Human gammadelta T cells as mediators of chimaeric-receptor redirected anti-tumour immunity

Human peripheral blood gammadelta T cells (Vgamma9(+) Vdelta2(+)) can be selectively expanded in vivo by the systemic administration of aminobisphosphonates without prior antigen priming. To assess the potential of human gammadelta T cells to serve as effector cells of specific anti-tumour immunity, we expanded peripheral blood-derived gammadelta T cells and transduced them with recombinant retrovirus encoding G(D2)- or CD19-specific chimaeric receptors. Flow cytometric analysis of T cells from four individual donors cultured in the presence of zoledronate at day 14 of culture showed selective enrichment of the gammadelta T cell population (Vgamma9(+) Vdelta2(+) CD3(+) CD4(-) CD8(-)) to 73-96% of total CD3(+) T cells. Retroviral gene transfer resulted in chimaeric receptor surface expression in 73 +/- 12% of the population. Transduced gammadelta T cells efficiently recognized antigen-expressing tumour cell targets, as demonstrated by target-specific upregulation of CD69 and secretion of interferon-alpha. Moreover, transduced gammadelta T cells efficiently and specifically lysed the antigen-expressing tumour targets. They could be efficiently expanded in vitro and maintained in culture for prolonged periods. Zoledronate-activated human gammadelta T cells expressing chimaeric receptors may thus serve as potent and specific anti-tumour effector cells. Their responsiveness to stimulation with aminobisphosphonates may enable the selective re-expansion of adoptively transferred T cells in vivo, permitting long lasting anti-tumour immune control.     

Resistance to regulatory T cell-mediated suppression in rheumatoid arthritis can be bypassed by ectopic foxp3 expression in pathogenic synovial T cells

Increasing evidence suggests that regulatory T cell (Treg) function is impaired in chronic inflammatory diseases such as rheumatoid arthritis (RA). Here we demonstrate that Tregs are unable to modulate the spontaneous production of TNF-α from RA synovial cells cultured from the diseased synovium site. Cytokine (IL-2, IL-6, TNF-α) activated T cells (Tck), cells we previously demonstrated to mimic the effector function of pathogenic RA synovial T cells, contained Tregs that survived and divided in this cytokine environment; however, the up-regulation of key molecules associated with Treg function (CTLA-4 and LFA-1) was impaired. Furthermore, Tregs were unable to suppress the function of Tcks, including contact-dependent induction of TNF-α from macrophages, supporting the concept that impaired Treg function/responsiveness contributes to chronicity of RA. However, ectopic foxp3 expression in both Tcks and pathogenic RA synovial T cells attenuated their cytokine production and function, including contact-dependent activation of macrophages. This diminished response to cytokine activation after ectopic foxp3 expression involved inhibited NF-κB activity and differed mechanistically from that displayed endogenously in conventional Tregs. These results suggest that diseases such as RA may perpetuate owing to the inability of Tregs to control cytokine-activated T-cell function. Understanding the mechanism whereby foxp3 attenuates the pathogenic function of synovial T cells may provide insight into the mechanisms of chronicity in inflammatory disease and potentially reveal new therapeutic candidates.     

Aryl hydrocarbon receptor deficiency in T cells suppresses the development of collagen-induced arthritis

The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis have not been elucidated. Here, we show that Ahr deficiency ameliorated collagen-induced arthritis, a mouse model of RA. Collagen-immunized Ahr KO mice showed decreased serum levels of such proinflammatory cytokines as IL-1β and IL-6. The Th17 and Th1 cell populations in lymph nodes from these mice decreased and increased, respectively, whereas the percentage of regulatory T cells was unchanged. Interestingly, a lack of Ahr specifically in T cells significantly suppressed collagen-induced arthritis development, whereas Ahr deficiency in macrophages had no effect. These finding indicate that the development of experimental autoimmune arthritis depends on the presence of Ahr in T cells, and that Th1/Th17 balance may be particularly important for this process.     

Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells

Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4⁺ T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β–induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.     

Adoptive transfer of syngeneic T cells in HIV-1 discordant twins indicates rapid regulation of T-cell homeostasis

The safety and efficacy of adoptive T-cell transfer (ATT) was tested in the context of viral suppression in syngeneic twins discordant for human immunodeficiency virus type 1 (HIV-1) infection. Human leucocyte antigen-matched T cells of seven HIV-negative twins were obtained by lymphapheresis and immediately transfused into the HIV-infected sibling. Four twins received 12 ATTs each, with a mean of 2.10 +/- 0.97 x 10(9) CD4(+) T cells and 1.74 +/- 0.81 x 10(9) CD8(+) T cells. Additional transfers were performed in three more twin pairs to study the short-term kinetics of transfused syngeneic T cells. Mean CD4(+) T-cell counts increased significantly, by 0.133 +/- 0.136 x 10(9) cells/l at 1 h and 0.144 +/- 0.12 x 10(9) cells/l at 3 h post-transfusion (P < 0.0001). Short-term kinetic studies suggested a rapid clearance of transferred T cells from the peripheral blood within minutes due to a distribution to marginal pools. After a mean follow up of 39 months, however, a sustained increase of the mean CD4(+) T-cell count was observed (from 0.232 x 10(9) to 0.523 x 10(9) cells/l) without changes of plasma viraemia. We conclude that ATT combined with highly active antiretroviral therapy is safe and leads to a considerable increase in CD4(+) T-cell numbers. The clearance kinetics of the transfused cells from peripheral blood indicates a very rapid regulation of T-cell homeostasis in HIV infection.     

Encounter with antigen-specific primed CD4 T cells promotes MHC class II degradation in dendritic cells

Major histocompatibility complex class II molecules (MHC-II) on antigen presenting cells (APCs) engage the TCR on antigen-specific CD4 T cells, thereby providing the specificity required for T cell priming and the induction of an effective immune response. In this study, we have asked whether antigen-loaded dendritic cells (DCs) that have been in contact with antigen-specific CD4 T cells retain the ability to stimulate additional naïve T cells. We show that encounter with antigen-specific primed CD4 T cells induces the degradation of surface MHC-II in antigen-loaded DCs and inhibits the ability of these DCs to stimulate additional naïve CD4 T cells. Cross-linking with MHC-II mAb as a surrogate for T-cell engagement also inhibits APC function and induces MHC-II degradation by promoting the clustering of MHC-II present in lipid raft membrane microdomains, a process that leads to MHC-II endocytosis and degradation in lysosomes. Encounter of DCs with antigen-specific primed T cells or engagement of MHC-II with antibodies promotes the degradation of both immunologically relevant and irrelevant MHC-II molecules. These data demonstrate that engagement of MHC-II on DCs after encounter with antigen-specific primed CD4 T cells promotes the down-regulation of cell surface MHC-II in DCs, thereby attenuating additional activation of naïve CD4 T cells by these APCs.     

Induction of antigen-specific regulatory T cells in wild-type mice: visualization and targets of suppression

Antigen-specific transplantation tolerance in the absence of immunosuppressive drugs is a rarely achieved goal. Immune responses to Y chromosome-encoded transplantation antigens (HY) can have life-threatening consequences in the clinic. Here, we have adopted a procedure developed in T cell antigen receptor (TCR)-transgenic mice to convert naïve T cells into male-specific Foxp3⁺ regulatory T cells (Tregs) in WT female mice. For this purpose, female mice were infused by osmotic minipumps with a single class II MHC-presented HY peptide and Tregs visualized by tetramer staining. As a result, animals developed Treg-mediated long-term tolerance to all HY transplantation antigens, irrespective of whether they were recognized by CD4 or CD8 T cells, on skin or hematopoietic grafts from male donors.     

Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis

The ultimate goal for the treatment of autoimmunity is to restore immunological tolerance. Regulatory T cells (Treg) play a central role in immune tolerance, and Treg functional abnormalities have been identified in different autoimmune diseases, including rheumatoid arthritis (RA). We have previously shown that natural Treg from RA patients are competent at suppressing responder T cell proliferation but not cytokine production. Here, we explore the hypothesis that this Treg defect in RA is linked with abnormalities in the expression and function of CTLA-4. We demonstrate that CTLA-4 expression on Treg from RA patients was significantly reduced compared with healthy Treg and is associated with an increased rate of CTLA-4 internalization. Regulation of T cell receptor signaling by CTLA-4 was impaired in RA Treg and associated with delayed recruitment of CTLA-4 to the immunological synapse. Artificial induction of CTLA-4 expression on RA Treg restored their suppressive capacity. Furthermore, CTLA-4 blockade impaired healthy Treg suppression of T cell IFN-γ production, but not proliferation, thereby recapitulating the unique Treg defect in RA. Our results suggest that defects in CTLA-4 could contribute to abnormal Treg function in RA and may represent a target for therapy for inducing long-lasting remission.     

Characterization of hepatitis B virus surface antigen-specific CD4+ T cells in hepatitis B vaccine non-responders

AIM: To study the mechanisms of hepatitis B vaccine non-response, we examined hepatitis B virus surface antigen (HBsAg)-induced proliferation of peripheral blood mononuclear cells (PBMC) obtained from hepatitis B (HB) vaccinees. METHODS: Subsequently, we have examined the features of HBsAg-reactive CD4+ T cells in HB vaccine non-responders (NR). Based on serum antibodies to HBsAg (anti-HBs) titers, we divided these vaccinees into three groups: high responder (HR), middle responder (MR) and non-responder (NR), and examined HBsAg-induced proliferation of their PBMC. RESULTS: We found that the in vitro response of PBMC to stimulation with HBsAg was correlated with their serum anti-HBs titer (mean stimulation index was 10.71 in HR, 4.37 in MR and 1.96 in NR). However, by the deletion of CD8+ T cells, the increased response was observed in two of four NRs. CONCLUSIONS: The present results have also shown that at least four distinct HBsAg-reactive CD4+ clones existed (variable gene of T cell receptor beta (V beta) 17 + clone restricted with HLA-DR locus (DR4), V beta 8 + clone restricted with HLA-DQ locust (DQ7), and both V beta 5.1 + clone and V beta 20 + clone restricted with either DR9 or DQ3) in NRs. The results demonstrated that heterogeneous HBsAg-reactive CD4 clones existed in some HB vaccine NRs.     

Circulating tumor antigen-specific regulatory T cells in patients with metastatic melanoma

Although it is accepted that regulatory T cells (T regs) contribute to cancer progression, most studies in the field consider nonantigen-specific suppression. Here, we show the presence of tumor antigen-specific CD4⁺ T regs in the blood of patients with metastatic melanoma. These CD4⁺ T regs recognize a broad range of tumor antigens, including gp100 and TRP1 (melanoma tissue differentiation antigens), NY-ESO-1 (cancer/testis antigen) and survivin (inhibitor of apoptosis protein (IAP) family antigen). These tumor antigen-specific T regs proliferate in peripheral blood mononuclear cells (PBMC) cultures in response to specific 15-mer peptides, produce preferentially IL-10 and express high levels of FoxP3. They suppress autologous CD4⁺CD25⁻ T cell responses in a cell contact-dependent manner and thus share properties of both naturally occurring regulatory T cells and type 1 regulatory T cells. Such tumor antigen-specific T regs were not detected in healthy individuals. These tumor antigen-specific T regs might thus represent another target for immunotherapy of metastatic melanoma.     

Killer cell Ig-like receptor (KIR) 3DL1 down-regulation enhances inhibition of type 1 diabetes by autoantigen-specific regulatory T cells

Both Foxp3(+) regulatory T cells (Tregs) and antigen-expanded Foxp3(-) Tregs play an important role in regulating immune responses as well as in preventing autoimmune diseases and graft rejection. Molecular mechanisms modulating Treg function remain largely unclear, however. We report here on the expression and function of an inhibitory killer cell Ig-like receptor, KIR3DL1, in a nonobese diabetic (NOD) mouse-derived autoantigen-specific Treg (2D2), which protects from type 1 diabetes (T1D) in adoptive transfer experiments. This gene is not expressed in T1D pathogenic T cells (Tpaths) or non-Tpath T cells. KIR genes are known to play an important role in regulating natural killer (NK) cell function, but their role in Tregs and T1D is unknown. To examine whether KIR3DL1 expression may modulate Treg function, we used shRNA to down-regulate KIR3DL1 expression (2D2-shKIR). We find that KIR3DL1 down-regulation enhances in vitro function, as measured by improved suppression of target cell proliferation. Antibody blockade of IL-10 but not IL-4 partially abrogated suppressive function. In vivo function is also improved. Adoptive transfer of 2D2-shKIR into 10-wk-old NOD mice prevented spontaneous insulitis and T1D, and the inhibitory effect was further improved if the cells were transferred earlier into 6-wk-old NOD mice. These studies indicate that KIR3DL1 expression may negatively regulate Treg function and suggest a previously undescribed target for improving immune tolerance for potential treatment of autoimmune diseases like T1D.     

Retroviral transfer of human CD20 as a suicide gene for adoptive T-cell therapy

The aim of adoptive T-cell therapy of cancer is to selectively confer immunity against tumor cells. Autoimmune side effects, however, remain a risk, emphasizing the relevance of a suicide mechanism allowing in vivo elimination of infused T cells. We investigated the use of human CD20 as suicide gene in T-lymphocytes. Potential effects of forced CD20 expression on T-cell function were investigated by comparing CD20- and mock-transduced cytomegalovirus (CMV) specific T cells for cytolysis, cytokine release and proliferation. The use of CD20 as suicide gene was investigated in CMV specific T cells and in T cells genetically modified with an antigen specific T-cell receptor. No effect of CD20 on T-cell function was observed. CD20-transduced T cells with and without co-transferred T-cell receptor were efficiently eliminated by complement dependent cytotoxicity induced by therapeutic anti-CD20 antibody rituximab. The data support the broad value of CD20 as safety switch in adoptive T-cell therapy.     

Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells

The goal of cancer immunotherapy is the generation of an effective, stable, and self-renewing antitumor T-cell population. One such approach involves the use of high-affinity cancer-specific T-cell receptors in gene-therapy protocols. Here, we present the generation of functional tumor-specific human T cells in vivo from genetically modified human hematopoietic stem cells (hHSC) using a human/mouse chimera model. Transduced hHSC expressing an HLA-A*0201–restricted melanoma-specific T-cell receptor were introduced into humanized mice, resulting in the generation of a sizeable melanoma-specific naïve CD8⁺ T-cell population. Following tumor challenge, these transgenic CD8⁺ T cells, in the absence of additional manipulation, limited and cleared human melanoma tumors in vivo. Furthermore, the genetically enhanced T cells underwent proper thymic selection, because we did not observe any responses against non–HLA-matched tumors, and no killing of any kind occurred in the absence of a human thymus. Finally, the transduced hHSC established long-term bone marrow engraftment. These studies present a potential therapeutic approach and an important tool to understand better and to optimize the human immune response to melanoma and, potentially, to other types of cancer.     

Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

Background

Cytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen.

Design and Methods

SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour ⁵¹chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (³H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34⁺ hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation.

Results

Cytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34⁺ progenitor cells, residual clonogenic activity was preserved.

Conclusions

Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.     

Development of rheumatoid arthritis after treatment of large granular lymphocyte leukemia with deoxycoformycin

The association of T-cell large granular lymphocyte (LGL) leukemia and rheumatoid arthritis is well described and it is now recognized that these patients and patients with Felty's syndrome represent different aspects of a single disease process. Most patients have rheumatoid arthritis at the time of diagnosis of LGL leukemia. This is the first detailed report of the development of rheumatoid arthritis after the diagnosis and treatment of LGL leukemia as well as the first report of rheumatoid arthritis that occurred in association with deoxycoformycin treatment. It is likely that the beneficial sustained normalization of neutrophil counts as a result of deoxycoformycin treatment played a significant role in the development of this complication. Hematological improvement occurred despite molecular genetic evidence of persistence of the abnormal T-cell clone. The role of the clonally expanded T cells in the pathogenesis of neutropenia and rheumatoid arthritis is discussed. Am. J. Hematol. 57:253–257, 1998. © 1998 Wiley-Liss, Inc.     

T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells

To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2–restricted T-cell recptor γ-chain alternate reading-frame protein (TARP)_4–13 epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2⁺ prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP_4–13 epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.     

Distinct CD4+ helper T cells involved in primary and secondary responses to infection

Helper T cells are critical for protective immunity, CD8(+) T-cell memory, and CD4(+) recall responses, but whether the same or distinct CD4(+) T cells are involved in these responses has not been established. Here we describe two CD4(+) T cells, LLO118 and LLO56, specific for an immunodominant Listeria monocytogenes epitope, with dramatically different responses to primary and secondary infection. Comparing in vivo responses, LLO118 T cells proliferate more strongly to primary infection, whereas surprisingly, LLO56 has a superior CD4(+) recall response to secondary infection. LLO118 T cells provide more robust help for CD8(+) T-cell responses to secondary infection than LLO56. We found no detectable differences in antigen sensitivity, but naive LLO118 T cells have much lower levels of CD5 and their T-cell receptor levels are dramatically down-regulated after their strong primary response. Thus, distinct CD4(+) helper T cells are specialized to help either in primary or secondary responses to infection.     

Entry of diabetogenic T cells into islets induces changes that lead to amplification of the cellular response

In an accompanying paper, we find specific localization of diabetogenic T cells only to islets of Langerhans bearing the specific antigen. Instrumental in the specific localization was the presence of intraislet dendritic cells bearing the β-cell-peptide-MHC complex. Here, we report that the entry of diabetogenic CD4 T cells very rapidly triggered inflammatory gene expression changes in islets and vessels by up-regulating chemokines and adhesion molecules. Vascular cell adhesion molecule-1 (VCAM-1) expression was notable in blood vessels, as was intercellular adhesion molecule-1 (ICAM-1). ICAM-1 was also found on β-cells. These expression changes induced the entry of nonspecific T cells that otherwise did not localize to the islets. In contrast to the entry of diabetogenic CD4 T cells, the entrance of nonspecific T cells required a chemokine response and VCAM-1 expression by the islets. IFN-γ was important for the early gene expression changes in the islets. By microarray analysis, we detected up-regulation of a group of IFN-inducible genes as early as 8 h post-T-cell transfer. These studies establish that entry of diabetogenic T cells induces a state of receptivity of islets to subsequent immunological insults.