Regulation of Human Cytotoxic T Lymphocytes Development by the Synergistic Effect of IL-7 and sCD23

The present study was undertaken to investigate the interaction of IL-7 and sCD23 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were stimulated with mitogen plus IL-2 and subcultured for 7 days with IL-7 and/or sCD23. The combination of IL-7 and sCD23 synergistically enhanced the proliferation of both CD4⁺and CD8⁺T cells. CD8⁺T cells, however, were usually more responsive to IL-7 and sCD23. This synergy was observed on both subsets of T cells. Furthermore, these cytokines synergistically augment the CTL activity of CD8⁺T cells in both mitogen- and antigen-activated T cells. MAbs anti-IL-2 or anti-IL-2R (CD25) and anti-IL-12 had no effect on T cell proliferation and CD8⁺cytotoxic activity induced by IL-7 and sCD23. We analyzed the effect on IFN-γ induction by CD8⁺T cells and found that IL-7 alone was incapable of inducing detectable levels of IFN-γ production, but together with sCD23 it enhanced the production of IFN-γ. We also found that IFN-γ was not required for enhanced CTL activity of CD8⁺T cells, because rabbit anti-IFN-γ did not block the synergistic effects of either cytokine. The data demonstrate that the synergistic stimulatory activity of IL-7 and sCD23 may be of significance in the human CTL development and provide an alternative mechanism of stimulating T cells for use in immunotherapy.     

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4⁺ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4⁺ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35^?/? and IL-12p40^?/?) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4⁺ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-γ (IFN-γ) and interleukin-4 (IL-4) in purified CD4⁺ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-γ and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-γ mRNA in CD4⁺ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-γ in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.     

Allergen-specific IL-10-secreting type I T regulatory cells,but not CD4CD25Foxp3 T cells,are decreased in peripheral blood of patients with persistent allergic rhinitis

We investigate the frequencies of CD4⁺CD25⁺Foxp3⁺ T cells and allergen-specific IL-10⁺IL-4^-, IFN-γ⁺IL-4^-, IL-4⁺IFN-γ^-CD4⁺ T cells (which display characteristics of nTreg, Tr1-, Th1- and Th2- cells, respectively) in peripheral blood mononuclear cells (PBMCs) of patients with AR and of healthy individuals. In addition, we estimated the suppressive effect of CD4⁺CD25⁺ Treg cells and allergen-specific, IL-10-secreting cells from both two groups. The frequency of CD4⁺CD25⁺Foxp3⁺ T cells is similar in 43 AR patients compared with 38 healthy subjects. CD4⁺CD25^high cells retain suppressive activity on allergen-stimulated cell proliferation and cytokine production of Th1 but not Th2 cells in both groups. However, the frequency of allergen-specific IL-10⁺IL-4^-CD4⁺ T cells is reduced in AR patients, and correlates inversely to clinical symptom scores. Allergen-specific, IL-10-secreting cells potently suppressed D. pteronyssinus major allergen 1-stimulated cell proliferation and cytokine production (IFN-γ and IL-4) in healthy individuals. Altogether our data indicate that the number and function of CD4⁺CD25⁺ Treg cells from allergic patients are not impaired. However, the deficiency of allergen-specific Tr1 cells may play a role in the development of AR.     

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.     

Influence of a mutation in IFN-γ receptor 2 (IFNGR2) in human cells on the generation of Th17 cells in memory T cells

The T cell subsets involved in inflammatory reactions are mainly the IFN-γ secreting Th1 cells and IL17-producing Th17 cells. Although Th17 cells are primed in the thymus, there is evidence that Th17 cells can be generated from effector memory CD4⁺ T cells. Cytokines as IL-6, TGF-β, IL-21 and IL-23 involved in development of Th17 cells are well described. Here we analyzed the impact of a mutation in the IFN-γ receptor 2 (IFN-γR2) on the induction of Th17 cells. By isolation of T cells and monocytes of a patient with this mutation we could demonstrate an inhibitory role of IFN-γ signaling as IFN-γR2-deficient monocytes induce a higher percentage of IL-17⁺ cells from both healthy and IFN-γR2-deficient CD4⁺ T cells. This data confirm the interference of these two T helper subsets and points to a balance of Th1 and Th17 cells obtained by their own cytokine production and their interplay with APCs.     

Schistosoma-specific helper T cell clones from subjects resistant to infection by Schistosoma mansoni are Th0/2

Although T helper cells play a critical role in human immunity against schistosomes, the properties of the T lymphocytes that govern resistance and pathogenesis in human schistosomiasis are still poorly defined. This work addresses the question as to whether human resistance to Schistosoma mansoni is associated with a particular T helper subset. Twenty-eight CD3⁺, CD4⁺, CD8^? parasite-specific T cell clones were isolated from three adults with high degree of resistance to infection by S. mansoni. The lymphokine secretion profiles of these clones were determined and compared to those of 21 CD3⁺, CD4⁺, CD8^? clones with unknown specificity, established from these same subjects in the same cloning experiment. Almost all parasite-specific clones produced interleukin (IL)-4 and interferon (IFN)-γ in large amounts. However, they generally produced more IL-4 than IFN-γ; variations in IL-4/IFN-γ ratios were accounted for by differences in IFN-γ production since IL-4 levels were comparable for the clones from the three subjects. T cell clones of unknown specificity produced significantly less IL-4 and more IFN-γ than parasite-specific T cell clones. Most clones produced IL-2, and IL-2 production did not differ between the two types of clones. Parasite-specific T cell clones from the resistant subjects were compared to specific T cell clones from a sensitized adult from a nonendemic area: T cell clones from this latter subject were the highest IFN-γ and the lowest IL-4 producers, compared to those of resistant subjects. Thus, parasite-specific T cell clones isolated from adults resistant to S. mansoni belong to the Th0 subset and produced more IL-4 than IFN-γ (Th0/2), whereas clones of a sensitized adult from a nonendemic area are also Th0, but produce more IFN-γ than IL-4 (Th0/1). These results support previous conclusions on the role of IgE in protection against schistosomes in humans, and may indicate that IFN-γ is required for full protection.     

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.     

IL-15 promotes regulatory T cell function and protects against diabetes development in NK-depleted NOD mice

IL-15, an anti-apoptotic cytokine, has been reported to promote the survival and function of NK cells and T cells, including regulatory T cells (Tregs). Here we examined the effect of repeated injections of IL-15 on the development of diabetes in NOD mice. Injection of recombinant murine IL-15, once a day for 2 weeks, neither inhibited nor accelerated diabetes development in untreated NOD mice. However, treatment with IL-15 significantly reduced the incidence and delayed the onset of diabetes in NOD mice that were depleted of NK cells, while NK cell depletion alone had no protection against the disease development. The protective effect in IL-15-treated, NK cell-depleted NOD mice was associated with an increase in immunosuppressive activity of CD4⁺CD25⁺ Tregs. IL-15 also enhanced Foxp3 expression in CD4⁺CD25⁺ cells in an in vitro culture system, and such an effect of IL-15 was abrogated by IL-15-activated NK cells. Inhibition of IL-15-induced Foxp3 expression by IL-15-activated NK cells likely resulted from their IFN-γ production, as recombinant IFN-γ, or the culture supernatant of IL-15-activated wild-type mouse NK cells but not of IL-15-activated IFN-γ-deficient NK cells, mediated a similar inhibition. IFN-γ also diminished the stimulatory effect of IL-15 on Treg function in vitro. These results indicate that IL-15 has the potential to promote Treg function and protect against diabetes development in NOD mice, but such an activity can be eliminated by simultaneous activation of NK cells in IL-15-treated mice.     

Quantity and Quality Reconstitution of NKG2A+ Natural Killer Cells Are Associated with Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation

The immune mechanism underlying graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (HSCT) remains unclear. Natural killer (NK) cells play a crucial role in mediating pathogen-specific immunity and are the first donor-derived lymphocytes reconstituted post-HSCT. However, NK cells vary at different stages after HSCT. Here, we found that the absolute NKG2A⁺ subset cell counts and the percentages of NKG2A⁺ among NK cells were significantly reduced in GVHD patients after HSCT compared with those from non-GVHD patients. Moreover, the reduction in NKG2A⁺ NK cells in post-HSCT GVHD patients was ascribed to increased apoptosis and a decreased proliferation capacity while retaining a strong graft-versus-leukemia effect. In vitro assays showed that co-culture of T cells with NKG2A⁺ NK cells significantly reduced IFN-γ secretion by T cells and increased IL-4 secretion. Moreover, the CD25 expression level was decreased, whereas the number of cells with the CD4⁺CD25⁺FOXP3⁺ phenotype was increased. In addition, the NKG2A⁺ NK cells induced T cell apoptosis and decreased T cell proliferation during the co-culture process. Importantly, NKG2A⁺ NK cells mainly regulated activated but not resting T cells. In vivo assays showed that the serologic IL-10 level was evidently lower in GVHD than in non-GVHD patients, whereas the IL-1β, IFN-γ, and tumor necrosis factor-α levels were higher in GVHD patients. Furthermore, the NKG2A⁺ NK cell ratio from GVHD patients was markedly increased by the presence of exogenous IL-10 but not by other cytokines. In contrast, the NKG2A⁺ cell ratio from non-GVHD patients was not increased by IL-10. Therefore, post-HSCT GVHD may be ascribed to the reduced induction of NKG2A⁺ NK cells by IL-10, which further overactivates T cells.     

Human complement C3 deficiency: Th1 induction requires T cell-derived complement C3a and CD46 activation

Human T helper type 1 (Th1) responses are essential in defense. Although T cell receptor (TCR) and co-stimulator engagement are indispensable for T cell activation, stimulation of additional receptor pathways are also necessary for effector induction. For example, engagement of the complement regulator CD46 by its ligand C3b generated upon TCR activation is required for IFN-γ production as CD46-deficient patients lack Th1 responses. Utilizing T cells from two C3-deficient patients we demonstrate here that normal Th1 responses also depend on signals mediated by the anaphylatoxin C3a receptor (C3aR). Importantly, and like in CD46-deficient patients, whilst Th1 induction are impaired in C3-deficient patients in vitro, their Th2 responses are unaffected. Furthermore, C3-deficient CD4⁺ T cells present with reduced expression of CD25 and CD122, further substantiating the growing notion that complement fragments regulate interleukin-2 receptor (IL-2R) assembly and that disturbance of complement-guided IL-2R assembly contributes to aberrant Th1 effector responses. Lastly, sustained intrinsic production of complement fragments may participate in the Th1 contraction phase as both C3a and CD46 engagement regulate IL-10 co-expression in Th1 cells. These data suggest that C3aR and CD46 activation via intrinsic generation of their respective ligands is an integral part of human Th1 (but not Th2) immunity.     

Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-γ) in CD28+ (‘cytotoxic’) and CD28− (‘suppressor’) CD8+ subsets

We have measured by flow cytometry the ability of subsets of CD8⁺ CD3⁺ lymphocytes within mononuclear cell preparations to make intracellular cytokines (IL-2, tumour necrosis factor-alpha (TNF-α) and IFN-γ) on stimulation in vitro with phorbol myristate acetate (PMA) and ionomycin for 16 h. These CD8⁺ subsets were defined by the presence or absence of CD28 or HLA-DR. Subsets of normal CD8⁺ cells were compared with cells from the antibody deficiency disease common variable immunodeficiency (CVID). In CVID there was a significant increase in the production of IFN-γ in the CD8⁺ CD28⁺ subset (‘cytotoxic’). This reflects a shift in this disease towards an excessive Th1 response away from B cell help. Paradoxically, some CVID patients also showed a reduction in IFN-γ production in the CD8⁺ CD28^? subset (‘suppressor’) which was associated with a failure of these cells to maintain a state of activation after a stimulus in vitro. The B cell problem in this disease is known to be related to a failure of T cell help shown by an inability to produce the antigen-specific CD4⁺ memory T cells needed for successful B cell maturation. The two pathological CD28 subsets of CD8⁺ cells we have found in CVID may both be detrimental to a normal CD4-dependent immune response. The CD28^? suppressor subset expands and is unable to maintain activation and cytokine secretion, and the CD28⁺ cytotoxic subset is over-producing the Th1 cytokine IFN-γ.     

Naturally activated CD4+ T cells are highly enriched for cytokine-producing cells

Most T cells in a normal non-immunized individual are in a resting state. However, a small proportion of splenic T cells are large activated cells both in specific pathogen-free and antigen-free mice. To further elucidate the effector functions associated with these “naturally” activated CD4⁺ T cells, we have characterized the expression of various membrane markers, cytokine production and T helper activity by these cells. We show that naturally activated CD4⁺ T cells express activation markers and contain tenfold higher proportions of cells producing IL-4, IL-10 and IFN-γ as compared to small CD4⁺ T cells. Despite the high proportion of IFN-γ producers, naturally activated CD4⁺ T cells still induce B cell proliferation and differentiation. These results are discussed in the context of normal physiological autoreactivity.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

IL-12 and IL-18 induce interferon-γ production and de novo CD2 expression in porcine γδ T cells

γδ T cells are highly abundant in the blood and spleen of pigs but little is known about their functional differentiation. In this study the potential of the type-1 polarizing cytokines IL-12 and IL-18 in combination with IL-2 and Concanavalin A (ConA) to stimulate porcine γδ T cells was investigated. Stimulation of purified γδ T cells with ConA and IL-2 induced a strong proliferation of CD2⁻ γδ T cells, whereas additional stimulation with IL-12 and IL-18 caused a stronger proliferation of CD2⁺ γδ T cells. IFN-γ could only be detected in supernatants of γδ T-cell cultures supplemented with IL-12 and IL-18. Experiments with sorted CD2/SWC5-defined γδ T-cell subsets revealed that CD2⁺SWC5⁻ γδ T cells are the main producers of IFN-γ following stimulation with IL-2/IL-12/IL-18. Additional stimulation with ConA led to an upregulation of CD2 within the CD2⁻ γδ T cell subsets, indicating a previously unnoticed plasticity of CD2-defined γδ T cell subsets.     

Low expression of CD40 and B7 on macrophages infiltrating UV-exposed human skin; role in IL-2Rα− T cell activation

After UV exposure of skin, epidermal Langerhans cells (LC) are depleted, whereas CD11b⁺ CD36⁺ CD1a⁻ monocytes/macrophages (UV-Mϕ) infiltrate. Different immunological outcomes in vivo are mediated by LC (sensitization) and UV-Mϕ (tolerance) which may be related to the distinct T cell activation states that these antigen-presenting cells (APC) induce. We previously demonstrated that CD4⁺ T lymphocytes activated by UV-Mϕ are, in contrast to LC-activated T cells, IL-2Rα deficient, and we hypothesize that this differential T cell activation is related to differences in co-stimulatory molecules between UV-Mϕ and LC. Using four-color flow cytometry, we found a reduced capacity to up-regulate expression of the important co-stimulatory molecules CD40, B7-1 and B7-2 by UV-Mϕ relative to LC. This alteration in co-stimulatory molecule expression was selective, because UV-Mϕ express equal levels of ICAM-1 and ICAM-3, and increased levels of LFA-1, relative to LC. After bidirectional signaling with T cells during alloantigen presentation, UV-Mϕ still exhibited less CD40 and B7-1 than LC. Addition of IFN-γ induced CD40 and B7-1 expression on UV-Mϕ and restored IL-2Rα expression on UV-Mϕ-activated T cells but had no effect on IL-2Rα on resting or LC-activated T cells. The restoration of IL-2Rα expression on UV-Mϕ-activated T cells by IFN-γ was inhibited (67 %, p = 0.005) by addition of neutralizing anti-CD40. Therefore, differences in co-stimulatory molecule expression, in particular CD40, on UV-Mϕ and LC are critical in determining the distinct T cell activation induced by these APC.     

Regulation of IFN-γ production in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF−/−) mice produce far lower serum levels of IFN-γ in response to LPS than GM-CSF+/+ mice. CD4⁺ and CD8⁺ T cells from LPS-injected GM-CSF−/− mice showed a deficiency in IFN-γ production and proliferative activity in response to IL-2 and IL-12, whereas IFN-γ production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF−/− mice do not have an intrinsic defect in IFN-γ production, because IL-12 injection induces the same high levels of IFN-γ in GM-CSF−/− and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF−/− T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-γ levels and T cell responses in LPS-injected GM-CSF−/− mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.