An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice

IL-4 receptor α chain (IL-4Rα)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Rα resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4⁺, CD62L-high, and CD62L-low T cell populations from uninfected IL-4Rα^−/− mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4Rα^−/−, β₂-microglobulin^−/− double homozygous mice produced less IL-4 than did either IL-4Rα^−/− or wild-type mice. These results indicate that an IL-4-independent, β₂-microglobulin-dependent pathway exists through which the CD62L-low CD4⁺ population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.     

Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-A^b_β⁰ mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.     

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4⁺ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8⁺ regulatory T cells that are specific for pathogenic CD4⁺ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8⁺ T cells emerge that preferentially lyse and regulate activated autologous CD4⁺ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β₂-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8⁺ T cells are also induced by TCV with activated CD4⁺ Vβ8⁺ T cells. These CD8⁺ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8⁺ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4⁺Vβ8⁺ T cell clone specifically recognize other CD4⁺ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1^a but not the allogeneic Qa-1^b molecule. Thus, Vβ-specific Qa-1-restricted CD8⁺ T cells are induced by activated CD4⁺ T cells. We suggest that these CD8⁺ T cells may function to specifically regulate activated CD4⁺ T cells during immune responses.     

Interferon-gamma and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells

The CD8 co-receptor can modulate CD8⁺ T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-γ and IL-4 exert opposing effects on the expression of CD8α mRNA and surface CD8 protein during CD8⁺ T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin (OVA)_257–264-specific TCR-transgenic OT-I CD8⁺ T cells activated with OVA_257–264-coated antigen presenting cells or polyclonal stimuli, and on wild type CD8⁺ T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-γ or IFN-γR gene. When WT or IFN-γ-deficient OT-I CD8⁺ T cells were analyzed 9 days after co-injection with control or IL-4-expressing OVA⁺ tumor cells into RAG-2^−/−γc^−/− mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-γ-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD8^low cells displayed markedly impaired binding of OVA_257–264/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-γ in tuning the CD8 co-receptor during primary CD8⁺ T cell activation both in vitro and in vivo.     

结核分枝杆菌Rv2941蛋白抗原表位集中区免疫原性研究

目的 分析结核分枝杆菌Rv2941抗原的T细胞表位集中区的免疫原性,探究其作为结核病疫苗候选抗原的潜力。方法 通过免疫表位数据库(Immune Epitope Database,IEDB)分析Rv2941抗原的T细胞表位区(命名为Rv2941p)并构建表达载体PET32a-Rv2941p,诱导表达并纯化Rv2941p。将其与免疫佐剂二甲基三十六烷基铵(DDA)和PolyI:C乳化后,皮下免疫BALB/c小鼠3次,每次免疫间隔10 d,末次免疫1周后处死小鼠,进行免疫效果评价。采用ELISA法检测免疫后小鼠血清中IgG、IgG1和IgG2a抗体滴度以及免疫后小鼠脾脏淋巴细胞分泌IL-4、IL-2、IL-6和IFN-γ的水平。同时,利用流式细胞技术检测淋巴细胞内CD4⁺T、CD8⁺T细胞增殖情况以及胞内细胞因子(IFN-γ、TNF-α和IL-4)表达水平。结果 Rv2941p可溶性表达,并获得高纯度的蛋白。Rv2941p诱导产生了高水平的特异性抗体IgG,与对照组相比,差异有统计学意义(P<0.001)。另外,Rv2941p提高了IgG2a/IgG1的比值。细胞因子检测结果显示,Rv2941p提高了IFN-γ和IL-6的分泌,与Ag85B组比较,差异有统计学意义(P<0.001)。同时,Rv2941p可以促进CD4⁺和CD8⁺T细胞的增殖分化,以及提高胞内IFN-γ和TNF-α的表达。结论 Rv2941p可以刺激小鼠产生较高的体液和细胞免疫,尤其Th-1类细胞免疫,可以作为结核病疫苗候选抗原,为结核病新型疫苗的开发奠定了基础。     

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes

Aims/hypothesis Preproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients.Methods A preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4⁺CD45RA^– and CD4⁺CD45RA⁺ T cell subfractions, representing memory and naive and recently primed T cells respectively.Results In CD4⁺ T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13–29 and insulin B-chain epitope B11–27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1–16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFN, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA⁺ and CD45RA^– subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFN content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4⁺ cells were characterised by increasd IFN, TNF, and IL-5.Conclusions/interpretation Our data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.Abbreviations PPI Preproinsulin - PBMC peripheral blood mononuclear cells - Th T helper cells - Ab+ autoantibody-positive - ICA islet cell antibodies - IA-2A islet thyrosine phosphatase - SI stimulation index - Tr T regulatory cells W. Karges and B.O. Boehm contributed equally to this article     

Pilot Study on Interferon-γ-producing T Cell Subsets after the Protective Vaccination with Radiation-attenuated Cercaria of Schistosoma japonicum in the Miniature Pig Model

CLAWN miniature pig has been shown to serve as a suitable host for the experimental infection of Schistosoma japonicum. In this study, we found that radiation-attenuated cercaria (RAC) vaccine gave CLAWN miniature pigs protective immunity against subsequent challenge infection with S. japonicum cercaria. To characterize the protective immune response of the pig model vaccinated by attenuated cercaria, flow cytometric analysis of the reactive T cell subsets was performed. The intracellular interferon (IFN)-γ and the cell surface markers revealed the peripheral blood CD3+ T-lymphocytes produced significant amounts of IFN-γ during the immunization period and after the challenge infection. CD4+ αβ-T cells as well as CD4+/CD8α^mid double positive and/or CD8α^high αβ-T cells were the major IFN-γ-producing CD3+ T cells. On the contrary, γδ T cells did not produce intracellular IFN-γ. Our results suggested that RAC-vaccinated miniature pigs showed effective protective immunity through the activation of αβ T cells bearing antigen specific T-cell receptors but not through the activation of γδ T cells.     

Identification of two major types of age-associated CD8 clonal expansions with highly divergent properties

CD8 memory T cells are tightly regulated in young, healthy individuals but are often perturbed in aged animals by the appearance of large CD8 T cell clones. These clones are associated with impaired immunity in the aged. The molecular basis of this phenomenon remains unclear. Here, it is shown that the issue is confused by the fact that the clones are heterogeneous. Some clones bear high, and others, low levels of integrin α₄ (itgα4). These subtypes differ by multiple criteria. They appear in mice of different ages, concentrate in different tissues, and have different stabilities in vivo and responses to stimulation in vitro. itgα4^high, but not itgα4^low, CD8 clonal expansions have several characteristics consistent with a chronically stimulated phenotype. These properties include lowered levels of CD8, decreased expression of some cytokine receptors, and elevated expression of various inhibitory receptors, including the programmed death-1 (PD1) receptor and the killer cell lectin-like receptor G1 (KLRG1). The characteristics of itgα4^high clonal expansions suggest that they may arise from age-dependent alterations in antigen expression and tolerance. These data redefine CD8 clonal expansions into at least two distinct entities and indicate that there are multiple mechanisms that drive age-related alterations of CD8 T cell homeostasis.     

Development and function of T cells in T cell antigen receptor/CD3 ζ knockout mice reconstituted with FcɛRI γ

Engagement of α–β T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and ζ–ζ or ζ–η dimers. Previous experiments using knockout (KO) mice that lacked ζ (ζKO) showed that ζ is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in ζKO mice, a subset of T cells in the gut of both ζKO and normal mice bore nearly normal levels of TCR on its surface. This was because ζ was replaced by the FcRI γ (FcRγ). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that ζ replacement by the FcRγ chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of ζ substitution by FcRγ for T cell development and function in vivo, we produced ζKO mice expressing FcRγ in all of their T cells (FcRγTG ζKO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcRγ⁺ lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcRγTG ζKO mice were lower than those of controls. This was particularly true for the CD4⁺ T cells. We conclude that FcRγ can replace the functions of ζ in T cell development in vivo but that TCR/CD3 complexes associated with FcRγ rather than ζ are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4⁺ and CD8⁺ T cells.     

The integrin α4β7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1

Both activated and resting CD4⁺ T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin α₄β₇ (α₄β₇), the gut mucosal homing receptor. We find that α₄β₇^high CD4⁺ T cells are more susceptible to productive infection than are α₄β₇^low-neg CD4⁺ T cells in part because this cellular subset is enriched with metabolically active CD4⁺ T cells. α₄β₇^high CD4⁺ T cells are CCR5^high and CXCR4^low; on these cells, α₄β₇ appears in a complex with CD4. The specific affinity of gp120 for α₄β₇ provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.     

Multivalent structure of an αβT cell receptor

Whether there is one or multiple αβT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRβ chains incorporate at least two αβTCRs in a single TCR/CD3 complex. Evidence for bispecific αβTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRβ-variable regions. Such (αβ)₂TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (αβ)₂TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.     

Specific requirement for CD3ɛ in T cell development

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3γ, -δ, -, and ζ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3γ, -δ, or ζ results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3^−/− mice, and thymocyte development is arrested at the early CD4⁻CD8⁻ stage. Although these results suggest that CD3 is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3γ and CD3δ genes also is reduced in CD3^−/− mice. Thus, it is unclear whether the phenotype of CD3^−/− mice reflects the collective effects of CD3γ, -δ, and - deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3 gene via Cre/loxP-mediated recombination, we generated mice that lack CD3 yet retain normal expression of the closely linked CD3γ and CD3δ genes. These (CD3^Δ/Δ) mice exhibited an early arrest in T cell development, similar to that of CD3^−/− mice. Moreover, the developmental defect could be rescued by expression of a CD3 transgene. These results identify an essential role for CD3 in T cell development not shared by the CD3γ, CD3δ, or ζ-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.     

Generation of CD8 suppressor factor and β chemokines, induced by xenogeneic immunization, in the prevention of simian immunodeficiency virus infection in macaques

Previous xenogeneic immunization experiments in rhesus macaques with simian immunodeficiency virus (SIV) grown in human CD4⁺ T cells consistently elicited protection from challenge with live SIV. However, the mechanism of protection has not been established. We present evidence that xenogeneic immunization induced significant CD8 suppressor factor, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP) 1α, and MIP-1β (P < 0.001 - P < 0.02). The concentrations of these increased significantly in protected as compared with infected macaques (P < 0.001). Xenogeneic stimulation in vitro also up-regulated CD8 suppressor factors (SF; P < 0.001) and the β chemokines which were neutralized by antibodies to the 3 β chemokines. Recombinant human RANTES, MIP-1α and MIP-1β which bind to simian CCR5, suppressed SIV replication in a dose-dependent manner, with RANTES being more effective than the other two chemokines. The results suggest that immunization with SIV grown in human CD4⁺ T cells induces CD8-suppressor factor, RANTES, MIP-1α and MIP-1β which may block CCR5 receptors and prevent the virus from binding and fusion to CD4⁺ cells.     

T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells

To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2–restricted T-cell recptor γ-chain alternate reading-frame protein (TARP)_4–13 epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2⁺ prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP_4–13 epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.     

Analysis of T Cell Receptor Vβ Diversity in Peripheral CD4+ and CD8+ T Lymphocytes Obtained From Patients With Chronic Severe Hepatitis B

Background:

The hepatitis B virus (HBV) antigen-induced cellular immune response plays an important role in HBV clearance. Changes in the diversity of complementarity determining region 3 (CDR3) and T-cell receptor (TCR) sequences are used to monitor the response of T cells to antigens.

Objectives:

The aim of the present study was to determine whether the TCR Vβ repertoire of patients with chronic severe hepatitis B (CSHB) undergoes increased stimulation, and to identify conserved motifs in specific TCR Vβ families.

Patients and Methods:

Peripheral blood mononuclear cells (PBMCs) from 18 patients with CSHB were sorted into CD4+ and CD8+ T subsets, using monoclonal antibody-coated magnetic beads. The TCR Vβ CDR3 was subsequently characterized using immune spectratyping. The TCR Vβ families exhibiting a CDR3 spectratype that underwent monoclonal expansion were sequenced.

Results:

The number of oligoclonal or monoclonal expansion TCR Vβ families detected in the analyzed CD8+ T cells was significantly higher than the number detected in CD4+ T cells. The CDR3 spectratype analysis showed predominant usage of TCR Vβ5, Vβ7, Vβ9, Vβ12, and Vβ18 families in CD8+ T cell subsets of CSHB patients. Furthermore, conserved amino acid motifs were found to be associated with the monoclonal expansion of CD8+ TCR Vβ families. In addition, JB1S1 and JB2S7 region genes were present at a high frequency.

Conclusions:

The CD4+ and CD8+ TCR Vβ gene families undergo clonal expansion in CSHB patients, and CD8+ T cells play a major role in the pathogenesis of CSHB. Moreover, the conserved motifs and limited use of joining region genes observed in the CSHB patients of this cohort indicated that similar antigenic epitopes are recognized.     

Rapid tolerization of virus-activated tumor-specific CD8+ T cells in prostate tumors of TRAMP mice

To study T cell responses to tumors in an autochthonous model, we expressed a CD8 T cell epitope SIYRYYGL (SIY) in the prostate of transgenic adenocarcinoma (TRAMP) mice (referred to as TRP-SIY), which spontaneously develop prostate cancer. Naïve SIY-specific CD8 T cells adoptively transferred into TRP-SIY mice became tolerized in the prostate draining lymph nodes. Vaccination of TRP-SIY mice intranasally with influenza virus that expresses the SIY epitope resulted in generation of SIY-specific effector T cells in the lung-draining lymph nodes. These effector T cells expressed TNFα and IFNγ, eliminated SIY peptide-loaded target cells in vivo, and infiltrated prostate tumors, where they rapidly lost the ability to produce effector cytokines. A population of these T cells persisted in prostate tumors but not in lymphoid organs and could be induced to re-express effector functions following cytokine treatment in vitro. These findings suggest that T cells of a given clone can be activated and tolerized simultaneously in different microenvironments of the same host and that effector T cells are rapidly tolerized in the tumors. Our model provides a system to study T cell-tumor interactions in detail and to test the efficacy of cancer immunotherapeutic strategies.