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γδ T cells are involved in the control of

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+signal that is crucial for T-cell function. The Ca2+channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived fromin vitrostudies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulationin vitro,Trpm2-/-and WT CD4+and CD8+T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation ofTrpm2-/-CD8+T cells and unimpaired differentiation of CD4+T cells into Th1, Th17, and Treg cells under specific polarizing conditions.In vivo,Trpm2-/-and WT CD8+T cells showed equal specific responses toListeria monocytogenesafter infection of WT andTrpm2-/-mice and after transfer of WT andTrpm2-/-CD8+T cells into infected recipients. CD4+T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT andTrpm2-/-CD4+T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

The transcription factor IRF4 is required for CD8(+) T cell activation, proliferation, and differentiation to effector cells and thus is essential for robust CD8(+) T cell responses. The function of IRF4 in memory CD8(+) T cells yet needs to be explored. To investigate the role of IRF4 for maintaining differentiation state and survival of CD8(+) memory T cells, we used a mouse model with tamoxifen-inducible Irf4 knockout to preclude effects due to inefficient memory cell differentiation in absence of IRF4. We infected mice with ovalbumin-recombinant listeria and induced Irf4 knockout after clearance of the pathogen. Loss of IRF4 resulted in phenotypical changes of CD8(+) memory T cells but did not cause a reduction of the total memory T cell population. However, upon reencounter of the pathogen, CD8(+) memory T cells showed impaired expansion and acquisition of effector functions. When compared to CD8(+) effector memory T cells, CD8(+) tissue-resident memory T cells (T(RM) cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8(+) T(RM)-cell populations, and tamoxifen-induced Irf4 deletion caused a reduction of this cell population. In conclusion, our results demonstrate that IRF4 is required for effective reactivation but not for general survival of CD8(+) memory T cells. Formation and maintenance of CD8(+) T(RM) cells, in contrast, appear to depend on IRF4.

In anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), antibodies and T cells directed against the Goodpasture antigen, the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1), provoke renal inflammation resulting in rapidly progressing crescentic GN. Interleukin 6 (IL-6) is a pleiotropic cytokine with both pro- and anti-inflammatory activities, and IL-6 blockade is successfully used for treatment of diseases associated with acute and chronic inflammation. However, the role of IL-6 in anti-GBM GN is unclear. Here, we use the mouse model of experimental autoimmune glomerulonephritis (EAG) to study the role of IL-6 in anti-GBM GN. DBA/1J mice were immunized with α3(IV)NC1 and developed fatal crescentic GN. Treatment of mice with neutralizing anti-IL-6 antibodies impaired the generation of α3(VI)NC1-specific TH1 and TH17 cells. However, despite lasting reduction of the TH17 cell response, antibody treatment did not prevent crescentic GN. Antibody treatment was also ineffective in a therapeutic setting with pre-existing autoantibodies and T cells. In conclusion, our results indicate that although the blockade of IL-6 impairs the development of autoimmunity against α3(VI)NC1, this treatment does not ameliorate crescentic GN both in a preemptive and a therapeutic approach.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (T

Rationale: CD38 is a target for the therapy of multiple myeloma (MM) with monoclonal antibodies such as daratumumab and isatuximab. Since MM patients exhibit a high rate of relapse, the development of new biologics targeting alternative CD38 epitopes is desirable. The discovery of single-domain antibodies (nanobodies) has opened the way for a new generation of antitumor therapeutics. We report the generation of nanobody-based humanized IgG1 heavy chain antibodies (hcAbs) with a high specificity and affinity that recognize three different and non-overlapping epitopes of CD38 and compare their cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. Methods: We generated three humanized hcAbs (WF211-hcAb, MU1067-hcAb, JK36-hcAb) that recognize three different non-overlapping epitopes (E1, E2, E3) of CD38 by fusion of llama-derived nanobodies to the hinge- and Fc-domains of human IgG1. WF211-hcAb shares the binding epitope E1 with daratumumab. We compared the capacity of these CD38-specific hcAbs and daratumumab to induce CDC and ADCC in CD38-expressing tumor cell lines in vitro and in patient MM cells ex vivo as well as effects on xenograft tumor growth and survival in vivo. Results: CD38-specific heavy chain antibodies (WF211-hcAb, MU1067-hcAb, JK36-hcAb) potently induced antibody-dependent cellular cytotoxicity (ADCC) in CD38-expressing tumor cell lines and in primary patient MM cells, but only little if any complement-dependent cytotoxicity (CDC). In vivo, CD38-specific heavy chain antibodies significantly reduced the growth of systemic lymphomas and prolonged survival of tumor bearing SCID mice. Conclusions: CD38-specific nanobody-based humanized IgG1 heavy chain antibodies mediate cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. These promising results of our study indicate that CD38-specific hcAbs warrant further clinical development as therapeutics for multiple myeloma and other hematological malignancies.

IL-6 is required for the response of mice against Listeria monocytogenes. Control of infection depends on classical IL-6 signaling via membrane IL-6Rα, but IL-6 target cells and protective mechanisms remain unclear. We used mice with IL-6Rα-deficiency in T cells (Il6rafl/fl×CD4cre) or myeloid cells (Il6rafl/fl×LysMcre) to define the role of these cells in IL-6-mediated protection. Abrogation of IL-6Rα in T cells did not interfere with bacteria control and induction of TH1 and CD8+ T-cell responses. IL-6Rα-deficiency in myeloid cells caused significant defects in listeria control. This defect was not associated with reduced recruitment of granulocytes and inflammatory monocytes, and both cell populations were activated and not impaired in cytokine production. However, IL-6Rα-deficient inflammatory monocytes displayed diminished expression of IL-4Rα and of CD38, a protein required for phagocytosis and innate control of listeria. In vitro studies revealed that IL-4 and IL-6 cooperated in induction of CD38. In listeria-infected mice, phagocytic activity of inflammatory monocytes correlated with CD38 expression levels on cells and inflammatory monocytes of Il6rafl/fl×LysMcre mice were significantly impaired in phagocytosis. In conclusion, we demonstrate that inhibition of classical IL-6 signaling in myeloid cells causes alterations in differentiation and function of these cells, which subsequently prevent effective control of L. monocytogenes.

The utility of nanobodies and conventional antibodies for in vivo imaging is well known, but optimum dosing and timing schedules for one versus the other have not been established. We aimed to improve specific tumor imaging in vivo with nanobodies and conventional antibodies using near-infrared fluorescence (NIRF) imaging. We used ARTC2 expressed on lymphoma cells as a model target antigen. ARTC2-specific nanobody s+16a and conventional antibody Nika102 were labeled with NIRF-dye AF680. In vivo NIRF-imaging of ARTC2-positive and ARTC2-negative xenografts was performed over 24 h post-injection of 5, 10, 25, or 50 µg of each conjugate. Specific target-binding and tissue-penetration were verified by NIRF imaging ex vivo, flow cytometry and fluorescence microscopy. NIRF-imaging of s+16a(680) in vivo revealed a six times faster tumor accumulation than of Nika102(680). Using 50 µg of s+16a(680) increased the specific signals of ARTC2-positive tumors without increasing background signals, allowing a tumor-to-background (T/B) ratio of 12.4 ± 4.2 within 6 h post-injection. Fifty micrograms of Nika102(680) increased specific signals of ARTC2-positive tumors but also of ARTC2-negative tumors and background, thereby limiting the T/B ratio to 6.1 ± 2.0. Ten micrograms of Nika102(680) only slightly reduced specific tumor signals but dramatically reduced background signals. Ex vivo analyses confirmed a faster and deeper tumor penetration with s+16a(680). Using nanobody s+16a allowed same-day imaging with a high T/B ratio, whereas antibody Nika102 gave optimal imaging results only 24 h post injection. Nanobody s+16a required a high dose, whereas antibody Nika102 had the best T/B-ratio at a low dose. Therefore, timing and dosage should be addressed when comparing nanobodies and conventional antibodies for molecular imaging purposes.