Antigen-induced T cell death is regulated by CD4 expression

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.     

Notch1 expression on T cells is not required for CD4+ T helper differentiation

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.     

Persistent expression of CD94/NKG2 receptors by virus-specific CD8 T cells is initiated by TCR-mediated signals

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.     

Inhibitory effect of interleukin-16 on interleukin-2 production by CD4+ T cells

Signalling through CD4 by human immunodeficiency virus (HIV)-1 envelope glycoprotein (gpl20) and/or anti-CD4 antibodies can promote T-cell activation and anergy. Interleukin (IL)-16 is a competence growth factor for CD4+ T cells that can induce a G0 to G1 cell cycle transition but cannot induce cell division. The receptor of this cytokine is thought to be the CD4 molecule, although the binding epitope of IL-16 differs from that of HIV. We have demonstrated that both HIV-1/gp120 and IL-16 induced CD4+ T-cell dysfunction, as indicated by suppression of mitogen-induced IL-2 production. Two anti-CD4 antibodies with different binding sites on CD4 also showed an inhibitory effect on IL-2 production. These results indicate that promotion of CD4+ T-cell anergy via the CD4 molecule does not depend on the binding sites of the CD4 ligands.     

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells

There is a growing body of evidence which suggests that CD8⁺ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4⁺ T cell responses to ovalbumin (OVA) by CD8⁺ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8⁺ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8⁺ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8⁺ T cells on days 12–18 greatly enhanced the IgE response, which rose to 26 μ/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 μ/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8⁺ T cells from animals immunized on day 12 completely inhib ited the IgE response. This effect was antigen specific; CD8⁺ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8⁺ T cell depletion decreased interferon (IFN)-γ production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4⁺ T cells. Furthermore, CD8⁺ T cell depletion and addition of anti-IFN-γ antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8⁺ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8⁺ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-γ production by CD4⁺ T cells.     

IFN-{gamma} expression in CD8+ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4+ T cell death

Infection with pathogens containing superantigens (Sags) canresult in massive excessive CD4⁺ T cell activation and deathin such conditions as toxic shock, food poisoning and autoimmunediseases. We here showed how enhancement of IL-6 signaling suppressesSag-mediated activated CD4⁺ T cell death. Sag-induced CD4⁺ Tcell death increased in IL-6 knockout (KO) mice, whereas itdecreased in mice characterized by enhanced IL-6–gp130–STAT3signaling. The serum concentration of IFN- was inversely correlatedwith the magnitude of IL-6 signaling, and IFN- deficiency inhibitedSag-induced activated CD4⁺ T cell death, suggesting that IL-6suppresses CD4⁺ T cell death via IFN- expression. Interestingly,depletion of activated CD8⁺ T cells inhibited Sag-mediated increasesin IFN- expression in IL-6 KO mice as well as the augmentedCD4⁺ T cell death. The results demonstrate that IL-6–gp130–STAT3signaling in activated CD8⁺ T cells contributes to Sag-inducedCD4⁺ T cell death via IFN- expression, highlighting this signalingaxis in CD8⁺ T cells as a potential therapeutic target for Sag-relatedsyndromes.     

Cooperation between CD4 and CD8 T cells for anti-tumor activity is enhanced by OX40 signals

The relative contribution of OX40 (CD134) to priming of CD8 T cells in complex systems where CD4 and CD8 cells respond and cooperate together is not clear. We previously found that OX40 expressed on tumor-reactive CD8 T cells controls their initial persistence when adoptively transferred in vivo and is required for delayed tumor growth. We now show that exogenous stimulation of OX40 with agonist antibody augments its ability to suppress the growth of new as well as established tumors, correlating with marked expansion of adoptively transferred CD8 T cells. Concomitantly, anti-OX40 strongly enhanced the number of tumor antigen-reactive CD4 T cells. Moreover, the augmented accumulation of CD8 T cells was prevented in animals lacking MHC class II or depleted of CD4 cells and did not occur in OX40-deficient animals receiving wild-type CD8 cells, demonstrating that non-CD8 cells are the major target of OX40 signals. These results suggest that while OX40 signaling to a CD8 T cell can control its expansion, OX40 expressed on non-CD8 cells strongly influences CD8 priming and in vivo activity. OX40 therefore represents an important signal for allowing effective cooperation between CD4 and CD8 cells and for promoting cell interplay and tumor rejection where CD8 activity is limiting.     

Notch signalling regulates cytokine production by CD8+ and CD4+ T cells

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.     

Interleukin (IL)-4 promotes T helper type 2-biased natural killer T (NKT) cell expansion, which is regulated by NKT cell-derived interferon-gamma and IL-4

CD1d-restricted natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and Th2 cytokines and also play regulatory or pathological roles in immune responses. NKT cells are able to expand when cultured with alpha-galactosylceramide (alpha-GalCer) and interleukin (IL)-2 in a CD1d-restricted manner. However, the expansion ratio of human NKT cells is variable from sample to sample. In this study, we sought to determine what factor or factors are responsible for efficient in vitro expansion of NKT cells from various inbred mouse strains. Although the proportion of NKT cells in the spleen was nearly identical in each mouse strain, the growth rates of NKT cells cultured in vitro with alpha-GalCer and IL-2 were highly variable. NKT cells from the B6C3F1 and BDF1 mouse strains expanded more than 20-fold after 4 days in culture. In contrast, NKT cells from the strain C3H/HeN did not proliferate at all. We found that cell expansion efficiency correlated with the level of IL-4 detectable in the supernatant after culture. Furthermore, we found that exogenous IL-4 augmented NKT cell proliferation early in the culture period, whereas interferon (IFN)-gamma tended to inhibit NKT cell proliferation. Thus, the ratio of production of IL-4 and IFN-gamma was important for NKT cell expansion but the absolute levels of these cytokines did not affect expansion. This finding suggests that effective expansion of NKT cells requires Th2-biased culture conditions.     

TcR-induced regulated secretion leads to surface expression of CTLA-4 in CD4+CD25+ T cells

In this study we show that CD4+ T cells develop a functional regulated secretory compartment after differentiation into effector cells, as shown by their increased expression and T-cell receptor-induced exocytosis of lysosomal and cytotoxic effector proteins. We tested the hypothesis that activation-induced surface cytotoxic T-lymphocyte-associated antigen (CTLA-4) expression in CD4+CD25+ regulatory T cells occurs via a similar regulated secretory pathway. Fluorescence microscopy showed that internal CTLA-4 in these cells was stored in a vesicular compartment distinct from lysosomal vesicles. Rapid activation-induced CTLA-4 surface expression in mouse CD4+CD25+ T cells is independent of protein synthesis and Rab-27a. When antigen-dependent T-cell-antigen-presenting cell (APC) conjugates were analysed for surface distribution of CD86 on APC, a higher concentration of CD86 molecules was observed in the synapse of APC conjugated to CD4+CD25+ cells than APC conjugated to CD4+CD25- cells. These results demonstrate that fast delivery of mediators by the regulated secretory pathway in CD4+ T cells can be used to perform other functions that are not involved in cytotoxic function but that can influence/regulate other cells.     

CD28 controls the development of innate‐like CD8+ T cells by promoting the functional maturation of NKT cells

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αβ NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger⁺ IL‐4⁺ NKT cells and upregulation of LFA‐1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28‐deficient mice is cell autonomous. Moreover, we show in both wild‐type C57BL/6 mice and in downstream of tyrosine kinase‐1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28‐mediated regulation of thymic IL‐4⁺ NKT cells promotes the differentiation of eomesodermin⁺ CD44^high innate‐like CD8⁺ T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT‐cell homeostasis and the size of the innate‐like CD8⁺ T‐cell pool.     

Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells

Interleukin-2-deficient mice (IL-2^?/?) crossed to a BALB/c genetic background develop a lymphoproliferative syndrome with severe hemolytic anemia and die within 5 weeks of age. The presence of autoantibodies of various specificities and inflammatory lesions in several organs are indicative of a generalized autoimmune disease. No alterations of the immune system were observed in 6-day-old animals, but 10-day-old mice already showed an increased proliferation and polyclonal activation of lymphocytes. The treatment of IL-2^?/? mice with anti-gp39(CD40L) antibody prevented the disease and indicated that the appearance of activated CD4⁺ T cells (CD44^high, CD69⁺) represents the first alteration of the immune system in IL-2^?/? mice. Collectively, our results suggest that an essential role of IL-2 in vivo, which is not compensated by other cytokines, is the maintenance of self tolerance.