Ilan Bank

TNF Activates a NF-κB–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-κB 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-κB target genes, such as the cytokines lymphotoxin-α 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-γ 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.

Aspergillosis in systemic lupus erythematosus.

Infection is the major cause of morbidity and mortality in systemic lupus erythematosus (SLE). Although various fungi account for a substantial number of these lethal infections, aspergillosis, an important opportunistic infection in immunosuppressed patients, is described rarely. Only 23 cases have been reported in the English-language medical literature. Risk factors for acquiring aspergillosis in these patients were high grade disease activity, granulocytopenia, use of steroids and other immunosuppressive treatment and presence of bacterial infection. The diagnosis in most patients was delayed and they died. Here, we describe three SLE patients with invasive aspergillosis. Features of our patients diseases were similar to those reported previously. Aspergillosis appeared while they had active SLE treated with high dose corticosteroids. In 2 patients the fungal infection was systemic and diagnosed post mortem. Both were leukopenic and had concurrent bacterial infection and one received amphotericin B prior to death. In the third, the infection was localized to a transplanted kidney and was cured by nephrectomy. Aspergillosis should be suspected in patients with active SLE, who are immunocompromised and sustain concomitant bacterial infections. The currently poor prognosis may be improved with more aggressive diagnostic investigation and treatment.

Role of γ/δ T cells in a patient with CD4+ CD3– lymphocytosis, hypereosinophilia, and high levels of IgE

Abstract Background: CD4 + CD3 – T cells have previously been shown to play a pathogenic role in the hypereosinophilic syndrome by secreting IL-5 and IL-4. Objectives: The goal of this study was to study the role of CD4 + CD3 – and other T-cell subsets in a patient with eosinophilia, dermatitis, and a high level of IgE (100,000 IU/mL) in the serum. Methods: We isolated PBMCs and performed flow cytometry, cell cultures, and in vitro assays of Ig, lymphokine production, and cell-mediated cytotoxicity. Results: Flow cytometric and immunohistochemical analysis of the PBMCs revealed a major population (consisting of approximately 85% of the CD4 + T cells) that lacked expression of CD3 and T-cell receptors on the cell surface (CD4 + CD3 – T cells), but did express CD3 peptides in the cytoplasm. Activation of the PBMCs in vitro resulted in a 100-fold greater than normal release of IL-4, whereas IFN-γ production was less than normal, suggesting a predominantly type 2 helper functional phenotype of the CD4 + CD3 – T cells. Importantly, both CD4 – CD8 low Vδ1 + T-cell receptor γδ + and CD4 + CD3 – T cells were cultured from the PBMCs. The former secreted IFN-γ exclusively, whereas the latter secreted both IL-4 and IFN-γ. Furthermore, only the T-cell receptor γδ + lymphocytes were cytotoxic to autologous B-lymphoblastoid cells and specifically inhibited IgE production in cultures of autologous polyclonally stimulated PBMCs. Conclusions: The results suggest that CD8 low Vδ1 + T-cell receptor γδ + clones functionally counteract IgE-inducing effects of type 2 CD4 + CD3 – helper cells in this patient with hypereosinophilic syndrome. (J Allergy Clin Immunol 1998;102:621-30.)

Vγ9+ γδ 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.

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.

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.

The Effect of Blockade of Tumor Necrosis Factor α on VLA-1+T-Cells in Rheumatoid Arthritis Patients

The α1β1 integrin, very late antigen (VLA)-1, characterizes collagen adherent interferon (IFN) γ producing memory T cells in inflamed synovium. We now report that the mean percentage of VLA-1+ T cells is significantly lower among peripheral blood mononuclear cells of rheumatoid patients responsive to antitumor necrosis factor (TNF) α therapy than of those with active disease not receiving therapy. Neutralization of TNFα during in vitro polyclonal activation of VLA-1− T cells reduced differentiation to expression of VLA-1 and inhibited secretion of IFNγ, but did not affect integrin expression on in vivo differentiated VLA-1+ T cells. Moreover, synovial fluids of patients relapsing during and after therapy were enriched in VLA-1+ T cells and lines derived from VLA-1+ T cells in peripheral blood of treated patients retained collagen binding and secreted IFN γ. Thus, whereas therapy decreases VLA-1+ T cells in rheumatoid arthritis patients, a subset is resistant and contributes to residual and recurring inflammation.

Immune-mediated syndromes following intravenous bisphosphonate therapy

ObjectivesIntravenous (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.MethodsRetrospective 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.ResultsThirteen patients had recieved zoledronate. In six, new-onset or exacerbation of a previous inflammatory/autoimmune disorder was diagnosed within 3xa0months following infusion. Of these, one patient developed new-onset rheumatoid arthritisxa0(RA), two polymyalgia rheumaticaxa0(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).ConclusionsIntravenous ABP may trigger induction of persistent autoimmune syndromes, especially when accompanied by an immediate adverse reaction or pre-existing malignancy.

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 4u2009days 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, CLu2009+u2009zol 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 CLu2009+u2009zol, whereas %IFNγ+ Vδ1+ T cells was lower only in the presence of CL or CLu2009+u2009zol. %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.