A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.     

Carbon monoxide‐treated dendritic cells decrease β1‐integrin induction on CD8+ T cells and protect from type 1 diabetes

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)‐treated DCs loaded with pancreatic β‐cell peptides protect mice from disease. This protection is peptide‐restricted, independent of IL‐10 secretion by DCs and of CD4⁺ T cells. Although no differences were observed in autoreactive CD8⁺ T‐cell function from gCO‐treated versus untreated DC‐immunized groups, gCO‐treated DCs strongly inhibited accumulation of autoreactive CD8⁺ T cells in the pancreas. Interestingly, induction of β1‐integrin was curtailed when CD8⁺ T cells were primed with gCO‐treated DCs, and the capacity of these CD8⁺ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO‐treated DCs appears to be an original strategy to control autoimmune disease.     

High frequency of CD8αα homodimer-bearing T cells in human fetal intestine

Immunohistochemistry on frozen sections was used to identify CD8αα cells and CD8αβ cells in human intestine. As observed previously, CD8αβ cells predominate (>95%) in tonsil and post-natal intestine. However in human fetal intestine (16–24 weeks gestation), almost half the CD8⁺ cells in the lamina propria are CD8αα, and many CD8αα cells can be identified in the epithelium. In contrast, in the T cell zones of the Peyer's patches, CD8αβ cells are dominant. The CD8αα cells are virtually all αβ T cell receptor positive. By analogy with the murine system, these CD8αα cells in the fetal gut may be directly derived from the marrow, undergoing thymus-independent differentiation in the gut mucosa.     

TGF‐β downregulates KLRG1 expression in mouse and human CD8+ T cells

The inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) and the integrin α_E (CD103) are expressed by CD8⁺ T cells and both are specific for E‐cadherin. However, KLRG1 ligation by E‐cadherin inhibits effector T‐cell function, whereas binding of CD103 to E‐cadherin enhances cell–cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8⁺ T cells from untreated and virus‐infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8⁺ T cells‐infiltrating hepatocellular carcinomas. As TGF‐β is known to induce CD103 expression in CD8⁺ T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF‐β signaling in mouse as well as in human CD8⁺ T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8⁺ T‐cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8⁺ T cells if lymphocytes from tissues expressing high levels of TGF‐β are analyzed.     

mTOR signaling promotes a robust and continuous production of IFN‐γ by human memory CD8+ T cells and their proliferation

Human CD8⁺ T cells are functionally heterogeneous and can be divided into phenotypically and functionally distinct subsets according to CCR7 and CD45RA expression levels. Among these, CCR7^lowCD45RA^low effector memory CD8⁺ T cells (Tem) and CCR7^lowCD45RA^high CD8⁺ T cells, which are designated as Temra and considered to be terminally differentiated cells, are Ag‐experienced T cells but show different functionalities. Here, we show that, while Tem proliferate vigorously and produce IFN‐γ persistently and robustly, Temra proliferate poorly and lose the ability to produce IFN‐γ over time after TCR stimulation. Temra showed impaired cell growth upon TCR stimulation, which was associated with defective activation of the mammalian target of rapamycin (mTOR) signaling. Furthermore, rapamycin, an inhibitor of mTOR signaling, interfered with the robust and continuous proliferation of and IFN‐γ production by Tem at later time points after TCR stimulation. Thus, these data collectively indicate that activation of mTOR signaling is required for the robust functions of Tem cells in humans and suggest that defective mTOR signaling in Temra contributes to their functional impairment.     

γδ T cells restrain extrathymic development of Foxp3+‐inducible regulatory T cells via IFN‐γ

Inducible Treg (iTreg) cells generated from Ag‐stimulated naïve CD4⁺ T cells in the periphery play an important role in regulating immune responses. TGF‐β is a key cytokine that promotes this conversion process; however, how this process is regulated in vivo remains unclear. Here, we report that γδ T cells play a crucial role in controlling iTreg generation and suppressor function. Ag‐induced iTreg generation was significantly enhanced in C57BL/6 mice in the absence of γδ T cells. Inhibition of iTreg conversion was mediated by IFN‐γ produced by activated γδ T cells but not by activated CD4⁺ T cells. BM chimera experiments further confirmed γδ‐derived IFN‐γ‐dependent mechanism in regulating iTreg generation in vivo. Lastly, human peripheral blood γδ T cells also interfere with iTreg conversion via IFN‐γ. Our results suggest a novel function of γδ T cells in limiting the generation of iTreg cells, potentially balancing immunity and tolerance.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

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.     

Secreted IL‐1α promotes T‐cell activation and expansion of CD11b+Gr1+ cells in carbon tetrachloride‐induced liver injury in mice

Interleukin‐1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL‐1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL‐1α in a mouse model of carbon tetrachloride‐induced acute liver injury. We show that secreted IL‐1α aggravates liver damage and membrane IL‐1α slightly protects mice from liver injury. Further studies showed that secreted IL‐1α promotes T‐cell activation. It also increased the expansion of CD11b⁺Gr1⁺ myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL‐1α induced IL‐6 production from hepatocytes. IL‐6 neutralization reduced the proliferation of CD11b⁺Gr1⁺ myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL‐1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b⁺Gr1⁺ myeloid cells. These results demonstrate that secreted and membrane IL‐1α play different roles in acute liver injury. Secreted IL‐1α could promote T‐cell activation and the recruitment and expansion of CD11b⁺Gr1⁺ myeloid cells through induction of CCL2, CXCL5, and IL‐6. The controlled release of IL‐1α could be a critical regulator during acute liver inflammation.     

Biphasic role of 4‐1BB in the regulation of mouse cytomegalovirus‐specific CD8+ T cells

The initial requirement for the emergence of CMV‐specific CD8⁺ T cells is poorly understood. Mice deficient in the cosignaling TNF superfamily member, 4‐1BB, surprisingly developed exaggerated early CD8⁺ T‐cell responses to mouse CMV (MCMV). CD8⁺ T cells directed against acute MCMV epitopes were enhanced, demonstrating that 4‐1BB naturally antagonizes these primary populations. Paradoxically, 4‐1BB‐deficient mice displayed reduced accumulation of memory CD8⁺ T cells that expand during chronic/latent infection. Importantly, the canonical TNF‐related ligand, 4‐1BBL, promoted the accumulation of these memory CD8⁺ T cells, whereas suppression of acute CD8⁺ T cells was independent of 4‐1BBL. These data highlight the dual nature of the 4‐1BB/4‐1BBL system in mediating both stimulatory and inhibitory cosignaling activities during the generation of anti‐MCMV immunity.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Expression of an avian CD6 candidate is restricted to αβ T cells,splenic CD8+ γδ T cells and embryonic natural killer cells

A candidate avian CD6 homolog is identified by the S3 monoclonal antibody. The S3 antigen exists in a phosphorylated glycoprotein form of 130 kDa and a nonphosphorylated form of 110 kDa. Removal of phosphate groups and N-linked carbohydrates indicates a 78-kDa protein core. During thymocyte differentiation, the γδ T cells do not express S3, whereas mature CD4⁺ and CD8⁺ cells of αβ lineage acquire S3 antigen. All αβ T cells in the blood and spleen express the S3 antigen at relatively high levels. In contrast, only the CD8⁺ sub-population of γδ T cells in the spleen expresses the antigen and neither αβ nor γδ T cells in the intestinal epithelium express the S3 antigen. The S3 antigen is also found on embryonic splenocytes with a phenotypic profile characteristic of avian natural killer cells. The biochemical characteristics and this cellular expression pattern imply that the S3 antigen is the chicken CD6 homolog.     

α4β1 integrin promotes accumulation of tissue‐resident memory CD8+ T cells in salivary glands

The salivary glands (SGs) of virus‐immune mice contain substantial numbers of tissue‐resident memory CD8⁺ T cells (T_RM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus‐specific CD8⁺ T cells to the SGs have not yet been defined. Here, we found that polyinosinic‐polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR‐transgenic CD8⁺ T_RM cells in SGs in an α₄β₁ integrin‐dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 T_RM cells in SGs and intestine but not in kidney was also α₄ integrin dependent. Blockade of α₄β₇ by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus‐induced accumulation of P14 T_RM cells in the intestine but not in SGs. In conclusion, our data reveal that α₄β₁ integrin mediates CD8⁺ T_RM accumulation in SGs and that poly(I:C) can be used to direct activated CD8⁺ T cells to this organ.     

Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell‐mediated anti‐tumour immunity in a murine mammary breast cancer model

Both invariant natural killer T (NK T) cells and CD4⁺CD25⁺ T regulatory cells (T_regs) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by α‐galactosylceramide (α‐GalCer) execute anti‐tumour activity by secreting cytokines. By contrast, T_regs intrinsically suppress antigen‐specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T_regs regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either α‐GalCer or anti‐CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T_regs boosted the anti‐tumour effect of α‐GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T_reg cell depletion with α‐GalCer to stimulate NK T cells for cancer therapy.     

Murine T Cell Determination of Pregnancy Outcome: I. Effects of Strain, αβ T Cell Receptor, γδ T Cell Receptor,and γδ T Cell Subsets

PROBLEM: T cells bearing αβ T cell receptor (TcR) and γδ TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, αβ, γδ, and CD8^+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of γδ T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted γδ T cells producing the abortogenic cytokines, TNF-α and γ-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-β2 (TGF-β2). Immunization also boosted the number of αβ T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-δ) depleted γδ T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-β) decreased the number of αβ T cells and led to 100% abortions. CD8⁺ T cells present in peri-implant decidua before onset of abortions were mostly αβ TcR⁺, although some were γδ⁺. Changes in γδ and αβ T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual γδ T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-β2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-α and γ-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine⁺ subset of γδ cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by γδ T cells may augment the cytokine pool. In contrast, αβ T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the γδ T cells in decidua.     

Preferentially expanding Vγ1+ γδ T cells are associated with protective immunity against Plasmodium infection in mice

γδ T cells play a crucial role in controlling malaria parasites. Dendritic cell (DC) activation via CD40 ligand (CD40L)‐CD40 signaling by γδ T cells induces protective immunity against the blood‐stage Plasmodium berghei XAT (PbXAT) parasites in mice. However, it is unknown which γδ T‐cell subset has an effector role and is required to control the Plasmodium infection. Here, using antibodies to deplete TCR Vγ1⁺ cells, we saw that Vγ1⁺ γδ T cells were important for the control of PbXAT infection. Splenic Vγ1⁺ γδ T cells preferentially expand and express CD40L, and both Vγ1⁺ and Vγ4⁺ γδ T cells produce IFN‐γ during infection. Although expression of CD40L on Vγ1⁺ γδ T cells is maintained during infection, the IFN‐γ positivity of Vγ1⁺ γδ T cells is reduced in late‐phase infection due to γδ T‐cell dysfunction. In Plasmodium‐infected IFN‐γ signaling‐deficient mice, DC activation is reduced, resulting in the suppression of γδ T‐cell dysfunction and the dampening of γδ T‐cell expansion in the late phase of infection. Our data suggest that Vγ1⁺ γδ T cells represent a major subset responding to PbXAT infection and that the Vγ1⁺ γδ T‐cell response is dependent on IFN‐γ‐activated DCs.     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.