IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

Recurrence of autoreactive antigen-specific CD4+ T cells in autoimmune diabetes after pancreas transplantation

CD4+ T cells specific for the diabetes-associated autoantigen GAD65 were analyzed using peripheral blood samples after pancreas transplantation in subjects with T1D with clinical evidence of recurrent autoimmune diabetes. MHC class II tetramers facilitated the identification and cloning of antigen-specific autoreactive cells, which were found at several time points over a multiyear span, in spite of chronic immunosuppression of the subjects. Comparisons of TCR clonotypes by cDNA sequencing revealed that identical T cells were present in the circulation, separated by long time intervals, consistent with a persistent memory response associated with recurrent autoimmunity.     

CD4+ suppressor cells of autoimmune encephalomyelitis respond to T cell receptor-associated determinants on effector cells by interleukin-4 secretion.

We have previously demonstrated that CD4+ suppressor T cells (Ts) inhibit the secretion of interferon (IFN)-gamma, but not interleukin (IL)-2, by effector cells of experimental autoimmune encephalomyelitis (EAE). Moreover, CD4+ Ts appear to regulate IFN-gamma by secretion of transforming growth factor-beta. We now show that CD4+ Ts produce a lymphokine with IL-4 activity in response to a determinant associated with EAE effector cells. CD4+ Ts do not proliferate or secrete IFN-gamma, IL-2, or IL-4 in response to myelin basic protein, nor do CD4+ Ts proliferate or secrete IL-2 when co-cultured with irradiated EAE effector cells. Rather, CD4+ Ts secrete IL-4 when co-cultured with either irradiated effector spleen cells or irradiated encephalitogenic line cells. CD4+ Ts do not secrete IL-4 in response to OVA-primed spleen cells, suggesting that the suppressor cells recognize a determinant specific to encephalitogenic T cells. Furthermore, CD4+ Ts secrete IL-4 when cultured with synthetic T cell receptor (TcR) V beta 8, but not TcR V beta 14 peptide, in the presence of antigen-presenting cells. This response is major histocompatibility complex class II restricted as demonstrated by inhibition of the response with anti-class II monoclonal antibody. These results suggest that CD4+ Ts recognize a determinant associated with TcR on the surface of EAE effector cells and respond by secreting IL-4, in a manner analogous to the Th2 lymphocyte subtype.     

Regulation of experimental autoimmune encephalomyelitis by CD4+, CD25+ and CD8+ T cells: analysis using depleting antibodies

Experimental Autoimmune Encephalomyelitis (EAE) can be induced in mice of the C57BL/6 strain by subcutaneous immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide p35-55 in CFA, administered twice at an interval of one week and supplemented with Bordetella pertussis toxin given IV. Here, we studied the effect on the induction of EAE of depleting antibodies to CD4, CD8, or CD25 administered before either the first or the second dose of MOG p35-55. We found that anti-CD4 abolished EAE when given before the first immunization; anti-CD4 did not affect the disease when it was given before the second immunization. Anti-CD8 enhanced EAE induction when given before either of the two immunizations. Anti-CD25 enhanced EAE to the same degree as anti-CD8 when given before the first immunization, but anti-CD25 was even more effective in enhancing EAE when given before the second immunization. The anti-CD25 treatment led to significantly enhanced IFNgamma production by T cells responding to MOG p35-55 and persisting anti-MOG antibodies detectable 56 days after the first immunization. Administration of anti-CD8 or anti-CD25 abolished the need for pertussis toxin to induce EAE. These findings are compatible with the idea that CD4 T cells are required for the initial induction of EAE and that the disease is down-regulated by T cells expressing CD8 or CD25. These regulatory T cells exist prior to MOG immunization, but the CD25+ regulators appear to be further amplified by immunization.     

MOG诱导小鼠实验性自身免疫性脑脊髓炎及其对CD4+CD25+调节性T细胞的影响

目的： 探讨采用分子克隆技术诱导表达出的含硫氧还蛋白基团的髓鞘少突胶质细胞糖蛋白细胞外Ig样结构域(MOGIgd-TrxA)融合蛋白免疫C57BL/6小鼠,建立多发性硬化的动物模型实验性自身免疫性脑脊髓炎的可行性;通过检测模型小鼠脾脏中CD4+CD25+调节性T细胞的数量,初步探讨CD4+CD25+调节性T细胞在实验性自身免疫性脑脊髓炎发病机制中的作用。方法: (1)分子克隆技术合成MOGIgd-TrxA融合蛋白,纯化、超滤浓缩后Bradford法测定蛋白浓度。(2)动物实验:C57BL/6小鼠,随机分为4组：MOGIgd-TrxA组（MOG组）、豚鼠脊髓匀浆组、硫氧还蛋白（TrxA）组及生理盐水正常对照组,每组动物12只,各组以相应抗原乳剂免疫小鼠制作EAE模型后评估其临床神经功能、组织病理(HE染色和髓鞘luxol fast blue染色)并评价模型质量。(3)流式细胞仪(FACS)检测小鼠脾脏中CD4+CD25+调节性T细胞的百分比。结果:(1)纯化浓缩后MOGIgd-TrxA蛋白纯度达98%左右,浓度约2.3 g/L。(2)MOG组小鼠与豚鼠脊髓匀浆组小鼠在临床神经功能评分等方面无显著差异(P>0.05)。2组发病动物组织切片HE染色和髓鞘染色均有不同程度的病理改变。(3)CD4+CD25+调节性T细胞占CD4+T细胞比例MOG组为(4.71±1.61)％,豚鼠脊髓匀浆组为(1.44±0.65)％,均明显低于生理盐水正常对照组（9.22±1.24）％和硫氧还蛋白（TrxA）组（8.97±1.20）％(P<0.01)。结论: (1)分子克隆技术合成的MOGIgd蛋白免疫C57BL/6小鼠能够成功诱导出EAE模型,且模型稳定、发病率高,为进一步研究MS免疫发病机制并采取有效治疗措施奠定基础;(2)CD4+CD25+调节性T细胞数量减少可能与EAE小鼠临床表现相关。     

Inconsistence between number and function of autoreactive T cells in the course of experimental autoimmune encephalomyelitis

Background: Mouse experimental autoimmune encephalomyelitis (EAE) is widely used model of multiple sclerosis (MS). The role of autoreactive CD4⁺ and CD8⁺ T cells in the development of mouse EAE has been demonstrated. However, little information is available about the relation between the frequency and reactivity of myelin antigen-reactive CD4⁺ and CD8⁺ T cells in secondary lymphoid organs and their relevance with the inflammation and pathological lesion of CNS during the course of EAE mouse model.

Methods: In this study, an EAE model with a clinical course containing acute onset, peak and chronic remission stages was established in C57BL/6J mice by myelin oligodendrocyte protein (MOG)_35–55 peptide immunization, and followed by the monitoring of clinical and pathological parameters and autoreactive T cells at different stages during the course.

Results: The dynamic changes of inflammatory infiltration, myelin loss, and astrocyte proliferation in brain and spinal cord were highly consistent with clinical severity observed in EAE course. However, the frequencies of both MOG-specific CD4⁺ and CD8⁺ T cells in secondary lymphoid organs presented different dynamic trends from the IFN-γ production by MOG-reactive T cells. Meanwhile, the IL-17 production by MOG-reactive CD4⁺ T cells was consistent with the proliferation of MOG-specific CD4⁺ T cells.

Conclusions: Both CD4⁺ and CD8⁺ T cells were most sensitive to MOG antigen stimulation for IFN-γ production during the early stage of EAE, but then rapidly lost the function despite their vigorous proliferation at the peak stage and later.     

Antigen-specific splenic CD4+ and CD8+ regulatory T cells generated via the eye, suppress experimental autoimmune encephalomyelitis either at the priming or at the effector phase

The injection of antigen into the ocular anterior chamber (AC) induces the generation of splenic CD4(+) and CD8(+) regulatory T (Treg) cells, specific for the antigen injected into the AC. These Treg cells inhibit the induction (CD4(+)) and also the expression (CD8(+)) of a delayed-type hypersensitivity response. The ability of AC-induced self-antigen-specific Treg cells in modulating autoimmunity is not well defined. Here we show that an injection of encephalitogenic myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide into the anterior chamber of the eye (AC-MOG), before the induction of or during established experimental autoimmune encephalomyelitis (EAE) induced by MOG(35-55), suppresses the induction or progression of EAE, respectively. CD4(+) or CD8(+) splenic Treg cells induced by an injection of AC-MOG prevent EAE either at the inductive (priming) or at the progressive (effector) phase, respectively. This suppression of EAE by an AC-MOG injection or by intravenous transfer of splenic regulatory cells induced by an AC-MOG injection is specific for the antigen injected into the AC. Additionally, our data suggest that splenic CD8(+) Treg cells that suppress active EAE may use a transforming growth factor (TGF)-β-dependent suppression mechanism while the suppression of the induction of EAE by the AC-induced CD4(+) Treg cells is independent of TGF-β. Thus, we show for the first time that regulation of EAE at the priming or the chronic phase requires different phenotypes of Treg cells. Hence, it is important to consider the phenotype of Treg cells while designing effective cell-based therapies against autoimmune disorders.     

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.     

Human keratinocyte induction of rapid effector function in antigen-specific memory CD4+ and CD8+ T cells

The ability of human keratinocytes to present antigen to T cells is controversial and, indeed, it has been suggested that keratinocytes may promote T cell hyporesponsiveness. Furthermore, it is unclear whether keratinocytes can process antigen prior to MHC class I and class II presentation. We tested the ability of keratinocytes to induce functional responses in epitope-specific CD4+ and CD8+ memory T cells using peptides, protein and recombinant expression vectors as sources of antigen. Keratinocytes were able to efficiently process and present protein antigen to CD4+ T cells, resulting in cytokine secretion (Th1 and Th2). This interaction was dependent on keratinocyte expression of HLA class II and ICAM-1, which could be induced by IFN-gamma. In addition, keratinocytes could present virally encoded or exogenous peptide to CD8+ T cells, resulting in T cell cytokine production and target cell lysis. Finally, T cell lines grown using keratinocytes as stimulators showed no loss of function. These findings demonstrate that keratinocytes are able to efficiently process and present antigen to CD4+ and CD8+ memory T cells and induce functional responses. The findings have broad implications for the pathogenesis of cutaneous disease and for transcutaneous drug or vaccine delivery.     

Urothelial antigen-specific CD4+ T cells function as direct effector cells and induce bladder autoimmune inflammation independent of CD8+ T cells

The role of CD4(+) T cells in bladder autoimmune inflammation has not been identified because of the lack of a proper animal model. We investigated CD4(+) T-cell responses to bladder urothelial ovalbumin (OVA), a model self-antigen (Ag), in transgenic URO-OVA mice. The expression of bladder urothelial OVA rendered mice unresponsive to OVA and resulted in quick clearance of Ag-specific CD4(+) T cells. Adoptive transfer of naive OVA-specific CD4(+) T cells led to exogenous T-cell proliferation, activation, and bladder infiltration but no inflammatory induction. In contrast, adoptive transfer of preactivated OVA-specific CD4(+) T cells induced bladder inflammation. Studies further demonstrated that CD4(+) T cells induced bladder inflammation in URO-OVA mice depleted of CD8(+) T cells or deficient in the recombinase activating gene-1 (Rag-1(-/-)). These results indicate that urothelial Ag-specific CD4(+) T cells can function as direct effector cells to induce bladder autoimmune inflammation independent of CD8(+) T cells.     

Soluble mannosylated myelin peptide inhibits the encephalitogenicity of autoreactive T cells during experimental autoimmune encephalomyelitis

We have previously shown that immunization with a mannosylated myelin peptide in complete adjuvant induces tolerance instead of disease in experimental autoimmune encephalomyelitis (EAE), a rodent model for multiple sclerosis. In this report we demonstrate that treatment with a soluble mannosylated epitope of proteolipid protein (M-PLP(139-151)) significantly inhibits disease mediated by autoreactive myelin-specific T cells during EAE. Treatment with M-PLP(139-151), applied in different EAE models, significantly reduced the incidence of disease and the severity of clinical symptoms. Delayed-type hypersensitivity responses were abolished after peptide treatment, emphasizing the impact on peripheral T-cell reactivity. Histological analysis of spinal cord tissue from mice treated with M-PLP(139-151) revealed the presence of only few macrophages and T cells. Moreover, little expression of interferon-gamma, interleukin-23, or major histocompatibility complex class II antigen was detected. Immune modulation by M-PLP(139-151) was primarily antigen-specific because an irrelevant mannosylated peptide showed no significant effect on delayed-type hypersensitivity responses or on the course of EAE. Therefore, mannosylated antigens may represent a novel therapeutic approach for antigen-specific modulation of autoreactive T cells in vivo.     

Tumor‐specific CD4+ T cells maintain effector and memory tumor‐specific CD8+ T cells

Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor‐specific CD4⁺ T cells enhance CD8⁺ T‐cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase‐related protein 1‐specific CD4⁺ transgenic T cells‐CD4⁺ T cells and pmel‐CD8⁺ T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p ≤ 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8⁺ T cells with tumor‐specific cytokine expression. When combined with CD4⁺ T cells, transfer of total (naïve and effector) or effector CD8⁺ T cells were highly effective, suggesting CD4⁺ T cells can help mediate therapeutic effects by maintaining function of activated CD8⁺ T cells. In addition, CD4⁺ T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8⁺ T cells recovered from mice treated with both CD8⁺ and CD4⁺ T cells had decreased expression of PD‐1 and PD‐1‐blockade enhanced the therapeutic efficacy of pmel‐CD8 alone, suggesting that CD4⁺ T cells help reduce CD8⁺ T‐cell exhaustion. These data support combining immunotherapies that elicit both tumor‐specific CD4⁺ and CD8⁺ T cells for treatment of patients with cancer.     

IL-21 modulates CD4+ CD25+ regulatory T-cell homeostasis in experimental autoimmune encephalomyelitis

Abstract The homeostasis of CD4 ⁺ CD25 ⁺  regulatory T cells (Tregs) depends on the cytokine interleukin (IL)-2. As IL-21 shares sequence homology with IL-2 and the IL-21 receptors contain a γ-chain common to IL-2, we hypothesized that IL-21 could also affect the homeostasis of Tregs. We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), an animal model of relapsing-remitting human multiple sclerosis. We show that blockade of IL-21 in SJL/J mice before and after the induction of EAE enhances the influx of inflammatory cells into the central nervous system (CNS). The blockade of IL-21 leads to proliferation of proteolipid peptide (PLP_139-151)-autoreactive CD4 ⁺  T cells, which are capable to cause severe EAE in adoptively transferred recipient mice. Conversely, Tregs from mice where IL-21 was blocked, lose their capacity to prevent EAE induced PLP_139-151-reactive T cells. Notably, direct effects of IL-21 on Tregs are confirmed by studies of blockade of IL-21 in mice expressing a green fluorescent protein 'knocked' into a Foxp3 allele, in which a reduction of the number of Tregs and a downregulation of their frequency and expression of Foxp3 are observed. These data suggest a role of the IL-21/IL-21R axis in the homeostasis of Tregs in CNS autoimmunity.     

The effector to memory transition of CD4 T cells

The small number of antigen-specific memory CD4 T cells surviving long-term after antigen or pathogen challenge are often characterized by a surprising degree of phenotypic and functional heterogeneity. We here propose that the immune system has evolved to express this diversity in memory T-cell populations, in order to provide flexibility in recall responses, via a rapid transition from heterogeneous effector cells into correspondingly heterogeneous memory cells. Little attention has been paid to another important transition—from resting memory cell to re-activated effector. We would suggest that superior functional attributes of secondary effectors arising from memory CD4 T cells, as compared to primary effectors arising from naïve precursors, play an important and underappreciated role in protective secondary immune responses.     

Hyperimmunoglobulinemia E syndrome associated with coronary artery aneurysms: deficiency of central memory CD4+ T cells and expansion of effector memory CD4+ T cells.

BACKGROUND: Hyperimmunoglobulinemia E syndrome is a primary immunodeficiency disorder characterized by elevated IgE levels, recurrent infections, pruritic rash, and skeletal and dental abnormalities. Autosomal dominant, autosomal recessive, and sporadic forms have been described. Coronary artery aneurysms and analysis of central (TCMs) and effector (TEMs) memory T cells have not been previously reported with this syndrome. OBJECTIVE: To describe a 30-year-old woman with hyperimmunoglobulinemia E syndrome who was found to have coronary artery aneurysms, deficiency in CD4+ TCMs, and expansion of CD4+ TEMs expressing CD45RA antigen (TEMRAs). METHODS: The patient presented to the clinic after hospitalization for chest pain. Coronary angiogram performed during the hospitalization revealed aneurysms in multiple coronary arteries with thrombus formation. In addition, she had a history of recurrent pneumonia, staphylococcal skin abscesses, and a pruritic facial rash. An extensive immunologic evaluation was performed. RESULTS: Immunologic studies revealed increased serum IgE levels (13,434 IU/dL), decreased proliferative responses to the soluble recall antigens tetanus toxoid and Candida albicans, and normal responses to mitogens. Analysis of lymphocyte subsets showed a deficiency of CD4+ TEMs and an increase in CD4+ TEMRAs. In addition, a decreased proportion and number of memory B cells and a deficiency in antibody response to pneumococcal antigens were observed. CONCLUSION: Hyperimmunoglobulinemia E syndrome may be associated with coronary artery aneurysms and with deficiency in CD4+ TEMs and expansion of CD4+ TEMRAs. Comprehensive immunologic evaluation should be performed in patients with this syndrome.     

Origins of CD4(+) effector and central memory T cells

Lineage-committed effector CD4(+) T cells are generated at the peak of the primary response and are followed by heterogeneous populations of central and effector memory cells. Here we review the evidence that T helper type 1 (T(H)1) effector cells survive the contraction phase of the primary response and become effector memory cells. We discuss the applicability of this idea to the T(H)2 cell, T(H)17 helper T cell, follicular helper T cell (T(FH) cell) and induced regulatory T cell lineages. We also discuss how central memory cells are formed, with an emphasis on the role of B cells in this process.     

Similarities and differences in CD4+ and CD8+ effector and memory T cell generation

Naive CD4+ and CD8+ T cells undergo unique developmental programs after activation, resulting in the generation of effector and long-lived memory T cells. Recent evidence indicates that both cell-intrinsic and cell-extrinsic factors regulate memory T cell differentiation. This review compares and contrasts how naive CD4+ and CD8+ T cells make the transition to effector and/or memory cells and discusses the implications of these findings for vaccine development.