Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in interferon-γ and in interleukin-4-expressing cells

In an immune response, effector functions are controlled by T helper (Th) 1 cytokines [interferon-γ (IFN-γ), interleukin (IL)-2 and tumor necrosis factor-β] and Th 2 cytokines (IL-4, IL-5 and IL-10). Here we analyze by multiparameter immunofluorescence to what extent IL-2, IL-4, IL-5, IL-10 and IFN-γ are co-expressed in individual normal murine Th cells upon activation in vitro with the bacterial superantigen Staphylococcus aureus enterotoxin B, presented in the context of major histocompatibility complex class II. IL-2 and IFN-γ are co-expressed by some, but not by other Th cells. Expression of IL-4 and IFN-γ is exclusive. IL-10 is co-expressed in individual cells either with IL-4 or with IFN-γ. No IL-5-expressing cells are detected. While IL-10- and IL-4-co-expressing Th cells correspond to classical Th 2 cells, cells co-expressing IL-10 and IFN-γ could be involved in negative-feedback regulation of a Th 1 response. Apart from such functional implications, our results show that IL-2, IL-4, IL-5, IL-10 and IFN-γ are expressed independently of each other in individual murine Th cells.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.     

A 150-kDa molecule of macrophage membrane stimulates interleukin-2 and interferon-γ production and proliferation of ovalbumin-specific CD4+ T cells

In the present study, we describe the potential co-stimulatory role of a macrophage membrane-associated protein of 150 kDa (M150). The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was found to be a single molecule on two-dimensional gel electrophoresis. The molecule was re-constituted in phosphatidyl choline vesicles and tested for its ability to promote the proliferation and the secretion of lymphokines from T helper (Th) cells. The reconstituted M150 induced a significant proliferation of anti-CD3 monoclonal antibody (mAb)-stimulated ovalbumin-specific CD4⁺ T cells. Further, Th cells activated with this molecule in the presence of anti-CD3 mAb mainly secreted interleukin (IL)-2 and interferon-γ but not IL-4. M150 could not promote the proliferation of Th cells, or lymphokine secretion in the absence of anti-CD3 mAb. These observations suggest that M150 acts by selectively activating a Th1-like immune response.     

Differential response of CD4+ V7+ and CD4+ V7− T cells to T cell receptor-dependent signals: CD4+ V7+ T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen

V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells. In the current study, CD4⁺ V7+ and CD4⁺ V7? T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4⁺ V7+ T cells alone produced IL-2 and proliferated vigorously. By contrast, CD4⁺ V7? cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28 antibody were added to the cultures. The enhancement of the V7? T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up-regulate such signals through their interaction with counter-receptors on T cells. To assess the role of V7 in a system independent of co-stimulation, CD4⁺ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.     

Defective lymphokine production by most CD8+ and CD4+ tumor-specific T cell clones derived from human melanoma-infiltrating lymphocytes in response to autologous tumor cells in vitro

Human melanomas are infiltrated by tumor-reactive T lymphocytes. However, the ability of these cells to elicit a specific anti-tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma-specific tumor-infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin-2 (IL-2), interferon-γ (IFN-γ) and interleukin-4 (IL-4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL-2, IL-4 or IFN-γ. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL-2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL-2 and IL-4, but not IFN-γ secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA-peptide complexes revealed that defective IL-2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen-presenting tumor cells. For this last type of clone, we further showed that defective IL-2 induction resulted from an LFA-3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma-reactive TIL to control tumor development. Interestingly both CD4⁺ and CD8⁺ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.     

Selective activation of resting human γδ T lymphocytes by interleukin-2

In rheumatoid arthritis and other inflammatory diseases we and others have found that γδ T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate γδ T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the γδ but not the αβ T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the γδ T cells and a corresponding reduction of the fraction of αβ T cells. Limiting dilution analysis revealed that approximately 1/60 of the γδ T cells responded to IL-2 in contrast to only 1/250 of the αβ T cells. Comparison of the expression of the IL-2 receptor (IL-2R) a and P chains showed that there was a similar expression of the α chain on γδ and αβ T cells whereas the relative density of the β chain was more than twice as high on γδ T cells. Both the IL-2-induced proliferation of γδ T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2Rβ monoclonal antibody (mAb) but not by an anti-IL-2Rα mAb. Expression of CD69 on γδ T cells was dependent neither on the presence of B cells, monocytes, nor αβ T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of γδ T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on γδ T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated αβ T cells.     

T cell response in murine Leishmanial mexicana amazonensis infection: production of interferon-γby CD8+ cells

The immune response to Leishmania major has been the subject of many investigations. However, Leishmania includes many species with different clinical manifestations. In this report, we studied the Tcell response to L. mexicana amazonensis, a New World species, in a murine model. We found that, similar to L. major, an Old World species, resistant C57BL/6 mice produced a high level of IFN-γ and a low level of IL-4. Conversely, susceptible BALB/c mice produced a much lower level of IFN-γ and higher level of IL-4. Although IFN-γ is one of the important lymphokines that mediate macrophage activation and thus the destruction of the intracellular parasites, which lymphocyte subsets are producing the IFN-γ is still a controversy. Much evidence including the isolation of protective, IFN-γ-producing, CD4⁺ cell lines have confirmed the participation of CD4⁺ Thl cells unequivocally. However, both CD4⁺ and CD8⁺ cells produced IFN-γ. Recently, an increasing body of evidence has appeared suggesting that CD8⁺ cells also play a role in the resolution of murine L. major infection. We found that in the L. m. amazonensis model, when CD8⁺ lymphocytes from resistant C57BL/6 mice were eliminated by anti-CD8 antibody and complement-mediated lysis, the IFN-γ production was reduced by 77%. This indicated that CD8⁺ cells produced a significant amount of the IFN-γ. However, our results also indicate that IFN-γ production by CD8⁺ cells was dependent on CD4⁺ cells.     

Patterns of lymphokine secretion amongst mouse γδ T cell clones

Although the patterns of lymphokine (LK) secretion by CD4 and CD8 αβ T cells have been extensively studied, the question of whether γδ T cells display patterns of restricted LK production and whether these patterns are the same as seen in conventional αδ T cells has not been previously addressed. In this study we generated panels of γδ T cell clones from normal C57BL/6 and BALB/c mice using a lectin-driven system and compared their patterns of secretion of nine LK with those of CD4 and CD8 αβ T cell clones generated in the same system. The results showed that γδ T cell clones displayed nonrandom patterns of highly restricted LK production with a strong bias towards the production of type 1 LK. The dominant pattern was one of high level secretion of interferon-γ and tumor necrosis factor (TNF), with variable production of interleukin (IL)-2, and little or none of the type 2 LK IL-4, IL-5, IL-6, and IL-10. This pattern differed significantly from that of CD4 Th1 clones in that γδ clones showed a striking deficiency in the production of IL-3 and granulocyte/macrophage colony-stimulating factor. A small subset of γδ clones displayed a novel pattern, in which the only LK produced in substantial quantity were TNF and variable amounts of IL-2. The bias of γδ T cells towards type 1 LK production was not an artefact associated with cloning because bulk populations of splenic γδ T cells behaved in the same way, even when activated in the presence of high concentrations of IL-4.     

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).     

CD3-mediated apoptosis of human medullary thymocytes and activated peripheral T cells: Respective roles of interleukin-1, interleukin-2, interferon-γ and accessory cells

Clonal deletion represents an important mechanism for the establishment of tolerance, by the elimination of autoreactive T cells. Deletion is accomplished by programmed cell death, termed apoptosis, induced by mobilization of the T cell receptor (TCR) on both thymocytes and mature T cells. The mechanism which drives T cells towards cell death or cell proliferation after TCR mobilization remains unclear. We show here that the mobilization of the CD3/TCR complex of both CD4⁺ and CD8⁺ single-positive medullary human thymocytes and human mature activated T cells, in the absence of accessory cells, leads to an activation-induced cell death process by apoptosis. In both cases, apoptosis was associated with interferon (IFN)-γ gene expression and secretion in the absence of interleukin (IL)-2 gene expression; and the addition of anti-IFN-γ antibody prevented cell death. Apoptosis could also be prevented by cyclosporin A (CsA) treatment and could be re-induced by the addition of IFN-γ to CsA-treated cells. Addition of IL-2 had two different effects, it prevented apoptosis and also allowed proliferation in response to CD3 monoclonal antibody. Addition of IL-1, which induces IL-2 gene expression and secretion or addition of accessory cells, had the same preventive effect. These results suggest that the uncoupling of IFN-γ and IL-2 gene expression following CD3/TCR mobilization initiates apoptosis of human T cells at several different stages during development and activation. We propose that co-signals provided by accessory cells allow a coupling of IL-2 gene and IFN-γ gene expression, and that an essential role for IL-2 secretion in T cell activation involves the inhibition of a death program induced by IFN-γ secretion.     

DX5+CD4+ T cells modulate cytokine production by CD4+ T cells towards IL‐10 via the production of IL‐4

CD4⁺ Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune‐modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5⁺CD4⁺ T cells, which upon adoptive transfer show potent regulatory properties in murine collagen‐induced arthritis as well as in delayed‐hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5⁺CD4⁺ T cells on other CD4⁺ T cells in vitro. Although proliferation of naïve CD4⁺ T cells upon antigenic triggering was not altered in the presence of DX5⁺CD4⁺ T cells, there was a striking difference in cytokine production. In the presence of DX5⁺CD4⁺ T cells, an IL‐10‐producing CD4⁺ T‐cell response was induced instead of a predominant IFN‐γ‐producing Th1 response. This modulation did not require cell–cell contact. Instead, IL‐4 produced by DX5⁺CD4⁺ T cells was primarily involved in the inhibition of IFN‐γ and promotion of IL‐10 production by CD4⁺ T cells. Together, our data indicate that DX5⁺CD4⁺ T cells modulate the outcome of Th‐responses by diverting Th1‐induction into Th responses characterized by the production of IL‐10.     

CD25+ CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+ CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+ CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL-protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+ CD4+ T cells further up-regulate the IL-2R alpha chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+ CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+ CD4+ T cells is not a passive phenomenon but instead initiates--in conjunction with signals through the TCR--their differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo.     

Increased interleukin-10 production by ASC-deficient CD4+ T cells impairs bystander T-cell proliferation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an important component of the inflammasome, functioning as an adaptor protein that facilitates the recruitment and activation of procaspases that in turn promote the maturation of interleukin-1β (IL-1β) and IL-18. Despite initial focus on the inflammatory properties of ASC there is emerging evidence that highlights the importance of ASC in facilitating adaptive immune responses. However, the cellular and molecular basis for the involvement of ASC in adaptive immunity remains largely unexplored. We have previously demonstrated that activated ASC-deficient T cells have dampened proliferative responses. We have therefore explored the underlying cellular mechanism(s) by which ASC regulates T-cell proliferation. We show that under activating conditions (anti-CD3/CD28 stimulation) in bulk T-cell cultures the presence of ASC(-/-) CD4(+) T cells is sufficient to suppress the proliferative responses of neighbouring T cells. Furthermore, ASC(-/-) CD4(+) T cells upon activation exhibit a suppressive cytokine profile, with elevated production of IL-10 and reduced secretion of T helper type 1 cytokines, interferon-γ and IL-2. This increase in IL-10 secretion within the activated ASC(-/-) CD4(+) T-cell compartment was not associated with a proportional increase in conventional Foxp3(+) regulatory T (Treg) cells. Interestingly, when equal numbers of fluorescence-activated cell sorted ASC(+/+) and ASC(-/-) Treg cells (CD4(+) CD44(intermediate/high) CD25(+)) were activated in vitro, the ASC(-/-) fraction produced significantly more IL-10 than their wild-type counterparts, suggesting that ASC(-/-) Treg cells have greater suppressive capacity. Collectively, these results imply that the ASC may influence the development and functioning of Treg cells.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

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.     

Functional and molecular characterization of B cell-responsive Vδ1+ γδ T cells

Cells expressing the Vδ1⁺ gene segment are a minor γδ T cell population in human peripheral blood but predominate in epithelia and (inflamed) tissues. The characteristic dendritic-like morphology of these γδ T cells is consistent with their putative immune surveillance role in epithelia. Their function, however, remains unknown. We and others previously reported that a subset of Vδ1⁺ γδ T cells proliferates after stimulation with Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL), but not with fresh peripheral blood-derived B cells. These responses were independent of the type of T cell receptor (TcR) γ chain co-expressed with the Vδ1 chain. The in vivo relevance of this LCL-mediated activation as well as the nature of the stimulatory ligand on the LCL is not well established. In this study, we tested the proliferative response of Vδ1⁺ LCL-responsive T cells against non-EBV-transformed B cells, activated through CD40 by murine EL4 B5 cells, and to a panel of B cell lines differing in the expression of EBV nuclear antigen proteins and adhesion/co-stimulatory molecules. The role of the Epstein-Barr virus-derived antigen in the induction of this response could be excluded as the activated (non-EBV-transformed) peripheral blood B cells were also able to induce a proliferative response in the LCL-responsive Vδ1⁺ T cells. Therefore, the stimulatory ligand on B cells is of cellular rather than of viral origin, and its expression is up-regulated upon activation of B cells. The expression of B7 and CD39 molecules on the surface of activated B cells appeared to be crucial since antibodies to these structures could block the induction of proliferation of the Vδ1⁺ T cells. Finally, we investigated the diversity of the responding Vδ1⁺ γδ T cell clones by sequence analysis of the TcRδ junctional regions. No restricted V-D-J sequences were found among the LCL-responsive Vδ1⁺ T cell clones, arguing strongly against a mono- or oligoclonal Vδ1⁺ γδ T cell response to LCL. These findings may explain the presence of polyclonally activated Vδ1⁺ T cells in inflamed tissues where activated B cells are often present.     

CD4+Foxp3+调节性T细胞表达升高提示免疫应答而非移植耐受

目的 探讨诱导CD4+Foxp3+调节性T细胞(regulatory T cells,Tregs)表达的机制及其在移植耐受中可能的作用.方法 构建新生移植耐受小鼠动物模型,分析移植耐受与同种异体反应性T细胞、Tregs表达及嵌合体的关系;运用过继转移实验、EGFP转基因小鼠,研究移植排斥过程中Tregs表达及抗原特异性.结果 新生耐受小鼠形成嵌合体,同种异体反应性T细胞被克隆清除,但Tregs表达与初始小鼠相同;同种异体反应性T细胞识别抗原诱导免疫反应,Tregs在移植排斥反应中表达升高,且不仅同种异体反应性Tregs表达升高,非同种异体反应性Tregs表达也升高.结论 Tregs在移植排斥中非特异性诱导产生,可能为一种负反馈免疫调控机制.     

Critical stoichiometric ratio of CD4+ CD25+ FoxP3+ regulatory T cells and CD4+ CD25− responder T cells influence immunosuppression in patients with B‐cell acute lymphoblastic leukaemia

Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self-antigens. The frequency and suppressing activity of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4⁺ CD25⁺ FoxP3⁺ Treg cells in B-cell acute lymphoblastic leukaemia (B-ALL). We have included patients at diagnosis (n = 54), patients in clinical remission (n = 32) and normal healthy individuals (n = 35). These diagnosed patients demonstrated a lower number of CD4⁺ CD25⁺ cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor-β and CD152/CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4⁺ CD25⁻ responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4⁺ CD25⁺ FoxP3⁺ Treg cells became high with the progression of malignancy in B-ALL. Relative distribution of Tresp and Treg cells was only ˜5 : 1 in B-ALL but ˜35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B-ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4⁺ CD25⁺ cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4⁺ CD25⁺ FoxP3⁺ Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B-ALL.     

The proliferative response of CD4 T cells to steady-state CD8+ dendritic cells is restricted by post-activation death

CD8(+) splenic dendritic cells (DCs) from steady-state mice are less effective than the CD8(-) DC subset in their capacity to stimulate CD4 T cell proliferation in culture. However, we found that the two DC subtypes were equally potent at activating CD4 T cells, based on up-regulation of CD69 and CD25 expression. Also, we found no difference in the rate of T cell death prior to entry into the first division. We then tracked carboxyfluorescein diacetate succinimidyl ester-labeled T cells and employed a quantitative model to assess in detail the CD4 T cell expansion process in response to stimulation with CD8(+) or with CD8(-) DCs. The time required for most T cells to replicate their DNA prior to the first division was similar in both DC cultures. However, progression of the CD4 T cell population through subsequent divisions was reduced in CD8(+) DCs compared with CD8(-) DC culture. This was associated with an increased loss of viable T cells at each division. Post-activation, division-associated T cell death is therefore a major factor in the reduced response of CD4 T cells to CD8(+) DCs.