Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease

Th17 is a newly identified T-cell lineage that secretes proinflammatory cytokine IL-17. Th17 cells have been shown to play a critical role in mediating autoimmune diseases such as EAE, colitis, and arthritis, but their role in the pathogenesis of graft-versus-host disease (GVHD) is still unknown. Here we showed that, in an acute GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient, IL-17(-/-) donor T cells manifested an augmented Th1 differentiation and IFN-gamma production and induced exacerbated acute GVHD. Severe tissue damage mediated by IL-17(-/-) donor T cells was associated with increased Th1 infiltration, up-regulation of chemokine receptors by donor T cells, and enhanced tissue expression of inflammatory chemokines. Administration of recombinant IL-17 and neutralizing IFN-gamma in the recipients given IL-17(-/-) donor cells ameliorated the acute GVHD. Furthermore, the regulation of Th1 differentiation by IL-17 or Th17 may be through its influence on host DCs. Our results indicate that donor Th17 cells can down-regulate Th1 differentiation and ameliorate acute GVHD in allogeneic recipients, and that treatments neutralizing proinflammatory cytokine IL-17 may augment acute GVHD as well as other inflammatory autoimmune diseases.     

CD8+ T Cells of Tc2 Phenotype Mediate a GVL Effect and Prevent Marrow Rejection

Donor T cells mediate both beneficial and detrimental immune reactions in the setting of allogeneic BMT (alloBMT). T cells mediate the GVL effect and prevent marrow rejection, but also induce GVHD. In an attempt to favorably influence the balance of these allogeneic responses, we have evaluated the effect of donor CD4⁺, Thl/Th2 and CD8⁺, Tcl/Tc2 functional T cell subsets in murine marrow transplantation models. Our studies have identified the CD8⁺Tc2 population (which is a cytolytic effector secreting the type II cytokines IL-4, IL-5, and IL-10) as a subset capable of mediating the GVL effect and preventing marrow rejection with reduced GVHD. We have also shown that the Tc2 subset can be generated in humans. These studies indicate that administration of donor CD8⁺ T cells of Tc2 phenotype represents a strategy for improving alloBMT outcome.     

Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo

Peripheral blood stem cell (PBSC) transplantation is successful in improving engraftment without increasing acute graft-versus-host disease (GVHD), despite much larger numbers of T cells in unmanipulated PBSCs than in bone marrow grafts. In mouse models and retrospective human studies, granulocyte colony-stimulating factor (G-CSF) therapy has been associated with less acute GVHD. We studied the effect of G-CSF on interferon (IFN)-gamma and IL-4 expression in CD4(+) lymphocytes. CD4(+) cells co-cultivated with G-CSF and stimulated with PHA or CD3 monoclonal antibodies showed significant decreases in IFN-gamma and increases in IL-4 expression (n = 13; P <. 01). G-CSF appeared to have a direct effect on CD4(+) cells independent of monocytes present in the culture because purified CD4(+) cells exposed to G-CSF, washed, and cocultivated with untreated monocytes demonstrated similar changes in IFN-gamma and IL-4 expression, whereas untreated CD4(+) cells cocultured with G-CSF-stimulated monocytes behaved as controls. We then studied peripheral blood mononuclear cells (PBMCs) from G-CSF-mobilized PBSC donors. When their PBMCs were cultured with PHA or CD3 monoclonal antibody, the percent of IFN-gamma-expressing cells decreased by a mean of 55% and 42%, respectively, whereas the percent of IL-4-containing cells increased by a mean of 39% and 58%, respectively, following G-CSF stimulation. Increased apoptosis of IFN-gamma-producing CD4(+) cells was not responsible for the shift in TH1/TH2 subsets. G-CSF-R mRNA was present in both CD4(+) and CD8(+) cells. These results suggest that G-CSF decreases IFN-gamma and increases IL-4 production in vitro and in vivo and likely modulates a balance between TH1 and TH2 cells, an effect that may be important in PBSC transplantation.     

CD3/CD28-costimulated T1 and T2 subsets: differential in vivo allosensitization generates distinct GVT and GVHD effects

Adoptive T-cell therapy using CD3/CD28 co-stimulation likely requires in vivo generation of antigen specificity. Because CD28 promotes TH1/TC1 (T1) or TH2/TC2 (T2) differentiation, costimulation may generate donor T1 or T2 cells capable of differentially mediating allogeneic graft-versus-tumor (GVT) effects and graft-versus-host disease (GVHD). Costimulation under T1 or T2 conditions indeed generated murine TH1/TC1 cells secreting interleukin-2/interferon-gamma (IL-2/IFN-gamma) or TH2/TC2 cells secreting IL-4/IL-5/IL-10. In vivo, allogeneic T1 cells expanded, maintained T1 secretion, and acquired allospecificity involving IFN-gamma and IL-5. In contrast, allogeneic T2 cells expanded less and maintained T2 secretion but did not develop significant allospecificity.Allogeneic, but not syngeneic, T1 cells mediated a GVT effect against host-type breast cancer cells, as median survival time (MST) increased from 25.6 +/- 2.6 (tumor controls) to 69.2 +/- 5.9 days (P < 1.2 x 10(-9)). This T1-associated GVT effect operated independently of fasL because T1 cells from gld mice mediated tumor-free survival. In contrast, allogeneic T2 cells mediated a modest, noncurative GVT effect (MST, 29 +/- 1.3 days; P <.0019). T1 recipients had moderate GVHD (histologic score, 4 of 12) that contributed to lethality after bone marrow transplantation; in contrast, T2 recipients had minimal GVHD (histologic score, 1 of 12). CD3/CD28 co-stimulation, therefore, generates T1 or T2 populations with differential in vivo capacity for expansion to alloantigen, resulting in differential GVT effects and GVHD.     

IL-17 contributes to CD4-mediated graft-versus-host disease

CD4(+) interleukin-17 (IL-17)(+) T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4(+) donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17(-/-) T-cell recipients. However, upon transfer of murine IL-17(-/-) CD4(+) T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4(+) T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17(-/-) CD4(+) T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-gamma-secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-gamma, IL-4, and IL-6) in recipients of IL-17(-/-) CD4(+) T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.     

Pretreatment of donor mice with granulocyte colony-stimulating factor polarizes donor T lymphocytes toward type-2 cytokine production and reduces severity of experimental graft-versus-host disease

The incidence and severity of acute graft-versus-host disease (GVHD) after allogeneic transplantation using peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor (G-CSF) appear to be no worse than those after bone marrow transplantation, despite the presence of large numbers of T cells in the donor infusion. Experimental studies have shown that type-1 T cells (secreting interleukin-2 [IL-2] and interferon-gamma) mediate acute GVHD, whereas type-2 T cells (secreting IL-4 and IL-10) can prevent acute GVHD. We tested the hypothesis that G-CSF modulates T-cell function toward a type-2 response and thus reduces the severity of acute GVHD. B6 mice were injected with G-CSF or diluent for 4 days, and their splenic T cells were stimulated in vitro with alloantigen or mitogen in the absence of G-CSF. T cells from G-CSF-treated mice showed a significant increase in IL-4 production, with a simultaneous decrease in IL-2 and interferon-gamma production in response to both stimuli. We also examined the effect of G-CSF pretreatment of donors in a GVHD model (B6- ->B6D2F1). Survival was significantly improved in recipients of G-CSF- treated donors. Concanavalin-A-induced cytokine production at day 13 after transplantation also showed an increase in IL-4 along with a decrease in IL-2 and IFN-gamma production by splenocytes from recipients of G-CSF-treated bone marrow and T cells. These data show that pretreatment of donors with G-CSF polarizes donor T cells toward the production of type-2 cytokines, which is associated with reduced type-1 cytokine production and reduced severity of acute GVHD.     

Interleukin-12 inhibits murine graft-versus-host disease

Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post- BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD- inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.     

Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation

Inducible costimulator (ICOS) is expressed on activated and memory T cells and is involved in the regulation of cytokine production. We studied the role of ICOS on alloreactive T cells in graft versus host disease (GVHD) and determined that ICOS expression was up-regulated on alloreactive T cells in recipients of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) with GVHD. We compared ICOS-/- T cells with wild-type (WT) T cells in 2 GVHD models. In both models, recipients of ICOS-/- T cells demonstrated significantly less GVHD morbidity and mortality, which was associated with less intestinal and hepatic GVHD but increased cutaneous GVHD. In addition, recipients of ICOS-/- donor T cells displayed a slight decrease in graft versus leukemia (GVL) activity. Further analysis of alloreactive ICOS-/- T cells showed no defect in activation, proliferation, cytotoxicity, and target organ infiltration. Recipients of ICOS-/- T cells had decreased serum levels of interferon-gamma (IFN-gamma), while interleukin-4 (IL-4) and IL-10 levels were increased, suggesting that alloreactive ICOS-/- T cells are skewed toward T helper-2 (Th2) differentiation. These data suggest a novel role for ICOS in the regulation of Th1/Th2 development of activated T cells. In conclusion, alloreactive ICOS-/- donor T cells induce less GVHD due to a Th2 immune deviation while GVL activity is slightly diminished.     

Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes

Tumor antigen-specific CD4+ and CD8+ T lymphocytes, especially interferon-gamma (IFN-gamma)-producing type-1 helper T (Th1) and type-1 cytotoxic T (Tc1) cells, play a crucial role in tumor eradication. Adoptive transfer using tumor-specific Th1 and Tc1 cells is a promising therapeutic strategy for tumor immunotherapy. However, its clinical application has been hampered because of difficulties in generating tumor-specific Th1 cells from patients with tumors. To overcome this problem, we have developed an efficient method to prepare tumor-specific Th1 and Tc1 cells. T-cell receptor (TCR) alpha and beta genes obtained from an HLA-A24-restricted, Wilms tumor 1 (WT1) peptide-specific Tc clone were lentivirally transduced to polyclonally activated Th1 and Tc1 cells. As expected, TCR gene-modified Tc1 cells showed cytotoxicity and IFN-gamma production in response to peptide-loaded lymphoblastoid cell lines, WT1 gene-transduced cells, and freshly isolated leukemia cells expressing both WT1 and HLA-A24. Surprisingly, we further demonstrated that Th1 cells transduced with HLA-class I-restricted TCR genes also showed both cytotoxicity and cytokine production in an HLA-A24-restricted manner. In contrast to gene-modified Tc1 cells, Th1 cells produced high amounts of interleukin-2 (IL-2) in addition to IFN-gamma, which is beneficial for induction of antitumor cellular immunity. Thus, TCR gene-modified HLA-class I-restricted Th1 and Tc1 cells are a powerful strategy for the application to adoptive immunotherapy of human cancer.     

Type I and type II T cell profiles in chronic myelogenous leukemia

T cell immunity is considered to play an important role in the control of cell growth in chronic myelogenous leukemia (CML) although information regarding the characteristics of T lymphocytes in CML patients is limited. Using flow cytometric analysis of intracellular cytokine expression at the single-cell level, we analyzed helper T and cytotoxic T subsets in 8 CML patients. The percentage of interferon-gamma (IFN-gamma) single-positive CD4 cells (Th1) and that of interleukin-4 (IL-4) single-positive CD4 cells (Th2) was markedly decreased in pretreated CML patients compared to normal controls. In addition, the percentage of IFN-gamma/IL-4 double-positive CD4 cells (Th0) was also reduced. Consequently, the percentage of IFN-gamma/IL-4 double-negative CD4 cells was markedly increased. Similarly, a reduction in IFN-gamma single-positive CD8 cells (Tc1) and IFN-gamma/IL-4 double-positive CD8 cells (Tc0) and an increase in IFN-gamma/IL-4 double-negative CD8 cells were observed in pretreated CML patients. Imbalance of these parameters was markedly improved following cytoreduction therapy. Our findings directly indicate anergic states in CML patients. Determination of the factors that affect Th and Tc profiles may lead to further understanding of immunological states and the development of effective immunotherapy.     

Cytokine and chemokine profiles in autologous graft-versus-host disease (GVHD): interleukin 10 and interferon gamma may be critical mediators for the development of autologous GVHD

Administration of the immunosuppressive drug cyclosporine A (CsA) following autologous stem cell transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with autoreactive CD8(+) T cells that recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain. To investigate the potential role of cytokines and chemokines in autologous GVHD, interleukin 2 (IL-2), IL-4, IL-10, interferon gamma (IFN-gamma), and macrophage inflammatory protein-1alpha (MIP-1alpha) gene expression in peripheral blood mononuclear cells (PBMCs) was determined in 36 patients treated with CsA following transplantation and correlated with the induction of cytolytic activity against autologous phytohemagglutinin-stimulated lymphocytes (PHA-blasts) and the breast cancer cell line (T47D). The determination of gene expression by real-time polymerase chain reaction (PCR) revealed that IL-10 mRNA levels by PBMCs in patients with autologous GVHD were 29-fold higher than in healthy individuals. IFN-gamma (4-fold), IL-2 (3-fold), and MIP-1alpha (44-fold) mRNA levels were also increased in GVHD-induced patients compared with healthy individuals. The ability of PBMCs to lyse autologous PHA-blasts and T47D tumor cells exhibited an identical temporal relationship with expression of IL-10 and IFN-gamma during autologous GVHD. Moreover, the susceptibility to autologous GVHD as assessed in 75 patients was significantly associated with the IL-10(-1082) G/G polymorphic alleles, allelic variants in the promoter region that govern IL-10 production. These findings indicate that IL-10 may play an unexpected but critical role in autologous GVHD and could be utilized to enhance a graft-versus-tumor effect after transplantation. Interestingly, polymorphisms in the IL-10 promoter region may also explain differences in the susceptibility of patients to autologous GVHD induction.     

Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo

To evaluate the direct effect of adenosine on cytokine-polarized effector T cells, murine type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1) and Th2/Tc2 cells were generated using an antigen-presenting cell (APC)-free method. Tc1 and Tc2 cells had similar adenosine signaling, as measured by intracellular cyclic AMP (cAMP) increase upon adenosine A(2A) receptor agonism by CGS21680 (CGS). CGS greatly reduced Tc1 and Tc2 cell interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) secretion, with nominal effect on interferon gamma (IFN-gamma) secretion. Tc2 cell IL-4 and IL-5 secretion was not reduced by CGS, and IL-10 secretion was moderately reduced. Agonist-mediated inhibition of IL-2 and TNF-alpha secretion occurred via A(2A) receptors, with no involvement of A(1), A(2B), or A(3) receptors. Adenosine agonist concentrations that abrogated cytokine secretion did not inhibit Tc1 or Tc2 cell cytolytic function. Adenosine modulated effector T cells in vivo, as CGS administration reduced CD4(+)Th1 and CD8(+)Tc1 cell expansion to alloantigen and, in a separate model, reduced antigen-specific CD4(+) Th1 cell numbers. Remarkably, agonist-mediated T-cell inhibition was abrogated by in vivo IL-2 therapy. Adenosine receptor activation therefore preferentially inhibits type I cytokine secretion, most notably IL-2. Modulation of adenosine receptors may thus represent a suitable target primarily for inflammatory conditions mediated by Th1 and Tc1 cells.     

Serum cytokine levels and type 1 and type 2 intracellular T cell cytokine profiles in mixed connective tissue disease

OBJECTIVE: To determine serum cytokine concentrations and intracellular cytokine production of CD4+ and CD8+ T cells in 20 patients with mixed connective tissue disease (MCTD). METHODS: Detailed analysis of cytokine production; 8 patients were in the active stage, 12 in the inactive stage of the disease. Serum concentrations of interferon-gamma (IFN-gamma), interleukin 4 (IL-4), and IL-10 were assessed by ELISA. Intracellular content of IFN-gamma, IL-4, and IL-10 in CD4+ and CD8+ peripheral blood T cell and lymphocyte subsets was determined by flow cytometry. RESULTS: Serum concentrations of both type 1 and type 2 cytokines were significantly higher in patients with MCTD than in healthy controls. IFN-gamma expression of CD4+ and CD8+ T cells did not differ comparing all patients to controls. In patients with active MCTD, the percentage of CD8+/IFN-gamma+ Tc1 cells was significantly increased compared to inactive disease or to controls (p < 0.05). IL-4 expression of CD4+ T cells was scarcely detectable in MCTD, while a subpopulation of CD8+ Tc2 cells produced IL-4. A higher percentage of these CD8+/IL-4+ Tc2 cells were detected in patients with MCTD, especially in active disease, compared to controls (p < 0.05). The percentage of IL-10-expressing CD4+ and CD8+ T cells was higher in patients than in controls (p < 0.05). Again, CD4+ and CD8+ T cells from patients with active MCTD produced significantly more IL-10 than cells in patients with inactive disease or in controls (p < 0.05). CONCLUSION: Our results suggest that MCTD is associated with increased production of both type 1 (IFN-gamma) and type 2 cytokines (IL-4, IL-10). Cytokine production is usually higher in active MCTD than in inactive disease. CD8+ T cells may produce more IFN-gamma and IL-10 in comparison to CD4+ T cells. Patients with active disease have more IL-4-expressing Tc2 cells and IL-10-expressing Th2 and Tc2 cells than patients with inactive MCTD or controls. In MCTD, increased IL-10 production by Th2 and Tc2 cells may be an attempt by the immune system to downregulate the inflammatory reaction, although this effect may not be sufficient to restore the physiological Th1/Th2 balance in MCTD.     

Selective T-cell subset ablation demonstrates a role for T1 and T2 cells in ongoing acute graft-versus-host disease: a model system for the reversal of disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality of allogeneic stem cell transplantation. Strategies to control GVHD while maintaining graft versus leukemia (GVL) include herpes simplex virus thymidine kinase (HSV-tk) gene transduction of donor T cells followed by treatment with ganciclovir (GCV). Alternatively, GVHD and GVL may be mediated by distinct processes. In this regard, whether cytokine polarization occurs and to what degrees various subsets of cytokine-producing T cells mediate GVHD or GVL has been an active area of research using cytokine or cytokine antibody infusion or genetically deficient mice. This study takes a different approach that allows simultaneous investigation into both the mechanisms underlying GVHD reactions and the efficacy of HSV-tk suicide gene-based T-cell deletion. A source of donor T cells, splenocytes from mice transgenic for HSV-tk controlled by elements of either the interleukin-2 (IL-2) or IL-4 promoters (IL-2-tk and IL-4-tk, respectively) was used, thus allowing investigation into the roles of T1 and T2 cells in ongoing GVHD reactions. To assess treatment rather than prevention of GVHD, GCV was started at peak disease. Remarkably, treatment at this late time point rescued mice from the clinical effects of GVHD caused by T cells expressing either transgene. Thus, both T1 and T2 cells play an important role in clinical GVHD in a minor histocompatibility antigen-mismatched setting. In addition, because clinical disease was reversible even at its maximum, these observations provide controlled evidence that this strategy of treating ongoing GVHD could be effective clinically.     

Production of IL-10 by alloreactive sibling donor cells and its influence on the development of acute GVHD

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation. Pretransplant conditioning regimes cause release of proinflammatory cytokines that stimulate alloreactive donor T cells to attack recipient tissues. IL-10 has been shown to directly downregulate CD4+ T cells by suppressing IL-2 secretion and a critical role played by regulatory T cells has been demonstrated in animal models. One defining cytokine profile for regulatory T cells is the production of IL-10. Release of specific cytokines (IL-10, IL-4 and IFN-gamma) was detected using ELISPOT technology, following stimulation of donor peripheral blood mononuclear cells by recipient (human leukocyte antigen-matched sibling) alloantigen or by mitogen. Correlation between the frequency of cytokine-releasing cells and the development of acute GVHD was investigated. A high frequency of donor cells producing IL-10 in response to recipient alloantigen stimulation correlated with absence of acute GVHD after bone marrow transplant (BMT), while low frequency was strongly associated with severe GVHD. This study presents strong evidence that estimating the frequency of donor alloreactive cells producing IL-10 in response to recipient antigens will provide valuable information prior to BMT regarding potential transplant outcome.     

Interleukin 18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation

We have recently shown that early administration of interleukin 18 (IL-18) after bone marrow transplantation (BMT) attenuates acute graft-versus-host disease (GVHD) in a lethally irradiated parent into F1 (B6-->B6D2F1) BMT model. In this study, we investigated whether IL-18 can maintain graft-versus-leukemia (GVL) effect in this context. B6D2F1 mice received transplants of T-cell-depleted (TCD) bone marrow (BM) and splenic T cells from either syngeneic (H2(b/d)) or allogeneic B6 (H2(b)) donors. Recipient mice were treated with recombinant murine IL-18 or the control diluent. Initial studies demonstrated that IL-18 treatment did not affect the proliferative responses or the cytolytic effector functions of T cells after BMT. In subsequent experiments, animals also received host-type P815 mastocytoma cells at the time of BMT. All syngeneic BM transplant recipients died from leukemia by day 18. The allogeneic BM transplant recipients effectively rejected their leukemia regardless of treatment and IL-18 significantly reduced GVHD-related mortality. Examination of the cytotoxic mechanisms with perforin-deficient donor T cells demonstrated that perforin is critical for the GVL effect. Taken together these data demonstrate that IL-18 can attenuate acute GVHD without impairing the in vitro cytolytic function or the in vivo GVL activity after allogeneic BMT.     

Up-regulated expression in nonhematopoietic tissues of the BCL2A1-derived minor histocompatibility antigens in response to inflammatory cytokines: relevance for allogeneic immunotherapy of leukemia

T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24-restricted mHag ACC-1 and the HLA-B44-restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Analysis of cytotoxicity and IFN-gamma production illustrated that ACC-2-specific T cells did not recognize untreated MSCs or IFN-gamma-treated MSCs but showed specific recognition and killing of MSCs treated with TNF-alpha plus IFN-gamma. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.     

Tc1 effector diversity shows dissociated expression of granzyme B and interferon-gamma in HIV infection

OBJECTIVE: To examine antigen specific cytotoxic effector T cell diversity in HIV infected individuals. DESIGN: We used a panel of previously defined HLA class I-restricted HIV peptides to stimulate CD8 cells in freshly isolated peripheral blood mononuclear cells of HIV infected patients, to determine cognate killing via the perforin-granzyme pathway and inflammation induced by secretion of interferon (IFN)-gamma. METHODS: ELISPOT assays were used to measure the secretion of granzyme B (GzB) and IFN-gamma at single cell resolution. RESULTS: In all nine patients only approximately 20% of the peptides triggered a canonical Tc1 response with simultaneous release of GzB and IFN-gamma. The majority of these peptides (approximately 80%) that elicited recall responses fell into the 'single positive' category with induction of either GzB or IFN-gamma alone. The GzB positive cells did not produce interleukin (IL)-4 or IL-5. CONCLUSION: The GzB positive but IFN-gamma negative CD8 cells are programmed to induce apoptosis mediated killing without inflammation while the GzB negative and IFN-gamma positive CD8 cells could mediate inflammation without killing. This Tc1 CD8 effector cell diversity and the understanding of these differentiation mechanisms may enable the precise implementation and fine-tuning of fundamentally different defense strategies against HIV and other infections.     

Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production

T cells with natural killer cell phenotype and function (NKT cells) have been described in both human and murine tissues. In this study, culture conditions were developed that resulted in the expansion of CD8(+) NKT cells from bone marrow, thymus, and spleen by the timed addition of interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and anti-CD3 monoclonal antibody. After 14 to 21 days in culture, dramatic expansion of CD3(+), CD8(+), alphabetaT-cell receptor(+) T cells resulted with approximately 20% to 50% of the cells also expressing the NK markers NK1.1 and DX5. The CD8(+) NKT cells demonstrated lytic activity against several tumor target cells with more than 90% lysis by day 14 to day 21 of culture. Cytotoxicity was observed against both syngeneic and allogeneic tumor cell targets with the greatest lytic activity by the cells expressing either NK1.1 or DX5. The expanded CD8(+) NKT cells produce T(H)1-type cytokines with high levels of IFN-gamma and tumor necrosis factor alpha. Expansion of the CD8(+) NKT cells was independent of CD1d. Ly49 molecules were expressed on only a minority of cells. A single injection of expanded CD8(+) NKT cells was capable of protecting syngeneic animals from an otherwise lethal dose of Bcl1 leukemia cells. Expanded CD8(+) NKT cells produced far less graft-versus-host disease (GVHD) than splenocytes across major histocompatibility barriers, even when 10 times the number of CD8(+) NKT cells as compared to splenocytes were injected. This reduction in GVHD was related to IFN-gamma production since cells expanded from IFN-gamma knock-out animals caused acute lethal GVHD, whereas cells expanded from animals defective in fas ligand, fas, IL-2, and perforin did not. These data indicate that CD8(+) NKT cells expanded in this fashion could be useful for preserving graft-versus-leukemia activity without causing GVHD.