelium was the critical layer necessary for UV-dependent tolerance induction. Furthermore, the initial exposure to UV irradiation must occur through the endothelium and not the epithelium. Thus, the single-cell layered corneal endothelium is the target for the immunomodulatory effects of UV irradiation on corneal allografts.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

IL-2 pathway blocking in combination with anti-CD154 synergistically establishes mixed macrochimerism with limited dose of bone marrow cells and prolongs skin graft survival in mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5x10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.     

IL-2协同抗CD45RB抗体调节Treg/Th17细胞的比例并延长小鼠移植皮肤存活时间

目的:研究IL-2在抗CD45RB抗体诱导免疫耐受中对Treg/Th17细胞分化的影响,进一步阐明抗CD45RB抗体诱导免疫耐受的机制。方法:用免疫磁珠分选C57BL/6小鼠脾脏中的CD4+T细胞,在抗CD45RB抗体与IL-2的作用下培养72小时后,流式检测Treg/Th17细胞的变化。以BALB/c小鼠为供体,C57BL/6小鼠为受体建立同种异基因皮肤移植模型,分别给予抗CD45RB抗体及IL-2等治疗,术后1、3、5、7、9天取受体鼠脾细胞,动态检测Treg/Th17细胞的变化;术后第9天取移植皮肤HE染色观察炎性细胞的浸润情况;观察并记录移植皮肤的存活时间。结果:CD4+T细胞在IL-2联合抗CD45RB抗体的作用下培养72小时后,Treg比例升高,Th17细胞比例下降;IL-2联合抗CD45RB抗体治疗后明显延长小鼠移植皮肤的存活时间。结论:IL-2可以明显增强抗CD45RB抗体诱导免疫耐受的形成,使Treg细胞上调,下调Th17细胞,有利于免疫耐受的形成。     

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.     

Early expression of proinflammatory cytokines interleukin-1 and tumor necrosis factor-alpha after corneal transplantation.

This study's aim was to determine the early postoperative expression of proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) by corneal grafts. BALB/c (n = 90) and C57BL/6 (n = 90) murine recipients were grafted with donor corneas from either syngeneic or allogeneic mice. At 7 and 14 days after surgery, corneal grafts were excised and the recipient rims separated from the donor tissue. Corneal segments were cultured and assayed for cytokines by enzyme-linked immunosorbent assay (ELISA). There was profound upregulation in expression of both IL-1alpha and TNF-alpha after corneal transplantation. Among both low-rejecting BALB/c and high-rejecting C57BL/6 hosts, levels of IL-1alpha were significantly (p < 0.01) more marked in allogeneic as compared to syngeneic grafts. TNF-alpha overexpression was similarly more marked in allogeneic as compared to syngeneic grafts in both BALB/c and C57BL/6 hosts, although the difference was generally more marked among high-rejecting C57BL/6 recipients. In the case of both IL-1alpha and TNF-alpha, the principal source of cytokine expression in the transplanted tissue was the recipient rim. There is significant overexpression of both IL-1alpha and TNF-alpha during the first 2 weeks after transplantation in both syngeneic and allogeneic orthotopic corneal grafts. However, whereas in syngeneic grafts cytokine expression generally decreases after the first postoperative week, significantly elevated cytokine levels are sustained in allogeneic grafts, implicating IL-1 and TNF-alpha as mediators of the alloimmune response in corneal transplantation.     

Tim-1-Fc suppresses chronic cardiac allograft rejection and vasculopathy by reducing IL-17 production

Previously, we demonstrated that Tim-1-Fc prevents acute cardiac graft rejection by inhibiting Th1 response. In the present report, we tackled the impact of Tim-1-Fc on Th17 cells in a model of cardiac chronic rejection. Administration of Tim-1-Fc did not result in a detectable impact on innate immunity and regulatory T cells, while it provided protection for Bm12-derive cardiac grafts against chronic rejection in B6 recipients, as manifested by the reduction of inflammatory infiltration along with less severity of vasculopathy. Studies in T-bet^-/- recipients by implanting Bm12-derived cardiac grafts further revealed that Tim-1-Fc significantly protected cardiac grafts from chronic rejection along with attenuated production of IL-17 producing T cells. Depletion of CD4 and CD8 T cells or blockade of IL-17 in T-bet^-/- recipients demonstrated that Tim-1-Fc selectively suppresses Th17 differentiation along with attenuated IL-17 secretion. Together, our data suggest that Tim-1-Fc protects cardiac grafts from chronic rejection by suppressing CD4 Th17 development and functionality. Therefore, Tim-1-Fc might be a potential immunosuppressive agent in the setting of cardiac transplantation.     

Role of Th1 and Th2 cells in anterior chamber-associated immune deviation.

The immunological privilege of the anterior chamber (AC) of the eye is due, at least in part, to a selective antigen-specific down-regulation of delayed-type hypersensitivity (DTH) and a normal induction of antibody responses: a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). This dichotomy in the systemic immune responses is suggestive of a T-helper type-2 (Th2)-dominated immune phenotype in which a Th2 cell population is preferentially activated and cross-regulates T-helper type-1 (Th1) effector elements. This hypothesis was tested by comparing the cytokine pattern of antigen-pulsed spleen cells from mice primed in the anterior chamber with antigens that induce ACAID with responses in hosts primed with antigens that do not induce ACAID. The results indicated that CD4+ spleen cells from hosts primed in the AC with antigens that induce ACAID produced significant quantities of interleukin-10 (IL-10) but insignificant levels of IL-2, IL-4 and interferon-gamma (IFN-gamma). In contrast, hosts primed in the AC with antigens that do not induce ACAID, but instead elicit normal DTH, displayed cytokine patterns indicative of a Th1 response significant quantities of IL-2 and IFN-gamma were produced while IL-4 and IL-10 secretion was insignificantly different from normal controls. The immunological phenotype of the AC-primed hosts could be altered by systemic treatment with antibodies against either a Th1 cytokine (IFN-gamma) or a Th2 cytokine (IL-10). Hosts treated with anti-IL-10 antibody and subsequently primed in the AC with ACAID-inducing antigens developed normal DTH responses, while hosts treated with anti-IFN-gamma antibody and primed in the AC with antigens that normally produce positive DTH responses failed to develop positive DTH collectively the results support the proposition that immune privilege in the AC of the eye is due to the selective activation of a Th2 population that cross-regulates Th1 responses.     

Changes in expression of CD45R during the development of Th1 and Th2 cell lines.

Previous studies using anti-CD45R monoclonal antibodies (mAb) have shown that normal CD4+ T cells can be separated into virgin and memory cells based on their level of expression of CD45R. CD45Rhi (virgin) T cells secrete interleukin (IL)-2 whereas CD45Rlo (memory) T cells secrete both IL-2 and IL-4. In contrast to these results, studies using cultured T cell lines have shown that IL-2-secreting T helper cell type 1 (Th1) cells are CD45Rlo and IL-4-secreting Th2 cells are CD45Rhi. To resolve this discrepancy, and to determine how the Th1 and Th2 cell lines relate to memory CD45Rlo and virgin CD45Rhi CD4+ T cells, we have isolated CD45Rlo cells from alloantigen-primed mice and developed allospecific Th1 and Th2 cell lines. The lymphokines secreted by these lines were then evaluated. Our results show that as CD45Rlo T cells develop into in vitro cell lines which secrete IL-4, they become CD45Rhi. In contrast, CD45Rlo cells that develop into lines which secrete IL-2 maintain their CD45Rlo phenotype. Thus, although both Th1 and Th2 cells arise initially from CD45Rlo cells, Th2 cells become CD45Rhi during prolonged in vitro culture but, Th1 cells do not.     

Interleukin-12 differentially regulates expression of IFN-gamma and interleukin-2 in human T lymphoblasts.

Interleukin-12 (IL-12) is known to upregulate expression of interferon-gamma (IFN-gamma) by activated T cells. However, the effects of IL-12 on production of other Th1-type cytokines are less well defined. In this study, we examined the effects of IL-12 on expression of several cytokines, including IFN-gamma, IL-2, tumor necrosis factor-alpha (TNF-alpha), and IL-10, by primary human CD3(+) T cells. Although purified resting T cells were largely nonresponsive to IL-12 stimulation, anti-CD3-activated T cell blasts were strongly responsive, as demonstrated by the ability of IL-12 to induce Stat4 DNA-binding activity. Restimulation of T lymphoblasts on immobilized anti-CD3 monoclonal antibodies (mAb) induced rapid expression of TNF-alpha mRNA and more gradual increases in mRNA levels for IL-2, IFN-gamma, and IL-10. IL-12 markedly upregulated expression of IFN-gamma and IL-10 but downregulated expression of IL-2 in a dose-dependent and time-dependent manner. The levels of IL-2 produced by IL-12-treated T cells correlated inversely with the levels of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalized IL-2 production by IL-12-treated T cells, confirming that the inhibition of IL-2 production by IL-12 was IL-10 mediated. Thus, IL-12 amplified expression of IFN-gamma and IL-10 and, via its ability to upregulate production of IL-10, inhibited expression of IL-2. These findings demonstrate that IL-12 differentially regulates expression of the Th1-type lymphokines, IFN-gamma and IL-2, in T lymphoblasts.     

Interleukin-10 (IL-10) ameliorates corneal disease in a mouse model of recurrent herpetic keratitis

Interleukin 10 (IL-10), a moderator of Delayed Type Hypersensitivity (DTH) responses, has been demonstrated to be present late in acute HSV corneal infection and may help limit blinding inflammatory lesions there. In contrast, IL-10 is present early in the development of recurrent herpetic stromal keratitis (HSK) lesions in mice. To determine the role of IL-10 and DTH responses in recurrent HSK, we examined DTH responses and disease parameters in latently infected IL-10 knock out (KO) mice, and latently infected normal mice that were untreated or received anti-IL-10 antibodies or recombinant IL-10 following ultraviolet-B stimulated ocular HSV recurrence. Low DTH responses were associated with less severe corneal disease while high DTH responses were associated with greater corneal disease. In IL-10 KO mice, and in normal mice given anti-IL-10 antibodies, corneal opacification was increased and DTH responses were significantly prolonged. Normal mice receiving rIL-10 by ocular and intra-peritoneal routes had less severe corneal lesions. Our results indicate that IL-10 and DTH responses play an important role in the pathogenesis of recurrent HSK in mice.     

beta-estradiol suppresses T cell-mediated delayed-type hypersensitivity through suppression of antigen-presenting cell function and Th1 induction

BACKGROUND: Although an immunomodulatory role for estrogens has long been demonstrated by experimental and clinical observations, the mechanism by which estrogens exert their effect on T cells has not been clearly defined. METHODS: In this study we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on the delayed-type hypersensitivity (DTH) to purified protein derivatives (PPD) and associated immune response in female mice. RESULTS: E2 treatment decreased PPD-specific DTH response, which coincided with a decrease in the leukocytes numbers in the draining lymph nodes (DLN) and spleen compared with control mice. E2 treatment also suppressed the in vitro PPD-specific proliferative response of DLN and spleen cells from PPD-primed mice. The analysis of production and gene expression of cytokines by DLN cells demonstrated that E2 treatment suppressed IL-2 and IFN-gamma production in response to PPD in vitro. In contrast, IL-4 and IL-10 gene expression by DLN cells of E2-treated mice, taken 24 h after in vivo restimulation of mice with PPD, was enhanced. Furthermore, we found that spleen APC from E2-treated mice failed to induce optimum proliferation of the PPD-primed T cells in response to PPD in vitro. The impaired APC function by E2 was not due to induction of suppressor cell activity because addition of the normal spleen APC to APC from E2-treated mice restored the proliferative response of the PPD-primed T cells in response to PPD. CONCLUSION: Our results suggest that the E2-mediated inhibition of DTH reaction is due to a combination of the down regulation of APC function and deviation of the immune response from Th1-type to Th2-type.     

Prevention of acute and chronic allograft rejection by combinations of tolerogenic dendritic cells

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DC) helps to reduce acute allograft rejection. However, this method cannot effectively prevent grafts from infiltration of inflammatory cells and fibrosis, and thus has minimal effect on chronic allograft rejection. In this study, we used mitomycin C (MMC) to generate tolerogenic DC and demonstrated that donor (Balb/c)-derived MMC-DC could induce hyporesponsiveness of recipient (C57BL/6) T cells in vitro, potentially by inducing T-cell apoptosis, decreasing IL-2 and IL-12 secretion, and increasing regulatory T-cell numbers and IL-10 secretion. Furthermore, anti-CD154 monoclonal antibody (mAb) treatment combined with donor-derived MMC-DC prolonged the survival of the allografts in vivo. The mechanisms were similar to those in vitro. Impressively, both acute and chronic rejection were prevented when donor and F1 generation (Balb/c × C57BL/6) derived MMC-DC were injected together with anti-CD154 mAb into recipients before heart allotransplantation. In summary, we showed that donor and F1-derived tolerogenic DC have a synergistic effect on induction and maintenance of T-cell regulation and the secretion of immunosuppressive cytokines. Moreover, adoptive transfer of these two types of DC could inhibit both acute and chronic transplant rejection in mice.     

Switch in Chemokine Receptor Phenotype on Memory T Cells without a Change in the Cytokine Phenotype

Th1 and Th2 cells as defined by their cytokine profile are associated with the expression of the chemokine receptors CCR5 and CCR3, respectively. In committed human memory Th1 cells the cytokine profile is irreversibly expressed. However, it is not known if the chemokine receptor phenotypes of Th1 and Th2 cells are permanently associated to the cytokine profile or if it can be changed. To analyze the possibility of inducing a switch in chemokine receptor phenotype on memory Th cells we used differentiated memory Th cells isolated from synovial tissue (ST) samples of patients with rheumatoid arthritis (RA). Freshly isolated T cells, T-cell lines and T-cell clones from these tissues were manipulated with Th1 (interleukin (IL)-12 + anti IL-4) or Th2 (IL-4 + anti IL-12) inducing conditions. The surface expression of CCR5 and CCR3 was analyzed by flowcytometry and interferon (IFN)-gamma and IL-4 production by ELISA. A Th1-inducing cytokine environment increased the expression of CCR5 in Th1 cells and induced the expression of CCR5 in Th2 cells as compared to culture condition with only IL-2. Induction of CCR5 expression on Th2 clones was associated with secretion of some IFN-gamma. Moreover, the Th2-associated chemokine receptor CCR3 could be expressed on both Th1-dominant cell lines, and clones of Th1 and Th0 type after culture conditions with IL-4. This expression of CCR3 was associated with a reduced IFN-gamma production, but no IL-4 production could be induced. The IL-4-treated Th1 clones had a reduced migratory capacity against chemokines produced by ST cells compared to nonmanipulated T-cell clones. In contrast, the same IL-12-treated Th1 clones showed an increased migratory potential. Induction of the Th2-associated marker CCR3 on memory Th1 cells demonstrates that a change in chemokine receptor phenotype related to the Th2 type can be induced on terminally differentiated Th1 cells, without a change in the cytokine profile.     

Blockade of CD70-CD27 Interaction Inhibits Induction of Allergic Lung Inflammation in Mice

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T-cell activation. In this study, we investigated the effects of neutralizing anti-CD70 monoclonal antibody (mAb) in a murine model of allergic lung inflammation to determine whether CD27 contributes to the development of pathogenic Th2 cells and pulmonary inflammation. BALB/c mice were immunized by an injection of ovalbumin (OVA) with alum adjuvant and challenged with aerosolized OVA in PBS. Some groups of mice were treated with anti-CD70 mAb or control rat IgG during the induction or effector phase. The administration of anti-CD70 mAb during the induction phase, but not the effector phase, reduced eosinophil infiltration in lung tissue compared with control IgG-treated mice. Treatment with anti-CD70 mAb also resulted in the decreased production of Th2 cytokines (IL-4, IL-5, and IL-13) in the bronchoalveolar lavage fluid and draining lymph node cell cultures. We further revealed that antigen-specific CD4 T cells were separated into CD27(+) and CD27(-) populations in the lymph nodes of OVA-immunized DO11.10/Rag-2(-/-) mice. The CD27(+) CD4 T cells produced a high concentration of IFN-γ, representing Th1 cells. In contrast, CD27(-) CD4 T cells produced high concentrations of IL-4, IL-5, and IL-13, representing Th2 cells. Moreover, the population of CD27(-) Th2 cells was significantly reduced by the anti-CD70 mAb treatment. These results indicate an important role for CD27 in the development of pathogenic Th2 cells in a murine model of allergic lung inflammation.     

Suppression of murine thyroiditis via blockade of the CD40-CD40L interaction.

The CD40 ligand (gp39) is transiently expressed on activated CD4+ T cells and mediates cognate helper function by interacting with CD40 on B cells. Increasing evidence suggests, however, critical involvement of gp39 not only in antibody-mediated responses but also in the development of effector T cells. Here, we have investigated the effect of in vivo gp39 blockade on the induction of murine experimental autoimmune thyroiditis (EAT), a T-cell-mediated disease. Over a 5-week period, EAT was induced in SJL mice with thyroglobulin (Tg) and adjuvant. Concomitantly, mice received intraperitoneal (i.p.) injections of MR1, a gp39-specific hamster monoclonal antibody (mAb), at 4-day intervals. Control mice were challenged with Tg but received equivalent doses of hamster immunoglobulin (HIg). It was observed that the control mice developed severe thyroiditis whereas the MR1-treated mice exhibited very low levels of infiltration that were mostly focal in nature. Blockade of gp39 was effective since the Tg-specific IgG titres were low or undetectable in all MR1-treated animals compared with the controls. In addition, upon restimulation with Tg in vitro, lymph node cells (LNC) from Tg-primed, MR1-treated mice proliferated less strongly and secreted significantly lower amounts of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) than LNC from untreated or HIg-treated controls. These results strongly suggest that in vivo blockade of gp39 suppresses EAT by inhibiting the priming of inflammatory Tg-specific T-helper type 1 cells.