Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia.

Autologous T cells engineered to express a chimeric antigen receptor (CAR) against the CD19 antigen are in the frontline of contemporary hemato-oncology therapies, leading to high remission rates in B-cell malignancies. Although effective, major obstacles involve the complex and costly individualized manufacturing process, and CD19 target antigen loss or modulation leading to resistant and relapse following CAR therapy. A potential solution for these limitations is the use of donor-derived γδT cells as a CAR backbone. γδT cells lack allogenecity and are safely used in haploidentical transplants. Moreover, γδT cells are known to mediate natural anti-tumor responses. Here, we describe a 14-day production process initiated from peripheral-blood mononuclear cells, leading to a median 185-fold expansion of γδ T cells with high purity (>98% CD3+ and >99% γδTCR+). CAR transduction efficacy of γδ T cells was equally high when compared to standard CAR-T cells (60.5 ± 13.2 and 65.3 ± 18.3%, respectively). CD19-directed γδCAR-T cells were effective against CD19+ cell lines in vitro and in vivo, showing cytokine production, direct target killing, and clearance of bone marrow leukemic cells in an NSG model. Multiple injections of γδCAR-T cells and priming of mice with zoledronate lead to enhanced tumor reduction in vivo. Unlike standard CD19 CAR-T cells, γδCAR-T cells were able to target CD19 antigen negative leukemia cells, an effect that was enhanced after priming the cells with zoledronate. In conclusion, γδCAR-T cell production is feasible and leads to highly pure and efficient effector cells. γδCAR-T cell may provide a promising platform in the allogeneic setting, and may target leukemic cells also after antigen loss.

The Role of Gamma Delta T Cells in Autoimmune Rheumatic Diseases.

Autoimmune rheumatic diseases (ARDs), affecting ~1-1.5% of all humans, are associated with considerable life long morbidity and early mortality. Early studies in the 1990s showed numerical changes of the recently discovered γδ T cells in the peripheral blood and in affected tissues of patients with a variety of ARDs, kindling interest in their role in the immuno-pathogenesis of these chronic inflammatory conditions. Indeed, later studies applied rapid developments in the understanding of γδ T cell biology, including antigens recognized by γδ T cells, their developmental programs, states of activation, and cytokine production profiles, to analyze their contribution to the pathological immune response in these disorders. Here we review the published studies addressing the role of γδ T in the major autoimmune rheumatic diseases, including rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, systemic lupus erythematosus and scleroderma, and animal models thereof. Due to their unique properties spanning adaptive and innate immune functions, the ever deeper understanding of this unique T cell population is shedding new light on the pathogenesis of, while potentially enabling new therapeutic approaches to, these diseases.

Dysregulated CD25 and Cytokine Expression by γδ T Cells of Systemic Sclerosis Patients Stimulated With Cardiolipin and Zoledronate.

Objectives: γδ T cells, a non-conventional innate lymphocyte subset containing cells that can be activated by lipids and phosphoantigens, are abnormally regulated in systemic sclerosis (SSc). To further evaluate the significance of this dysregulation, we compared how exposure to an autoantigenic lipid, cardiolipin (CL), during co-stimulation with an amino-bisphosphonate (zoledronate, zol), affects the activation and cytokine production of SSc and healthy control (HC) γδ T cells. Methods: Expression of CD25 on Vγ9+, Vδ1+, and total CD3+ T cells in cultured peripheral blood mononuclear cells (PBMCs), their binding of CD1d tetramers, and the effect of monoclonal antibody (mAb) blockade of CD1d were monitored by flow cytometry after 4 days of in vitro culture. Intracellular production of IFNγ and IL-4 was assessed after overnight culture. Results: Percentages of CD25+ among CD3+ and Vδ1+ T cells were elevated significantly in short-term cultured SSc PBMC compared to HC. In SSc but not HC, CL and zol, respectively, suppressed %CD25+ Vγ9+ and Vδ1+ T cells but, when combined, CL + zol significantly activated both subsets in HC and partially reversed inhibition by the individual reagents in SSc. Importantly, Vδ1+ T cells in both SSc and HC were highly reactive with lipid presenting CD1d tetramers, and a CD1d-blocking mAb decreased CL-induced enhancement of %SSc CD25+ Vδ1+ T cells in the presence of zol. %IFNγ+ cells among Vγ9+ T cells of SSc was lower than HC cultured in medium, CL, zol, or CL + zol, whereas %IFNγ+ Vδ1+ T cells was lower only in the presence of CL or CL + zol. %IL-4+ T cells were similar in SSc and HC in all conditions, with the exception of being increased in SSc Vγ9+ T cells in the presence of CL. Conclusion: Abnormal functional responses of γδ T cell subsets to stimulation by CL and phosphoantigens in SSc may contribute to fibrosis and immunosuppression, characteristics of this disease.

Immune-mediated syndromes following intravenous bisphosphonate therapy.

OBJECTIVES: Intravenous (IV) infusion of aminobisphosphonates (ABP) induces cytokine release by peripheral blood Vγ9δ2 T cells, resulting in an immediate short-term inflammatory response in up to 50% of patients. We evaluated possible long-term pro-inflammatory effects of IV ABP. METHODS: Retrospective case-series study from one rheumatology specialist's clinic. 2261 electronic charts were reviewed for administration of 'zoledronate' or different brand names of zoledronic acid, and relevant clinical data was retrieved for patients who had received the infusion. RESULTS: Thirteen patients had recieved zoledronate. In six, new-onset or exacerbation of a previous inflammatory/autoimmune disorder was diagnosed within 3 months following infusion. Of these, one patient developed new-onset rheumatoid arthritis (RA), two polymyalgia rheumatica (PMR), two suffered a flare of Crohn's disease-related and aromatase inhibitor-induced arthralgias, and one patient acquired autoimmune hemophilia. Pre-existing malignancy and immediate inflammatory response following zoledronate were more frequent in patients experiencing new or worsening immunologic manifestations (3/6 vs. 0/7, and 5/6 vs. 2/7, respectively). CONCLUSIONS: Intravenous ABP may trigger induction of persistent autoimmune syndromes, especially when accompanied by an immediate adverse reaction or pre-existing malignancy.

The Role of γδ T Cells in Fibrotic Diseases.

Inflammation induced by toxins, micro-organisms, or autoimmunity may result in pathogenic fibrosis, leading to long-term tissue dysfunction, morbidity, and mortality. Immune cells play a role in both induction and resolution of fibrosis. γδ T cells are an important group of unconventional T cells characterized by their expression of non-major histocompatibility complex restricted clonotypic T cell receptors for non-peptide antigens. Accumulating evidence suggests that subsets of γδ T cells in experimentally induced fibrosis following bleomycin treatment, or infection with Bacillus subtilis, play pro-inflammatory roles that instigate fibrosis, whereas the same cells may also play a role in resolving fibrosis. These processes appear to be linked at least in part to the cytokines produced by the cells at various stages, with interleukin (IL)-17 playing a central role in the inflammatory phase driving fibrosis, but later secretion of IL-22, interferon γ, and CXCL10 preventing pathologic fibrosis. Moreover, γδ T cells appear to be involved, in an antigen-driven manner, in the prototypic human fibrotic disease, systemic sclerosis (SSc). In this paper we review in brief the scientific publications that have implicated γδ T cells in fibrotic diseases and their pro- and anti-fibrotic effects.

Anti-fibrotic characteristics of Vγ9+ γδ T cells in systemic sclerosis.

OBJECTIVES: γδ T cells of the Vγ9Vδ2 subtype secrete anti-fibrotic cytokines upon isopentenyl pyrophosphate (IPP) stimulation. In this study, we sought to compare IPP and Zoledronate, an up-regulator of IPP, effects on proliferation and cytokine secretion of Vγ9+ T cells from systemic sclerosis (SSc) patients and healthy controls (HCs). We also examined the effect of IPP-triggered peripheral blood mononuclear cells (PBMC) on fibroblast procolla- gen secretion. METHODS: PBMC from SSc patients and HCs were stimulated by increasing concentrations of Zoledronate, with or without IPP, and Vγ9+ T cell percentages were calculated using FACScan analysis. Subsequently, PBMC were cultured with IPP or toxic shock syndrome toxin-1 (TSST-1), and contents of the anti-fibrotic cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ were measured by ELISA kits. Finally, supernatants of IPP-triggered Vγ9+ T cells from SSc patients were added to fibroblast cultures, and relative intensities of procollagen α1 chains were determined by densinometry. RESULTS: Higher concentrations of Zoledronate were required for maximal proliferation of Vγ9+ T cells in 9 SSc patients compared to 9 HCs, irrespective of exogenous IPP. When compared to stimulation by TSST-1, a non-Vγ9+ selective reagent, secretion of the anti-fibrotic cytokines TNF-α and IFN-γ in response to IPP was relatively diminished in SSc but not in HCs. Reduction of procollagen secretion by fibroblasts cultured with supernatants of IPP-stimulated PBMC was observed only in some SSc patients. CONCLUSIONS: Activated Vγ9+ T cells could act as anti-fibrotic mediators in SSc, although decreased responsiveness to IPP may play a role in the pathological fibrosis of this disease.

Gamma delta T cells in non-immune patients during primary schistosomal infection.

The mevalonate pathway is critical for the survival of Schistosoma. γδ T cells, a small subset of peripheral blood (PB) T cells, recognize low molecular weight phosphorylated antigens in the mevalonate pathway, which drive their expansion to exert protective and immunoregulatory effects. To evaluate their role in schistosomiasis, we measured γδ T cells in the PB of non-immune travelers who contracted Schistosoma hematobium or Schistosoma mansoni in Africa. The maximal level of γδ T-cells following infection was 5.78 ± 2.19% of the total T cells, versus 3.72 ± 3.15% in 16 healthy controls [P = 0.09] with no difference between S. hematobium and S. mansoni in this regard. However, among the nine patients in the cohort who presented with acute schistosomiasis syndrome (AS), the level (3.5 ± 1.9%) was significantly lower than in those who did not (8.6 ± 6.4%, P < 0.05), both before and after therapy. Furthermore, γδ T cells increased significantly in response to praziquantel therapy. In a patient with marked expansion of γδ T cells, most expressed the Vδ2 gene segment, a hallmark of cells responding to cognate antigens in the mevalonate pathways of the parasite or the human host. These results suggest an immunoregulatory role of antigen responsive γδ T cells in the clinical manifestations of early schistosomal infection.

A Novel Prothrombotic Pathway in Systemic Sclerosis Patients: Possible Role of Bisphosphonate-Activated γδ T Cells.

OBJECTIVES: Infusions of aminobisphonates (ABP) activate Vγ9δ2T cells in vivo and induce an acute inflammatory response in 30% of patients treated for osteoporosis. Following the observation of digital thrombosis in a systemic sclerosis (SSc) patient after treatment with an intravenous ABP, zoledronate (Zol), we evaluated whether patient and control peripheral blood (PB) mononuclear cell (MC, PBMC) acquire a prothrombotic phenotype in response to Zol. RESULTS: Vγ9δ2T cells of both patients and healthy donors (HD) upregulated the CD69 activation antigen and secreted tumor necrosis factor (TNF)α in response to Zol in vitro. In addition, exposure to either Zol or lipopolysaccharide (LPS), or to both additively, induced expression of the highly procoagulant, tissue factor (TF)-1 on CD14+ monocytes. Importantly, only Zol-induced TF-1 was blocked by a monoclonal antibody to TNFα. Interestingly, we found that SSc, but not HD, Vδ1+ T cells were concurrently activated by Zol to produce interleukin (IL)-4. Addition of plasma from the blood of the SSc patient who developed critical digital ischemia after infusion of Zol, but neither plasma from a second patient with no adverse clinical response to Zol infusion nor of a HD, strongly enhanced Zol-induced monocyte TF-1, which could still be blocked by anti-TNFα. CONCLUSION: Aminobisphonates induced secretion of TNFα by Vγ9δ2+ T cells may lead to TNFα-dependent induction of procoagulant TF-1 induction on monocytes. In certain clinical settings, e.g., SSc, TF-1+ monocytes could play a role in triggering clinically relevant thrombosis.

Acute coronary syndromes are associated with a reduction of VLA-1+ peripheral blood T cells and their enrichment in coronary artery plaque aspirates.

Memory T cells producing interferon (IFN)γ and expressing very late antigen-1 (VLA-1) integrin collagen receptors are found in carotid atherosclerotic plaques, suggesting their involvement in coronary artery disease (CAD) as well. To determine the role of VLA-1+ T cells in CAD percent of CD3+ T cells binding monoclonal antibodies (mAb) to VLA-1 in peripheral blood (PB), and in coronary plaque material aspirated during coronary arterography and arterial blood, were analyzed in a cohort of 117 patients with CAD and 34 controls without CAD. % VLA-1+ T cells in PB was 0.63 ± 0.09% in controls compared to 0.96 ± 0.95% in patients with CAD (p<0.009). The increase was due to a marked elevation of % VLA-1+ T cells in stable CAD (1.6 ± 0.27%) whereas % VLA-1+ T cells during acute coronary syndromes (ACS) and in patients with ischemia by thalium SPECT scan had significantly lower levels. % VLA-1+ T cells in coronary artery plaque material aspirated during therapeutic angiography in patients with ACS was significantly higher than in arterial blood (1.39 ± 0.96% vs 0.75 ± 0.84%, p<0.035, n=3). Thus, % VLA-1+ T cells increases in the PB during stable CAD but decreases in ACS. The finding of their enrichment in coronary blood containing atherosclerotic plaque aspirates suggests that a shift of VLA-1+ T cells from blood to atherosclerotic plaques may play a role in plaque instability in patients with ACS.

Quantitative peripheral blood perturbations of γδ T cells in human disease and their clinical implications.

Human γδ T cells, which play innate and adaptive, protective as well as destructive, roles in the immune response, were discovered in 1986, but the clinical significance of alterations of the levels of these cells in the peripheral blood in human diseases has not been comprehensively reviewed. Here, we review patterns of easily measurable changes of this subset of T cells in peripheral blood from relevant publications in PubMed and their correlations with specific disease categories, specific diagnoses within disease categories, and prognostic outcomes. These collective data suggest that enumeration of γδ T cells and their subsets in the peripheral blood of patients could be a useful tool to evaluate diagnosis and prognosis in the clinical setting.

Cellular interactions of synovial fluid γδ T cells in juvenile idiopathic arthritis.

The pathogenesis of juvenile idiopathic arthritis (JIA) is thought to involve multiple components of the cellular immune system, including subsets of γδ T cells. In this study, we conducted experiments to define the functional roles of one of the major synovial fluid (SF) T cell subsets, Vγ9(+)Vδ2(+) (Vγ9(+)) T cells, in JIA. We found that as opposed to CD4(+) T cells, equally high percentages (∼35%) of Vγ9(+) T cells in SF and peripheral blood (PB) produced TNF-α and IFN-γ. Furthermore, stimulation with isopentenyl pyrophosphate (IPP), a metabolite in the mevalonate pathway, which is a specific potent Ag for Vγ9Jγ1.2(+) T cells, similarly amplified cytokine secretion by SF and PB Vγ9(+) T cells. Significantly, the SF subset expressed higher levels of CD69 in situ, suggesting their recent activation. Furthermore, 24-h coculturing with SF-derived fibroblasts enhanced CD69 on the SF > PB Vγ9(+) T cells, a phenomenon strongly augmented by zoledronate, a farnesyl pyrophosphate synthase inhibitor that increases endogenous intracellular IPP. Importantly, although Vγ9(+) T cell proliferation in response to IPP was significantly lower in SF than PBMC cultures, it could be enhanced by depleting SF CD4(+)CD25(+)FOXP3(+) cells (regulatory T cells). Furthermore, coculture with the Vγ9(+) T cells in medium containing zoledronate or IPP strongly increased SF-derived fibroblasts' apoptosis. The findings that IPP-responsive proinflammatory synovial Vγ9(+) T cells for which proliferation is partly controlled by regulatory T cells can recognize and become activated by SF fibroblasts and then induce their apoptosis suggest their crucial role in the pathogenesis and control of synovial inflammation.

GammadeltaT cells in juvenile idiopathic arthritis: higher percentages of synovial Vdelta1+ and Vgamma9+ T cell subsets are associated with milder disease.

OBJECTIVE: To analyze γδT cell subsets in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA), and to correlate γδT cell subsets with clinical characteristics. METHODS: γδT cell subsets as percentages of CD3+ T cells in samples of PB (n = 25) and SF (n = 93) were analyzed by flow cytometry in 93 JIA patients. The percentage of Vγ9+ γδT cells after 10 days of in vitro expansion with either interleukin 2 (IL-2) or isopentenyl pyrophosphate (IPP) plus IL-2 was determined. RESULTS: Both Vδ1+ and Vγ9+ γδT cell subsets were detected in SF of all patients, but only the percentage of Vδ1+ cells was higher in SF compared to PB (p < 0.01). The distribution of γδT cell subsets was similar in different JIA subgroups, whereas antinuclear antibody (ANA)-positive patients had a higher percentage of SF Vδ1+ T cells than ANA-negative patients (p < 0.01). The percentage of SF Vδ1+ T cells was inversely associated with age at onset, recurrence of synovitis, and erythrocyte sedimentation rate; and that of SF Vγ9+ T cells was inversely correlated with age at onset and was higher in patients who recovered from disease (n = 15). IPP-induced expansion of SF Vγ9+ T cells correlated with disease remission, whereas the expansion of SF Vγ9+ T cells in media with IL-2 alone was significantly greater in patients with uveitis. CONCLUSION: The percentage of Vδ1+ and Vγ9+ γδT cells among the SF T cells and their ability to respond to IPP or IL-2 correlated with specific outcomes of JIA, suggesting their role in the immunopathogenesis of this disease.

TNF activates a NF-kappaB-regulated cellular program in human CD45RA- regulatory T cells that modulates their suppressive function.

Emerging data suggest that regulatory T cell (Treg) dysfunction and consequent breakdown of immunological self-tolerance in autoimmunity can be mediated by factors that are not Treg-intrinsic (e.g., cytokines). Indeed, recent studies show that in rheumatoid arthritis the proinflammatory cytokine TNF reduces the suppressive function of Tregs, whereas in vivo TNF blockade restores this function and accordingly self-tolerance. However, until now a coherent mechanism by which TNF regulates the Treg has not been described. In this paper, we show that TNF induces preferential and significant activation of the canonical NF-kappaB pathway in human Tregs as compared with CD25(-) conventional T cells. Furthermore, TNF induced primarily in CD45RA(-) Tregs a transcription program highly enriched for typical NF-kappaB target genes, such as the cytokines lymphotoxin-alpha and TNF, the TNFR superfamily members FAS, 4-1BB, and OX-40, various antiapoptotic genes, and other important immune-response genes. FACS analysis revealed that TNF also induced upregulation of cell surface expression of 4-1BB and OX40 specifically in CD45RA(-)FOXP3(+) Tregs. In contrast, TNF had only a minimal effect on the Treg's core transcriptional signature or on the intracellular levels of the FOXP3 protein in Tregs. Importantly, TNF treatment modulated the capacity of Tregs to suppress the proliferation and IFN-gamma secretion by conventional T cells, an effect that was fully reversed by cotreatment with anti-TNFR2 mAbs. Our findings thus provide new mechanistic insight into the role of TNF and TNFR2 in the pathogenesis of autoimmunity.

Vgamma9+ gammadelta T cells in systemic sclerosis patients are numerically and functionally preserved and induce fibroblast apoptosis.

Vdelta1 expressing gammadelta T cells are oligoclonally expanded in systemic sclerosis (SSc) (scleroderma) and thought to play an immunopathogenic role, whereas that of Vgamma9+ gammadelta T cells is unclear. In studies of 16 patients and 16 healthy controls (HCs) we found that whereas the percent of Vdelta1+ gammadelta T cells was significantly elevated among the peripheral blood T cells in patients without radiographic evidence of interstitial lung disease (n=7), Vgamma9+ T cells were equally and persistently represented irrespective of pulmonary disease or cyclophosphamide treatment, at levels similar to healthy controls. Furthermore, ex vivo triggering of patient Vgamma9+ T cells with isopentenyl pyrophosphate plus interleukin-2-induced dose-dependent expansion, secretion of tumor necrosis factor alpha, and contact-dependent apoptosis of co-cultured fibroblasts similarly to Vgamma9+ T cells of controls. Fully functional Vgamma9+ T cells persisting in the peripheral blood of patients with progressive systemic sclerosis could potentially play an immunopathogenic role in vivo by secreting cytokines and inducing death of fibroblasts in a contact-dependent manner when activated by specific antigens.

A case of murine typhus associated with large vessel infarct of the spleen.

We present a case of Rickettsia typhi infection (the causative agent of endemic typhus) associated with an isolated splenic infarction. Large vessel infarction is a rare complication of murine typhus, unlike infections caused by Rickettsia rickettsii and Rickettsia conorii (the spotted fever group rickettsias) which are known to cause hemostatic changes that lead to coagulopathies and thrombotic events. This case adds to the scarce data in the literature on the association between large vessels infarction and endemic typhus and also highlights the importance of considering rickettsial infection in the differential diagnosis in the appropriate clinical settings.

Synovial VLA-1+ T cells display an oligoclonal and partly distinct repertoire in rheumatoid and psoriatic arthritis.

VLA-1 integrin expressing T cells are more frequent in inflammatory synovial fluids (SF) compared to peripheral blood. Recent studies suggest that VLA-1 expression mainly marks IFNgamma+ T cells while excluding both IL-4+ and regulatory FoxP3+ T cells. To further characterize the TCR repertoire of the potentially pathogenic VLA-1+ IFNgamma+ T cells, isolated from SF of adult patients with rheumatoid and psoriatic arthritis, we determined the complementarity determining region (CDR)3 spectratypes. Here we show in a cohort of 9 patients that VLA-1+ T cells display a perturbed repertoire that, moreover, differs from that of VLA-1- synovial T cells and even VLA-1+ PB T cells. Importantly, random sequencing of the CDR3 region of the TCR variable beta (BV) 6.1 gene of both VLA-1+ and VLA-1- synovial T cells, in one patient, revealed that their sequences were by and large different (29 out of 33 clones). Thus, our results imply that VLA-1+ T cells that infiltrate into inflamed joints represent a partly distinct and highly oligoclonal population of Th1 cells, probably selected by unique antigens.

Detection of alpha1 integrin in urine of patients with immunoglobulin A nephropathy.

BACKGROUND: The alpha1beta1 integrin is a cell surface membrane heterodimer composed of noncovalently linked alpha1 and beta1 polypeptides that is up-regulated on activated and proliferating mesangial cells. METHODS: A double-sandwich enzyme-linked immunosorbent assay that detects alpha1 integrin in a specific and dose-dependent manner at concentrations greater than 150 ng/mL was used to evaluate whether intact alpha1 polypeptides are secreted in the urine samples of 29 patients with various kidney diseases and in those of 5 healthy individuals. RESULTS: alpha1 Integrin was detected in 8 of the 29 patients including 3 of 3 patients with biopsy-proven immunoglobulin A nephropathy and 3 of 3 clinically suspected but non-biopsy-proven immunoglobulin A nephropathy with evidence of active nephritis. No alpha1 integrins were found in samples of 5 healthy controls. CONCLUSIONS: alpha1 Integrin polypeptides can be detected in human urine, particularly in immunoglobulin A nephropathy. Further extensive studies are required to clarify the significance of secretion of alpha1 integrins in urine of patients with kidney disease.