Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire

Healthy liver, intestine, lung, and skin harbor resident lymphocytes with conventional and unconventional phenotypes. Lymphocytes also have been detected in healthy mice kidneys; however, these cells have not been well studied and have been largely overlooked. To better characterize the intra-renal lymphocytes, we extensively perfused C57BL/6J mice with PBS and then isolated mononuclear cells for flow cytometry analysis. We observed T cells, B cells, and NK cells in normal mice kidneys after extensive perfusion. Approximately 50% of kidney T lymphocytes expressed intermediate levels of CD3 (CD3int T cells). Similar to liver and lung, a high percentage of unconventional CD3+CD4(-)CD8(-) double-negative T cells was observed in normal mice kidneys, from which 11% expressed B220 antigen. Unlike the spleen and blood, the classic CD4+ and CD8+ T lymphocytes in the kidney had a high proportion of activated CD69+ and effector/memory CD44- CD62L ligand phenotypes. Also, a small percentage of CD4+CD25+forkhead box p3+ and NKT cells was observed in perfused and exanguinated kidneys. In addition, a distinct TCR repertoire was found on intra-renal conventional and unconventional T cells compared with those from the spleen. Finally, after 24 h of renal ischemia reperfusion injury (IRI), increased production of cytokines IFN-gamma and TNF-alpha by CD4+ and CD8+ T cells, isolated from perfused kidneys, was observed. These data suggest that some of these cells harbored in the kidney could be implicated in the immune response of the IRI pathogenic process.     

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.     

CML患者CD4+和CD8+T细胞的TCR Vβ基因谱系和克隆性分析

目的：了解慢性粒细胞白血病慢性期（CML-CP）患者外周血CD4^＋和CD8^＋T细胞中TCR Vβ亚家族T细胞的基因表达和克隆性.方法：利用RT-PCR分别扩增19例CML-CP患者外周血单个核细胞（PBMCs）、CD4^＋和CD8^＋细胞TCR Vβ亚家族基因的CDR3,阳性产物进一步经基因扫描分析其克隆性.结果：CML患者外周血CD4^＋和CD8^＋细胞分别表达部分（1～21个）Vβ亚家族,均以Vβ13的表达率最高,其次为Vβ9,多数患者的一些Vβ亚家族T细胞呈克隆性增殖,主要出现于CD8^＋细胞中,分别以Vβ21（CD4^＋）和Vβ11（CD8^＋）亚家族的克隆性增殖多见.结论：CML患者外周血CD4^＋和CD8^＋细胞的TCR Vβ谱系均存在限制性分布,患者存在的克隆性增殖CD4^＋和CD8^＋T细胞可能与宿主抗CML抗原反应有关,它们可能在抗CML效应中起主要作用.     

Maintenance of CD4+ T cell TCR Vbeta repertoire heterogeneity is characteristic of apathogenic SIV infection in non-human primate model of AIDS

A PCR-based assay was established to screen for potential changes in the heterogeneity of Vbeta TCR expressing CD4+ T cells from rhesus macaques (RM) and sooty mangabeys (SM) prior to and following SIV infection. The rationale for these studies was to determine whether the progressive CD4+ T cell loss in SIV-infected disease susceptible RM and the moderate CD4+ T cell loss in disease resistant SM leads to the depletion of select Vbeta TCR families of CD4+ T cells. Results show that whereas SIV infection leads to the loss of Vbeta TCR heterogeneity in disease susceptible RM, the CD4+ T cells from SM retain their degree of Vbeta TCR heterogeneity, suggesting that the mechanism(s) of SIV induced CD4+ T cell loss maybe distinct in these 2 species and contribute to the differences in the clinical outcome.     

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8⁺ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP^?/? mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP^?/? Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8⁺ T cell development and repertoire selection. In comparing SLAP^?/? OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP^?/? Vβ5 mice. We have found that SLAP^?/? OT-1 mice have fewer CD8⁺ thymocytes but have increased CD5 expression. SLAP^?/? OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8⁺ splenocytes upon tetramer staining. Our data demonstrate that SLAP^?/? Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8⁺ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8⁺ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8⁺ T cell development influences repertoire selection.     

TCR repertoire of suppressor CD8+CD28- T cell populations.

The cellular basis of graft rejection and the development of strategies for specific suppression of T cell responses against allogeneic and xenogeneic transplants represents an area of active investigation. Recently, a population of MHC-class I restricted CD8+CD28- T suppressor cells (Ts) which are able to inhibit specifically the proliferative response of allospecific, xenospecific and nominal-antigen specific CD4+ T helper cells (Th) has been identified. We have studied the TCR V beta gene repertoire expressed by CD8+CD28- Ts isolated from allospecific, xenospecific, and nominal antigen-specific T cell lines (TCL). A limited V beta repertoire has been found in all TCLs studied. The most restricted TCR V beta usage was observed within the population of Ts from xenospecific TCLs. The TCR V beta usage within the Ts subset of TCL differs from the TCR repertoire expressed by the CD4+ Th subset of the same TCL. This is consistent with the fact that Ts and Th cells recognize distinct MHC/ antigen complexes. The finding that the TCR repertoire used by Ts is limited opens new avenues for studying the mechanisms of transplant rejection.     

Immune memory in CD4+ CD45RA+ T cells.

This study addresses the question of whether human peripheral CD4+ CD45RA+ T cells possess antigen-specific immune memory. CD4+ CD45RA+ T cells were isolated by a combination of positive and negative selection. Putative CD4+ CD45RA+ cells expressed CD45RA (98.9%) and contained < 0.1% CD4+ CD45RO+ and < 0.5% CD4+ CD45RA+ CD45RO+ cells. Putative CD45RO+ cells expressed CD45RO (90%) and contained 9% CD45RA+ CD45RO+ and < 0.1% CD4+ CD45RA+ cells. The responder frequency of Dermatophagoides pteronyssinus-stimulated CD4+ CD45RA+ and CD4+ CD45RO+ T cells was determined in two atopic donors and found to be 1:11,314 and 1:8031 for CD4+ CD45RA+ and 1:1463 and 1:1408 for CD4+ CD45RO+ T cells. The responder frequencies of CD4+ CD45RA+ and CD4+ CD45RO+ T cells from two non-atopic, but exposed, donors were 1:78031 and 1:176,903 for CD4+ CD45RA+ and 1:9136 and 1:13,136 for CD4+ CD45RO+ T cells. T cells specific for D. pteronyssinus were cloned at limiting dilution following 10 days of bulk culture with D. pteronyssinus antigen. Sixty-eight clones were obtained from CD4+ CD45RO+ and 24 from CD4+ CD45RA+ T cells. All clones were CD3+ CD4+ CD45RO+ and proliferated in response to D. pteronyssinus antigens. Of 40 clones tested, none responded to Tubercule bacillus purified protein derivative (PPD). No difference was seen in the pattern of interleukin-4 (IL-4) or interferon-gamma (IFN-gamma) producing clones derived from CD4+ CD45RA+ and CD4+ CD45RO+ precursors, although freshly isolated and polyclonally activated CD4+ CD45RA+ T cells produced 20-30-fold lower levels of IL-4 and IFN-gamma than their CD4+ CD45RO+ counterparts. Sixty per cent of the clones used the same pool of V beta genes. These data support the hypothesis that immune memory resides in CD4+ CD45RA+ as well as CD4+ CD45RO+ T cells during the chronic immune response to inhaled antigen.     

人外周血CD4~+ IL-21~+记忆T细胞的特征

目的:探讨人外周血中白细胞介素21(IL-21)的产生细胞及其特征。方法:分离人外周血单个核细胞(PBMC),分为不刺激或anti-CD3(OKT3)、OKT3+anti-CD28、PMA+ionomycin刺激四个组,流式细胞术(FCM)检测产生IL-21的细胞亚群。PMA+ionomycin刺激PBMC、纯化CD4+、CD4+CD45RA-、CD4+CD45RA+细胞、脐带血单个核细胞(CB-MC),FCM分析产生IL-21细胞的表型特征和IL-21与Th1、Th2、Th17和Th22细胞因子之间的关系。结果:与OKT3、OKT3+anti-CD28相比,PMA+ionomycin能诱导最高量的IL-21产生。产生IL-21的主要细胞为CD4+T细胞,少数CD8+T细胞。CD4+IL-21+T细胞表达CD45RO,不表达CD45RA,其中部分细胞表达CCR6、CCR7或CXCR5。CD4+CD45RA-细胞表达IL-21远高于CD4+CD45RA+细胞。进一步研究表明,PBMC产生IL-21,而CBMC不产生。此外,大约24%的CD4+IL-21+细胞表达IFN-γ,小于10%CD4+IL-21+细胞表达IL-4、IL-17或IL-22。结论:人PBMC在多克隆刺激的条件下,可以诱导IL-21的产生。产生IL-21的主要细胞亚群具有记忆CD4+T细胞的表型。其中一部分CD4+IL-21+T细胞的表型独立于Th1、Th2、Th17和Th22细胞亚群。     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Post-thymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults

In spite of thymic involution early in life, the numbers of naive CD4(+) T cells only slowly decline in ageing humans implying peripheral post-thymic naive CD4(+) T cell expansion. This proliferation may compensate for continuous activation and death of naive CD4(+) T cells but may also have negative consequences for protective immunity. Here we show that naive CD4(+) T cells that have proliferated in the periphery are characterized by a highly restricted oligoclonal TCR repertoire. Additionally these cells, which constitute the majority of naive CD4(+) T cells in the elderly, display signatures of recent TCR engagement. Our results demonstrate for the first time that peripheral post-thymic proliferation of naive CD4(+) T cells in healthy human individuals causes a significant contraction of the peripheral TCR repertoire. This age-dependent deterioration of CD4(+) T cell immunity could entail ageing-associated autoimmunity, increased susceptibility to infection or cancer and decreased efficiency of vaccination.     

Sialyl lewisx antigen-expressing human CD4+ T and CD8+ T cells as initial immune responders in memory phenotype subsets

Cytokine production by memory T cells in secondary immune responses has a critical role in host defenses. Previously, we had demonstrated that a unique antigen composed of sialyl lewis(x) (sLe(x)) was expressed on CD45RO(+) memory-phenotype subsets of human T cells. Here, we found that the sLe(x) antigen was up-regulated on CD45RA(+) na?ve human CD4(+) T and CD8(+) T cells by TCR stimulation. In addition, sLe(x) antigen-expressing CD4(+) T and CD8(+) T cells in human PBMCs were activated immediately by cytokine stimulations composed of IL-2 plus IL-12 or IL-15 in an antigen-independent manner. Moreover, the sLe(x)-positive human CD8(+) T cells significantly enhanced reverse antibody-dependent cellular cytotoxicity compared with a sLe(x)-negative population. These findings clearly indicate that sLe(x) antigen-expressing memory phenotype CD4(+) T and CD8(+) T cells contribute to early-stage immunity by providing a source of IFN-gamma and cytotoxicity, suggesting that they would be a key immunomodulator in host defenses.     

Regulation of allergen-induced bone marrow eosinophilopoiesis: role of CD4+ and CD8+ T cells

BACKGROUND: The mechanisms of the distant stimulation of the bone marrow (BM) after airway allergen exposure remain largely obscure. T cells have been implicated in allergic airway inflammation but their role in allergen-induced BM eosinophilopoiesis is poorly understood. The aim of this study was to determine the role of CD4(+) and CD8(+) T cells in allergen-induced BM eosinophilopoiesis. METHODS: Ovalbumin (OVA)-sensitized wild type (WT), CD4 knockout (CD4-/-) and CD8 knockout (CD8-/-) mice were exposed intranasally to OVA or saline. Bromo-deoxyuridine (BrdU) was used to label newly produced cells. Bone marrow, blood and bronchoalveolar lavage (BAL) were sampled 24 h after the final exposure. Immunostaining for newly produced eosinophils (i.e. BrdU(+)/MBP(+)) and BM eosinophil progenitor [CD34(+)/CD45(+)/interleukin-5 (IL-5)Ralpha(+)] cells was performed. RESULTS: The number of newly produced BM eosinophils (BrdU(+)/MBP(+) cells) was significantly reduced in allergen exposed CD4-/- or CD8-/- mice compared with allergen exposed WT mice, which was followed by a subsequent decrease in newly produced blood and airway eosinophils. Furthermore, BM eosinophil progenitors were significantly reduced in allergen exposed CD4-/- and CD8-/- mice compared with WT mice. Finally, serum IL-5 and Bronchoalveolar lavage fluid eotaxin-2 levels were abolished in allergen exposed CD4-/- mice to levels seen in saline exposed WT mice. CONCLUSIONS: These data suggests that both CD4(+) and CD8(+) T cells have a regulatory role in allergen-induced BM eosinophilopoiesis, whereas CD4(+) T cells are obligatory for allergen-induced airway eosinophilia. The subsequent traffic of eosinophils to the airways is likely to be at least partly regulated by a CD4(+) T-cell-dependent local airway eotaxin-2 production.     

Functional and Vbeta repertoire characterization of human CD8+ T-cell subsets with natural killer cell markers,CD56+ CD57- T cells,CD56+ CD57+ T cells and CD56- CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK-type T cell); CD56 single positive (CD56)-T cells, CD56/CD57 double positive (DP)-T cells and CD57 single positive (CD57)-T cells in the peripheral blood. All NK-type T-cell populations expressed CD122 and intermediate levels of T-cell receptor (TCR; regular CD8+ T cells are CD122- and express high levels of TCR). The number of both DP-T cells and CD57-T cells, but not CD56-T cells, gradually increased with age. All NK-type T-cell populations produced larger amounts of interferon-gamma than did regular CD8+ T cells after stimulation with interleukin (IL)-2, IL-12 and IL-15. However, CD56-T cells and CD57-T cells but not DP-T cells showed a potent antitumour cytotoxity to NK-sensitive K562 cells, whereas only CD56-T cells showed a potent cytotoxity to NK-resistant Raji cells. Furthermore, although NK-type T cells produced large amounts of soluble Fas-ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VbetaT cells were found in each NK-type T-cell population. The non-variant CDR3 region(s) for the TCRbeta chain(s) showed CD57-T cells and CD56-T cells to be derived from distinct origins, while the DP-T cell population consisted of a mixture of the clones seen in both CD56-T cells and CD57-T cells. Our results suggest that CD57-T cells and CD56-T cells are functionally and ontogenically different populations while DP-T cells appear to originate from both CD56-T cells and CD57-T cells.     

Age-related differences in phenotype and function of CD4+ T cells are due to a phenotypic shift from naive to memory effector CD4+ T cells

Based on the combined expression of CD27 and CD28, a putative model of T cell differentiation has been previously proposed. We used CD27 and CD28 expression in order to comparatively study the size, cytokine production capacity and proliferative response of CD4+ T cell sub-populations from healthy young and elderly volunteers. Elderly persons had a lower percentage of CD27+CD28+ but a higher percentage of CD27-CD28+ and CD27-CD28-CD4+ T cells than the young persons. CD27-CD28-CD4+ T cells were present, although at relatively low numbers, in the vast majority of the healthy elderly donors but were only sporadically detected in young persons. Each CD4+ T cell sub-population exhibited a distinct phenotype and cytokine production profile, which were not affected by age. When purified CD27+CD28+ were stimulated by staphylococcal enterotoxin B, they proliferated to a greater extent than CD27-CD28+ and CD27-CD28-CD4+ T cells. However, we did not observe age-related differences in proliferative response of each sub-population. We concluded that although the size of the different sub-populations differed between the young and the old group, the functional characteristics of each sub-population were the same in both age groups. This suggests that on a per cell basis there is no functional impairment of CD4 memory T cells in elderly persons. Consequently, potential differences in the function of the total CD4+ T cell population are most likely due to different composition of repertoire.     

小鼠脾细胞CD4+CD25+调节性T细胞的表型特征

观察CD4+CD25+T和CD4+CD25-T细胞的表型和细胞因子的表达。自小鼠脾脏制备单个细胞悬液,分离CD4+T细胞、CD4+CD25+和CD4+CD25-T细胞,进行细胞表面标记,激活后进行细胞内细胞因子染色,利用流式细胞仪在单个细胞水平上分析细胞表面分子、转录因子和细胞因子表达之间的关系。结果:在CD4+T细胞中,约有7.8%的细胞同时表达CD25分子。与CD4+CD25-T细胞相比,CD4+CD25+T细胞CD44的表达略有增加,CD45RB的表达明显下降,CTLA-4和Foxp3明显增加。以同时表达CTLA-4和Foxp3的细胞为主,其次为单独表达Foxp3的细胞。细胞因子的研究结果表明,与CD4+CD25-T细胞相比,CD4+CD25+T细胞IL-2、IFN-γ明显减少,而只产生IL-10的细胞略有增加。CD4+CD25+调节性T细胞无论在表型、转录因子的表达以及细胞因子表达方面均于非调节性T细胞不同。     

Chronic lymphocytic leukaemia cells drive the global CD4+ T cell repertoire towards a regulatory phenotype and leads to the accumulation of CD4+ forkhead box P3+ T cells

Advanced chronic lymphocytic leukaemia (CLL) is associated with profound immunodeficiency, including changes in T regulatory cells (T(regs)). We determined the pattern of expression of forkhead box P3 (FoxP3), CD25, CD27 and CD127 and showed that the frequency of CD4+ FoxP3+ T cells was increased in CLL patients (12% versus 8% in controls). This increase was seen only in advanced disease, with selective expansion of FoxP3-expressing cells in the CD4+ CD25(low) population, whereas the number of CD4+ CD25(high) FoxP3+ cells was unchanged. CD4+ CD25(low) cells showed reduced expression of CD127 and increased CD27, and this regulatory phenotype was also seen on all CD4 T cells subsets in CLL patients, irrespective of CD25 or FoxP3 expression. Incubation of CD4+ T cells with primary CLL tumours led to a sixfold increase in the expression of FoxP3 in CD4+ CD25- T cells. Patients undergoing treatment with fludarabine demonstrated a transient increase in the percentage of CD4+ FoxP3+ T cells, but this reduced to normal levels post-treatment. This work demonstrates that patients with CLL exhibit a systemic T cell dysregulation leading to the accumulation of CD4+ FoxP3+ T cells. This appears to be driven by interaction with malignant cells, and increased understanding of the mechanisms that are involved could provide novel avenues for treatment.     

TCRalphabeta repertoire diversity of human naturally occurring CD4+CD25+ regulatory T cells

We examined the alphabeta T cell receptor (TCR) repertoire of naturally occurring CD4+CD25+ regulatory T (Treg) cells isolated from healthy human blood. Three-color FACS analysis demonstrated that the usage of variable region segments of TCRbeta chains by CD4+CD25+ cells did not differ from those of CD4+CD25- cells. Complementarity-determining region 3 (CDR3) size distribution analyses demonstrated that the repertoire diversity of CDR3beta was almost identical between CD4+CD25+ and CD4+CD25- T cell subsets, and that there was no skewing of the CDR3beta repertoire of CD4+CD25+ T cells. In contrast, in vitro activated CD4+CD25+ T cells by cytomegalovirus-derived antigens showed a skewed CDR3 size distribution pattern. These findings support the hypothesis that naturally occurring CD4+CD25+ T cell subset in humans is 1argely composed of a T cell lineage positively selected in the thymus as a consequence of the interaction between self-peptides and TCRs and not derived from recent activation by a limited array of antigens.