Effect of cyclosporin A on T cell immunity. I. Dose-dependent suppression of different murine T helper cell pathways

Cyclosporin A (CsA) is used as a clinical immunosuppressive agent. Despite its immunosuppressive potential, some studies involving in vivo administration of cyclosporin have failed to verify the immunosuppressive activity of this agent. The present study investigates the effect of different concentrations of CsA added in vitro, or of different doses of CsA administered in vivo, on the ability of murine spleen cells to produce interleukin 2 and to generate cytotoxic T lymphocytes in vitro when stimulated with TNP-self or H-2 alloantigens. The results indicate that self Ia-restricted T helper (Th) cells are more sensitive to lower doses of CsA than Th cells that are allorestricted. Thus, doses of CsA were found (15-30 mg/kg) that inactivated self Ia-restricted Th function, but not other Th or effector function. This Th cell defect could be partially corrected in vitro by addition of Th cell factors to the sensitization cultures. A higher dose (75 mg/kg) of CsA inactivated all detectable T cell responses, and this defect was not corrected by addition of Th cell factors. T cell function returned to normal levels within two weeks of cessation of CsA at all three doses of CsA tested. The selective loss of L3T4 Th function at the lower doses of CsA was associated with a radiosensitive, Ly-2 suppressor T cell that was selective in its action on self Ia-restricted Th cell function. Loss of all T cell function at the higher dose of CsA was associated with a radioresistant non-T suppressor cell that inactivated all T cell function tested. These results are discussed with respect to the selective dose-dependent effects of CsA on Th subsets, on the activation of suppressor cells with similar selectivity, and the implications of these findings on the use of CsA to prevent rejection of tissue allografts.     

Class II major histocompatibility complex-restricted T cell function in CD4-deficient mice

Previously, we and others have demonstrated that CD4-deficient mice have a normal number of T cells and B cells with a significant population of CD4^-8^-TcRαβ⁺ T cells. Surprisingly, however, these mice lacking CD4 show in vivo immunoglobulin isotype class switching from IgM to IgG in response to sheep erythrocytes and vesicular stomatitis virus. In this study we have depleted various subpopulations of T cells in vivo and shown that the population of CD4^-8^-TcRαβ⁺ T cells is responsible for providing “help” in the antibody response of CD4-deficient mice to vesicular stomatitis virus infection. We have used antigen-specific proliferation assays and blocking studies with class I and II major histocompatibility complex (MHC)-specific purified antibodies to show that these cells are class II MHC-restricted in responses against the T cell-dependent antigen keyhole limpet hemocyanin (KLH). Finally, phenotypic analysis of the CD4^- CD8^-thymocytes in CD4-deficient mice shows that these cells have a more mature phenotype than the CD4^-8^- thymocytes in wild type mice. These results indicate that CD4 is not absolutely necessary for positive selection or effector function of class II MHC-restricted helper T cells.     

CD4+ helper T cell depression in autism

CD4+ (helper) T cells are a heterogenous population of lymphocytes including at least two distinct subpopulations. To investigate the possibility that immune abnormalities in some subjects with autism may involve abnormal distributions of CD4+ and/or CD8+ cells, (suppressor) T cells, peripheral blood lymphocytes of 25 autistic subjects were characterized with monoclonal antibodies and flow cytometry. The autistic subjects had a significantly lower percentage and number of CD4+ cells, a lower number of T cells (CD2+ cells) and B cells (CD20+ cells), and a lower percentage and number of total lymphocytes than siblings and normal subjects. The level of blood values for female subjects appeared lower than those for males as compared to normal subjects of the same sex. These results suggest that a decrease in CD4+ cells is associated with autism.     

Self-reactive T cells are present in the peripheral lymphoid tissues of cyclosporin A-treated mice.

Cyclosporin A (CsA) prevents most immature thymocytes from progressing to a mature phenotype and blocks the deletion of T cells that express self-specific TCR in the small population of cells that achieve maturity. The latter effect may explain the paradoxical observation that this immunosuppressive drug can induce autoimmunity in irradiated rodents and humans if administered while new T cells are developing in the thymus. This study shows that the repopulation of the periphery with mature T cells is delayed in irradiated CsA-treated mice, presumably because CsA blocks T cell development in the thymus. The peripheral repertoire of these mice contained self-reactive IL-2 producing T cells that could be detected in a sensitive limiting dilution assay. In addition, self-reactive T cell hybridomas were isolated from the IL-2 receptor+ population present in CsA-treated mice. One of these hybridomas expressed a TCR V beta chain that is normally expressed on thymocytes that are deleted via recognition of self-antigens. Despite the presence of self-reactive T cells that had escaped clonal deletion, CsA-treated mice rarely developed lethal autoimmune disease, implying that a peripheral mechanism of tolerance can prevent the onset of autoimmune disease.     

Transcutaneous immunization with CD40 ligation boosts cytotoxic T lymphocyte mediated antitumor immunity independent of CD4 helper cells in mice

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin‐associated lymphatic tissue to induce immune responses. Employing T‐cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod‐containing vaccination platform (IMI‐Sol) rendering superior primary CD8⁺ and CD4⁺ T‐cell responses. However, it has been unclear whether IMI‐Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI‐Sol and CD40 ligation unleashes fullblown specific T‐cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4⁺ T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI‐Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI.     

CD4+ T cell matters in tumor immunity

CD4+ T cells have been shown to be able to affect tumor growth through both direct and indirect means. In addition, a requirement has been demonstrated for CD4+ T cells in the regulation and induction of T cell memory, and CD4+ suppressor T cells have been identified, stressing a role for CD4+ T cells in the induction and maintenance of antitumor immune responses. A review of the involvement of CD4+ T cells at different stages of tumor immunity is provided, and based on these data we discuss how CD4+ T cell response induction could be incorporated into tumor immunotherapy strategies.     

T helper 1/t helper 2 cell immunity in preeclamptic twin pregnancy.

Preeclampsia has been classified into two types on the basis of the T helper (Th)1/Th2 balance: Th1-predominant type and Th2 predominant type. In this study, we examined the Th1/Th2 ratio in peripheral Th cells in 11 patients with preeclamptic twin pregnancies, 11 normal (nonpreeclamptic) twin pregnancies, 11 preeclamptic singleton pregnancies, and 11 normal singleton pregnancies. The average Th1/Th2 ratio in the patients with preeclamptic twin pregnancy was 8.3 +/- 3.4 (mean +/- SD), which was similar to those in patients with normal singleton and twin pregnancies and significant lower than that in patients with preeclamptic singleton pregnancies (p = 0.003). The present results suggest that the mechanisms of preeclampsia differ between singleton and twin pregnancies.     

CD4+FoxP3+ regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection

The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4⁺FoxP3⁺ Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild‐type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4⁺FoxP3⁺ Treg cells by application of diphtheria toxin. In Treg cell‐depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA‐3⁺ T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)‐4, IL‐5, and IL‐13. In contrast, only a mild increase in the Th1‐associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.     

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.     

Altered aging-related thymic involution in T cell receptor transgenic, MHC-deficient, and CD4-deficient mice

During aging in mice and humans, a gradual decline in thymus integrity and function occurs (thymic involution). To determine whether T cell reactivity or development affects thymic involution, we compared the thymic phenotype in old (12 months) and young (2 months) mice transgenic for rearranged alphabeta or beta 2B4 T cell receptor (TCR) genes, mice made deficient for CD4 by gene targetting (CD4(-/-)), mice made deficient for major histocompatibility complex (MHC) class I (beta2M-/-) or class II genes (A(beta)(b-/-) on C57Bl/6 background) or both. The expected aging-related reductions in thymic weights were observed for all strains except those bearing disruption of both class I and class II MHC genes. Therefore, disruption of MHC class I and class II appeared to reverse or delay aging-related thymic atrophy at 12 months. Immunohistochemical analysis of aging-associated alterations in thymic morphology revealed that TCR alphabeta transgenes, CD4 disruption, and MHC class II disruption all reduced or eliminated these changes. All strains examined at 12 months showed alterations in the distribution of immature thymocyte populations relative to young controls. These results show that aging-associated thymic structural alterations, size reductions, and thymocyte developmental delays can be separated and are therefore causally unrelated. Furthermore, these results suggest that the T cell repertoire and/or its development play a role in aging-related thymic involution.     

Studies on CD4 T cell immunity using somatic transgene immunization

This review presents and discusses our recent data on the use of somatic transgene immunization in the induction of CD4 T cell responses in vivo. Somatic transgene immunization is a process that targets B lymphocytes resident in the spleen with DNA coding for antigenized immunoglobulin H chain genes. After transgenesis B lymphocytes function as antigen-producing and antigen-presenting cells. The studies reviewed herein describe the characteristics of the primary and memory CD4 response against a dominant Th cell determinant. In addition, they show how ad hoc modifications of the transgene result in the induction of a CD4 T cell response against Th cell determinants against which a response is normally not obtained. The new concept Th-Th cooperation is discussed.     

The role of CD4+ T cells in BKV-specific T cell immunity

Reactivation of polyomavirus BK (BKV) infection represents a severe complication in kidney transplant (KTX) patients. We previously reported an association between a declining BK viral load and the reconstitution of CD4⁺ T cell BKV-specific immunity in patients following kidney transplantation. However, the specific contribution of CD4⁺ T cells in the regulation of BKV-replication is unknown. Nevertheless, in vitro enrichment of BKV-specific T cells and subsequent adoptive T cell transfer may improve the restoration of immune competence in KTX patients with BKV infection. To date, strategies to capture human BKV-specific T cells with the ensuing expansion to clinically useful numbers are lacking. Here, we demonstrated a comprehensive flow cytometric analysis of the BKV-specific T cell response that permits access to the majority of T cells specific for immunodominant BKV antigens. A full-spectrum evaluation of the BKV-specific T cell response was performed by stimulating peripheral blood mononuclear cells (PBMC) with a mixture of BKV immunodominant peptide pools at varying concentrations and measuring activation marker expression and cytokine secretion. We also examined the effects of co-stimulation and PBMC resting time prior to activation. We defined the narrow range of stimulation conditions that permit the capture and expansion of functional BKV-specific T cell lines. The generated BKV-specific T cell lines showed the highest specificity and functionality when the T cells were captured according to IFNγ-secretion. This study highlights the multifunctional and cytolytic BKV-specific CD4⁺ T cells as a dominant population within the generated T cell product. This method offers a novel approach for the generation of BKV-specific T cell lines for adoptive immunotherapy and underscores the critical role of CD4⁺ T cells in the clearance of BKV.     

CD4 T cell priming in dendritic cell-deficient mice

Bone marrow (BM) chimeras (BMC) generated from mice carrying a null (-/-) mutation in the relB gene of the NF-kappaB family represent an ideal model for in vivo studies on the role of dendritic cells (DC) in the adaptive immune response. The spleen and lymph nodes (LN) of relB(-/-) BMC contain a small number of residual DC, mainly CD8alpha(+), that fail to up-regulate MHC class II and co-stimulatory molecules after stimulation in vitro. Moreover, residual spleen DC of relB(-/-) BMC have a 4-fold decrease in the ability to uptake and process soluble model antigen, ovalbumin (OVA), and failed to prime CD4 and CD8 T cells in vitro and in vivo. In addition, they also failed to present OVA peptide to OT-II transgenic T lymphocytes at a normal 1:10 (stimulator:responder) cell ratio. In spite of these multiple DC defects, relB(-/-) BMC immunized with plasmid DNA targeted to the spleen as the site of immune induction develop a specific CD4(+) T cell response comparable to that of relB competent mice. These data demonstrate that CD4( +) T cells can be primed in the absence of functional DC and suggest that relB may gauge the T cell response in vivo.     

Effect of cyclosporin A on immunity to Listeria monocytogenes.

The effect of the immunosuppressive drug cyclosporin A (CS-A) on immunity to the facultative intracellular bacterium Listeria monocytogenes was investigated in unprimed and primed mice. Different treatment protocols were followed to evaluate the time dependence of CS-A-mediated immune suppression and the effect of CS-A on immunological memory to L. monocytogenes. The effect of CS-A was observed only during and after activation of T cell-mediated immunity, whereas early resistance exerted by macrophages assessed 6 and 70 min after challenge remained unaffected. CS-A suppressed efficient elimination of L. monocytogenes even when given after day 3 of a primary infection. This contrasts with findings in other models, including viral infections, where CS-A must be administered very early in an immune response to suppress it. CS-A suppressed antibacterial resistance in mice primed at various times before challenge; suppression of protection was time dependent and was virtually complete in livers, whereas CS-A-resistant memory persisted in spleens for up to 10 months.     

香烟烟雾暴露对肺气肿小鼠CD4+IL-17+辅助性T细胞的影响

目的 探讨香烟烟雾暴露对肺气肿小鼠肺实质、外周血中CD4+IL-17+辅助性T细胞(Th17)的影响。方法 将40只雄性BALB/c小鼠按随机数字表法分为4组：对照12周组(C12)、对照24周组(C24)、烟雾暴露12周组(S12)、烟雾暴露24周组(S24),每组10只。香烟烟雾暴露法建立小鼠肺气肿模型。HE染色观察小鼠肺气肿的改变,计算平均内衬间隔(Lm)和肺泡破坏指数(DI);酶联免疫吸附法检测小鼠支气管肺泡灌洗液中IL-17、干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α水平及肺实质中IFN-γ和TNF-α水平;流式细胞术检测小鼠肺实质、外周血中CD4+ IL-17+T(Th17)细胞占CD4+T细胞的百分比;免疫荧光PCR法检测小鼠肺实质、外周血中RORγt和IL-17的mRNA表达,并分析这些指标的相互关系。结果 (1)S12组和S24组Lm为(39.19±3.51) μm和(46.87±7.16) μm,DI为39.13±1.57和45.16±3.13,均明显高于C12组和C24组(32.60±3.21) μm和(32.38±3.73) μm及28.23±1.62和28.86±2.07,以S24组增高更为明显,差异均有统计学意义(均P＜0.05)。(2) S12组和S24组BALF中IL-17、IFN-γ及TNF-α水平[(119.72±10.72) ng/L和(296.40±14.00) ng/L、(129.7±22.2) ng/L和(251.1±62.4) ng/L,(17.35±1.60) ng/L和(36.35±1.43) ng/L]较C12组和C24组[(52.06±4.70) ng/L和(51.89±6.82) ng/L、(85.8±26.8) ng/L和(88.9±11.5) ng/L,(6.41±0.90) ng/L和(5.85±0.92) ng/L]明显增高;在肺实质中,S12组和S24组IFN-γ及TNF-α水平[(1124.3±174.4) ng/L和(1342.7±206.1) ng/L,(77.2±13.7) ng/L和(101.7±19.0) ng/L]亦较C12组和C24组[(946.2±81.9) ng/L和(1027.2±188.3) ng/L,(62.1±16.1) ng/L 和(64.4±15.1) ng/L]明显增高;且以S24组增高更为明显,差异均有统计学意义（均P＜0.05）。(3)S12组和S24组肺实质中Th17细胞比例[(3.27±1.12)％和(7.19±2.24)％]明显高于C12组和C24组[(1.80±0.75)％和(1.99±0.59)％];S12组和S24组外周血中Th17细胞比例[(1.96±0.61)％和(3.82±1.26)％]亦明显高于C12组和C24组[(0.90±0.37)％和(0.97±0.32)％];且以S24组增高更为明显,差异均有统计学意义（均P＜0.05）。(4) S12组和S24组肺实质中RORγt和IL-17 mRNA表达量（1.73±0.35和4.52±1.15,4.00±0.87和83.43±21.01）较C12组和C24组（0.83±0.21和0.90±0.36,0.80±0.22和0.64±0.31）明显增高,以S24组增高更为明显,差异均有统计学意义(均P＜0.05);外周血中,S12组和S24组RORγt和IL-17 mRNA表达量(1.48±0.32和2.75±0.79,201.25±5.49和416.22±99.64)亦较C12组和C24组(0.59±0.25和0.75±0.36,24.90±5.49和22.28±4.24)明显增高,以S24组增高更为明显,差异均有统计学意义（均P＜0.05）。(5)烟雾暴露组中,外周血和肺实质的Th17细胞比例均与Lm、DI值显著正相关(r值分别为0.767、0.772;0.722、0.706,均P＜0.05)。结论 Th17细胞在香烟暴露肺部炎症、肺气肿小鼠外周血及肺内表达增高,并伴活性的增强,且随烟雾暴露时间延长而增强,提示香烟暴露肺气肿小鼠Th17细胞上调可能是导致小鼠肺内及全身炎症反应放大的原因之一。     

Humoral immunity reflects altered T helper cell bias in Borrelia burgdorferi-infected gamma delta T-cell-deficient mice

In murine Lyme borreliosis, the absence of gamma delta T lymphocytes augments the T helper cell 2 type humoral response but does not alter disease susceptibility. Arthropod transmission of Borrelia burgdorferi spirochetes results in similar antibody isotypes when gamma delta T cells are present, suggesting that vector effects can negate gamma delta T-cell functions in vivo.     

环孢霉素A对OVA免疫TCR转基因DO11.10小鼠CD4+CD25+T细胞的影响

目的：研究免疫抑制剂Csh对处于免疫应答状态的小鼠脾脏调节性CD4^＋CD25^＋T细胞影响。方法：采用流式细胞术检测卵白蛋白（OVA）免疫的DO11．10小鼠脾脏CD4^＋CD25^＋T细胞及其中Foxp 3^＋T细胞的变化。结果：OVA免疫后脾脏CD4^＋CD25^＋T细胞占CD4^＋T细胞百分数及Foxp 3^＋T细胞占CD4^＋CD25^＋T细胞百分数均增加，显示CsA可明显减少正常及免疫应答状态下小鼠脾脏CD4^＋CD25^＋T细胞及CD4^＋CD25^＋Foxp3^＋T细胞百分数。结论：CsA在抑制免疫应答的同时也可能抑制了免疫耐受的诱导。     

Effect of reduced EPHB4 expression in thymic epithelial cells on thymocyte development and peripheral T cell function

The Eph kinase (EPH) and ephrin (EFN) families are involved in a broad range of developmental processes. Increasing evidence is demonstrating the important roles of EPHBs and EphrinBs in the immune system. In this study on epithelial cell-specific Ephb4 knockout (KO) mice, we investigated T-cell development and function after EPHB4 deletion. KO mice presented normal thymic weight and cellularity. Their thymocyte subpopulation percentages were in the normal range. KO mice had normal T-cell numbers and percentages in the spleen, and T cells were activated and proliferated normally upon TCR ligation. Furthermore, naïve spleen CD4 cells from KO and wild type mice were capable of differentiating, in a comparable manner, into Th1, Th17 and Treg cells. In vivo, KO mice mounted effective delayed type hypersensitivity responses, indicating that thymocytes develop normally in the absence of TEC EPHB4, and T cells derived from EPHB4-deleted thymic epithelian cells (TEC) have normal function. Our data suggest that heavy redundancy and promiscuous interaction between EPHs and EFNs compensate for the missing EPHB4 in TECs, and TEC EPHB4's role in T cell development might only be revealed if multiple EPHs are ablated simultaneously. We cannot exclude the possibility that (1) some immunological parameters not examined in this study are affected by the deletion; (2) the deletion is not complete due to the leaky Cre-LoxP system, and the remaining EPHB4 in TEC is sufficient for thymocyte development; or (3) EPHB4 expression in TEC is not required for T cell development and function.