T cells expressing the gamma delta T-cell receptor potentiate coxsackievirus B3-induced myocarditis.

Initial studies determined whether intraperitoneal (i.p.) injection of BALB/c mice with 0.1, 1.0, and 10 mg of adriamycin (a cardiotoxic anthracycline antibiotic) for times ranging between 1 and 9 weeks prior to i.p. injection of 10(5) PFU of coxsackievirus B3 (CVB3) would alter the severity of virus-induced myocarditis. Prior adriamycin exposure enhanced pathogenicity of a poorly pathogenic CVB3 variant (H310A1) but had no effect on myocarditis produced by the pathogenic variant (H3). Cardiac virus concentrations were equivalent in H3- and H310A1-infected mice irrespective of adriamycin treatment. BALB/c mice treated with either 0.1 ml of complete Freund's adjuvant (CFA), 10 mg of adriamycin, or 10(5) PFU of H3 and H310A1 i.p. developed cytolytic Thy 1.2+ lymphocytes (CTL) to H3-infected myocytes 7 days later. CFA-, adriamycin-, and H3-treated mice developed CTL expressing the gamma delta+ T-cell receptors, while H310A1-infected animals did not. Only H3- and H310A1-infected mice developed alpha beta+ CTL. Treatment of BALB/c mice with 0.1 ml of CFA 5 days prior to H310A1 infection dramatically increased myocarditis. Selective depletion of gamma delta+ T cells abrogated this effect. The ability of gamma delta+ T cells to augment the pathogenicity of H310A1 infection was confirmed by adoptive transfer of CFA-stimulated T cells depleted of either gamma delta- or gamma delta+ cells into H310A1-infected recipients.     

Immortalization of human T cells expressing T-cell receptor gamma delta by herpesvirus saimiri.

Herpesvirus saimiri (HVS) has recently been shown to immortalize human CD4+ and CD8+ T cells expressing T-cell receptor alpha beta (TCR-alpha beta) with the maintenance of their original phenotypes and functional properties. However, the immortalization of human T cells expressing TCR-gamma delta by HVS has not been successful. Here we report that HVS can also infect and immortalize human T cells expressing TCR-gamma delta. Two human TCR-gamma delta+ T-cell clones, which continuously proliferated in interleukin-2-containing culture medium without any exogenous stimulation or addition of feeder cells for more than 8 months, were established by HVS infection. Morphologically, the HVS-transformed TCR-gamma delta+ T-cell clones were granular lymphocytes which exhibited wide-range HLA-unrestricted cytotoxicity as untransformed TCR-gamma delta+ T cells. Their phenotypes and cytotoxic activities were not altered during long-term culture. The immortalization of human TCR-gamma delta+ T cells by HVS infection would be useful for functional analysis of this lymphocyte population, which is believed to play an important role in protection against various infectious diseases.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.     

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4 + CD25 + T regulatory cells are able to limit the effectiveness of CD4 + T cells and are able to favor the maintenance and the progression of B. abortus infection.     

Characterization of an acute T lymphoblastic leukemia expressing the gamma/delta T-cell receptor

Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the gamma/delta receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a "natural killer"-like activity can be induced in these cells by IL-2.     

Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.     

Poxvirus CD8+ T-cell determinants and cross-reactivity in BALB/c mice

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+ T-cell determinants for poxviruses in H-2d-haplotype mice. CD8+ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.     

香菇多糖对急性弓形虫感染过程中CD4+CD25+Foxp3+调节性T细胞的调节作用研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染BALB/c小鼠CD4+ CD25+ Foxp3+调节性T细胞(Tregs)数量和功能的调节作用.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的Lent预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,FACS检测Tregs细胞数量的动态变化,ELISA法检测脾细胞培养上清中IL-10的分泌水平.结果 感染前6 d 1 mg/kg Lent用药组与药物未处理组相比显著提高了弓形虫感染小鼠的生存率;具有免疫抑制功能的Tregs数量于感染后8d达峰值,同时免疫应答中关键细胞因子IL-10的分泌水平也于感染后8d和10d显著增加.结论 对急性弓形虫感染的BALB/c小鼠采用Lent预处理之后能有效的调节Tregs的数量和功能,从而调控Th1/Th2之间的动态平衡达到治疗弓形虫的作用,为弓形虫病的临床治疗提供的新的理论依据.     

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.     

Plasmodium chabaudi AS: Distinct CD4+CD25+Foxp3+ regulatory T cell responses during infection in DBA/2 and BALB/c mice

Malaria infections display variation patterns of clinical course and outcome. Although CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells play an essential role in immune homeostasis, the immune regulatory roles involved in malaria infection remains to be elucidated. Herein, we compared the disparity in Treg cells response during the course of blood stage Plasmodium chabaudi chabaudi AS (P. c chabaudi AS) infection in DBA/2 and BALB/c mice. BALB/c mice initiated a Th1/Th2 profile respond to P. c chabaudi AS infection, but DBA/2 mice failed to control P. c chabaudi AS infection and almost of them died post-peak parasitemia. At the peak parasitemia, we found that higher proportion of Treg cells with elevated Foxp3 expression in DBA/2 than in BALB/c mice. We used anti-CD25 mAb to deplete Treg cells and found that the survival time and rate were prolonged in DBA/2 mice treated with anti-CD25 mAb. Treatment with anti-CD25 mAb in vivo led to enhanced pro-inflammation responses and Foxp3 expression decline on Treg cells. In contrast, after DBA/2 was treatment with anti-IL-10R mAb, IL-10R blockade in vivo caused excessive pro-inflammation responses and Foxp3 expression loss on CD4⁺CD25⁺ T cells. Earlier death was found in all of DBA/2 mice with anti-IL-10R mAb. It suggested that IL-2 and IL-10 signal involved in maintaining Foxp3 expression on Treg cells. In all, the moderate suppressive activity of Treg cells may facilitate resistance to P. c chabaudi AS infection.     

Necroinflammatory liver disease in BALB/c background,TGF-beta 1-deficient mice requires CD4+ T cells

The etiology of autoimmune liver disease is poorly understood. BALB/c mice deficient in the immunoregulatory cytokine TGF-beta1 spontaneously develop necroinflammatory liver disease, but the immune basis for the development of this pathology has not been demonstrated. Here, we show that BALB/c-TGF-beta1(-/-) mice exhibit abnormal expansion in hepatic mononuclear cells (MNCs) compared with wild-type littermate control mice, particularly in the T cell and macrophage lineages. To test whether lymphocytes of the adaptive immune system are required for the spontaneous development of necroinflammatory liver disease, BALB/c-TGF-beta1(-/-) mice were rendered deficient in B and T cells by crossing them with BALB/c-recombinase-activating gene 1(-/-) mice. BALB/c-TGF-beta1(-/-)/recombinase-activating gene 1(-/-) double-knockout mice showed extended survival and did not develop necroinflammatory liver disease. The cytolytic activity of BALB/c-TGF-beta1(-/-) hepatic lymphocytes was assessed using an in vitro CTL assay. CTL activity was much higher in BALB/c-TGF-beta1(-/-) hepatic MNCs compared with littermate control hepatic MNCs and was particularly pronounced in the CD4(+) T cell subset. Experimental depletion of CD4(+) T cells in young BALB/c-TGF-beta1(-/-) mice prevented the subsequent development of necroinflammatory liver disease, indicating that CD4(+) T cells are essential for disease pathogenesis in vivo. These data definitively establish an immune-mediated etiology for necroinflammatory liver disease in BALB/c-TGF-beta1(-/-) mice and demonstrate the importance of CD4(+) T cells in disease pathogenesis in vivo. Furthermore, TGF-beta1 has a critical role in homeostatic regulation of the hepatic immune system, inhibiting the development or expansion of hepatic cytolytic CD4(+) T cells.     

Relaxed DM requirements during class II peptide loading and CD4+ T cell maturation in BALB/c mice

Current ideas about DM actions have been strongly influenced by studies of mutant strains expressing the H-2(b) haplotype. To evaluate DM contributions to class II activities in BALB/c mice, we generated a novel mutation at the DMa locus via embryonic stem cell technology. Unlike long-lived A(b)/class II-associated invariant chain-derived peptide (CLIP) complexes, mature A(d) and E(d) molecules are loosely occupied by class II-associated invariant chain-derived peptide and are SDS unstable. BALB/c DM mutants weakly express BP107 conformational epitopes and toxic shock syndrome toxin-1 superantigen-binding capabilities, consistent with partial occupancy by wild-type ligands. Near normal numbers of mature CD4(+) T cells fail to undergo superantigen-mediated negative selection, as judged by TCR Vbeta usage. Ag presentation assays reveal consistent differences for A(d)- and E(d)-restricted T cells. Indeed, the mutation leads to decreased peptide capture by A(d) molecules, and in striking contrast causes enhanced peptide loading by E(d) molecules. Thus, DM requirements differ for class II structural variants coexpressed under physiological conditions in the intact animal.     

Analysis of signal transducing mechanisms in CD3+ CD4- CD8- cells expressing the putative T cell receptor gamma gene product

The signal transducing mechanisms, involved in the activation of CD3+ WT31- cells bearing the putative products of T cell receptor gamma genes, have been investigated. After stimulation with phytohemagglutinin or with monoclonal antibodies directed against CD2 or CD3 surface molecules, a rapid increase in free cytoplasmic Ca2+ concentration rise was detected in one representative CD3+ WT31- clone and in PEER cell line. Experiments performed in the presence of the Ca2+ chelator EGTA indicated that the free cytoplasmic Ca2+ concentration rise was consequent to an early release of Ca2+ from internal stores, followed by a sustained Ca2+ influx from the extracellular compartment. Moreover, increased levels of inositol-3-phosphate (the putative mobilizer of Ca2+ from the intracellular stores) were observed after stimulation, thus suggesting that activation of this cell subset occurs via the classical inositol-lipid metabolic pathway.     

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.     

CD28 costimulation induces delta opioid receptor expression during anti-CD3 activation of T cells

Previous studies have demonstrated that naive splenic mouse T cells express no or only very low levels of the delta-type opioid receptor (delta OR), but stimulation of mouse splenocytes with Con A results in induction of delta OR mRNA and protein. In this report we have shown that stimulation of highly purified populations of naive mouse T cells with anti-CD3 mAb alone results in T cell activation, as evidenced by sustained IL-2 secretion and cell proliferation, but fails to elicit delta OR expression. However, delta OR expression is induced by costimulation of these very pure T cells with anti-CD3 and anti-CD28 mAbs. The delta OR induction by anti-CD3 and anti-CD28 costimulation was completely blocked by inhibition of phosphatidylinositol 3-kinase with wortmannin. Because phosphatidylinositol 3-kinase activation in T cells is linked to costimulation, these results suggest that induction of delta OR expression during T cell activation is strictly dependent on costimulation. It also appears that costimulatory receptors other than CD28 can provide the signaling required for delta OR expression because delta OR mRNA was induced by Con A stimulation of splenocytes from CD28-deficient mice.     

Hormonal regulation of CD4(+) T-cell responses in coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infection causes significant cardiac inflammation in male, but not female, B1.Tg.Ealpha mice. This gender difference in disease susceptibility correlates with selective induction of CD4(+) Th1 (gamma interferon-positive) cell responses in animals with testosterone, whereas estradiol promotes preferential CD4(+) Th2 (interleukin-4 positive [IL-4(+)]) cell responses. Differences in immune deviation of CD4(+) T cells cannot be explained by variation in B7-1 or B7-2 expression. Infection significantly upregulated both molecules, but no differences were detected between estradiol- and testosterone-treated groups. Significantly increased numbers of activated (CD69(+)) T cells expressing the gammadelta T-cell receptor were found in male and testosterone-treated male and female mice. In vivo depletion of gammadelta+ cells by using monoclonal antibodies inhibited myocarditis and resulted in a shift from a Th1 to Th2 response phenotype. Taken together, our results indicate that testosterone promotes a CD4(+) Th1 cell response and myocarditis by promoting increased gammadelta+ cell activation.     

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.