IRF4 is required for migration of CD4+ T cells to the intestine but not for Th2 and Th17 cell maintenance.

The transcription factor Interferon Regulatory Factor 4 (IRF4) is central in control of T cell activation and differentiation. Deficiency of IRF4 results in severe immune deficiency and affects maturation and function of most if not all T cell subsets. Here we use mouse infection models for Citrobacter rodentium and Strongyloides ratti to analyze the function of IRF4 in T helper (Th) 17 and Th2 cell responses, respectively. IRF4 deficient mice were impaired in the control of both pathogens, failed to mount Th17 and Th2 cell responses and showed impaired recruitment of T helper cells to the intestine, the infection site of both pathogens. Compromised intestinal migration was associated with reduced expression of the intestinal homing receptors α4ß7 integrin, CCR9 and GPR15. Identification of IRF4 binding sites in the gene loci of these receptors suggests a direct control of their expression by IRF4. Competitive T cell transfer assays further demonstrated that loss of one functional Irf4 allele already affected intestinal accumulation and Th2 and Th17 cell generation, indicating that lower IRF4 levels are of disadvantage for Th2 and Th17 cell differentiation as well as their migration to the intestine. Conversion of peripheral CD4+ T cells from an Irf4 wildtype to an Irf4 heterozygous or from an Irf4 heterozygous to a homozygous mutant genotype after C. rodentium or S. ratti infection did not reduce their capacity to produce Th17 or Th2 cytokines and only partially affected their persistence in the intestine, revealing that IRF4 is not essential for maintenance of the Th2 and Th17 phenotype and for survival of these T helper cells in the intestine. In conclusion, we demonstrate that the expression levels of IRF4 determine Th2 and Th17 cell differentiation and their intestinal accumulation but that IRF4 expression is not crucial for Th2 and Th17 cell survival.

Cell-Autonomous Constitutive gp130 Signaling in T Cells Amplifies TH17 Cell Responses and Causes Severe Lung Inflammation.

IL-6 plays a fundamental role in T cell differentiation and is strictly controlled by surface expression and shedding of IL-6R. IL-6 also acts on other cells that might affect T cell maturation. To study the impact of cell-autonomous and uncontrolled IL-6 signaling in T cells, we generated mice with a constitutively active IL-6R gp130 chain (Lgp130) expressed either in all T cells (Lgp130 × CD4Cre mice) or inducible in CD4+ T cells (Lgp130 × CD4CreERT2 mice). Lgp130 × CD4Cre mice accumulated activated T cells, including TH17 cells, in the lung, resulting in severe inflammation. Tamoxifen treatment of Lgp130 × CD4CreERT2 mice caused Lgp130 expression in 40-50% of CD4+ T cells, but mice developed lung disease only after several months. Lgp130+ CD4+ T cells were also enriched for TH17 cells; however, there was concomitant expansion of Lgp130- regulatory T cells, which likely restricted pathologic Lgp130+ T cells. In vitro, constitutive gp130 signaling in T cells enhanced but was not sufficient for TH17 cell differentiation. Augmented TH17 cell development of Lgp130+ T cells was also observed in Lgp130 × CD4CreERT2 mice infected with Staphylococcus aureus, but gp130 activation did not interfere with formation of TH1 cells against Listeria monocytogenes. Lgp130+ CD4+ T cells acquired a memory T cell phenotype and persisted in high numbers as a polyclonal T cell population in lymphoid and peripheral tissues, but we did not observe T cell lymphoma formation. In conclusion, cell-autonomous gp130 signaling alters T cell differentiation. Although gp130 signaling is not sufficient for TH17 cell differentiation, it still promotes accumulation of activated T cells in the lung that cause tissue inflammation.

Kidney-resident innate-like memory γδ T cells control chronic Staphylococcus aureus infection of mice.

γδ T cells are involved in the control of Staphylococcus aureus infection, but their importance in protection compared to other T cells is unclear. We used a mouse model of systemic S. aureus infection associated with high bacterial load and persistence in the kidney. Infection caused fulminant accumulation of γδ T cells in the kidney. Renal γδ T cells acquired tissue residency and were maintained in high numbers during chronic infection. At day 7, up to 50% of renal γδ T cells produced IL-17A in situ and a large fraction of renal γδ T cells remained IL-17A+ during chronic infection. Controlled depletion revealed that γδ T cells restricted renal S. aureus replication in the acute infection and provided protection during chronic renal infection and upon reinfection. Our results demonstrate that kidney-resident γδ T cells are nonredundant in limiting local S. aureus growth during chronic infection and provide enhanced protection against reinfection.

Neutralization of IL-6 inhibits formation of autoreactive TH17 cells but does not prevent loss of renal function in experimental autoimmune glomerulonephritis.

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.

Interferon regulatory factor 4 controls effector functions of CD8+ memory T cells.

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 (TRM cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8+ TRM-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+ TRM cells, in contrast, appear to depend on IRF4.

TRPM2 Is Not Required for T-Cell Activation and Differentiation.

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 from in vitro studies 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 stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- 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 to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- 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 and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

Pathogen-induced tissue-resident memory TH17 (TRM17) cells amplify autoimmune kidney 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 (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.

Nanobody-based CD38-specific heavy chain antibodies induce killing of multiple myeloma and other hematological malignancies.

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.

Role of regulatory T cells in experimental autoimmune glomerulonephritis.

Anti-glomerular basement membrane (anti-GBM) disease is characterized by antibodies and T cells directed against the Goodpasture antigen, the noncollagenous domain of the α3-chain of type IV collagen [α3(IV)NC1] of the GBM. Consequences are the deposition of autoantibodies along the GBM and the development of crescentic glomerulonephritis (GN) with rapid loss of renal function. Forkhead box protein P3 (Foxp3)+ regulatory T (Treg) cells are crucial for the maintenance of peripheral tolerance to self-antigens and the prevention of immunopathology. Here, we use the mouse model of experimental autoimmune GN to characterize the role of Treg cells in anti-GBM disease. Immunization of DBA/1 mice with α3(IV)NC1 induced the formation of α3(IV)NC1-specific T cells and antibodies and, after 8-10 wk, the development of crescentic GN. Immunization resulted in increased frequencies of peripheral Treg cells and renal accumulation of these cells in the stage of acute GN. Depletion of Treg cells during immunization led to enhanced generation of α3(IV)NC1-specific antibodies and T cells and to aggravated GN. In contrast, depletion or expansion of the Treg cell population in mice with established autoimmunity had only minor consequences for renal inflammation and did not alter the severity of GN. In conclusion, our results indicate that in anti-GBM disease, Treg cells restrict the induction of autoimmunity against α3(IV)NC1. However, Treg cells are inefficient in preventing crescentic GN after autoimmunity has been established.

Control of Listeria monocytogenes infection requires classical IL-6 signaling in myeloid cells.

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 role of ADAM17 in the T-cell response against bacterial pathogens.

ADAM17 is a member of the A Disintegrin And Metalloproteinase family of proteases. It is ubiquitously expressed and causes the shedding of a broad spectrum of surface proteins such as adhesion molecules, cytokines and cytokine receptors. By controlled shedding of these proteins from leukocytes, ADAM17 is able to regulate immune responses. Several ADAM17 targets on T cells have been implicated in T-cell migration, differentiation and effector functions. However, the role of ADAM17 in T-cell responses is still unclear. To characterize the function of ADAM17 in T cells, we used Adam17fl/fl×CD4cre+ mice with a T-cell restricted inactivation of the Adam17 gene. Upon stimulation, ADAM17-deficient CD4+ and CD8+ T cells were impaired in shedding of CD62L, IL-6Rα, TNF-α, TNFRI and TNFRII. Surprisingly, we could not detect profound changes in the composition of major T-cell subsets in Adam17fl/fl×CD4cre+ mice. Following infection with Listeria monocytogenes, Adam17fl/fl×CD4cre+ mice mounted regular listeria-specific CD4+ TH1 and CD8+ T-cell responses and were able to control primary and secondary infections. In conclusion, our study indicates that ADAM17 is either not required in T cells under homoeostatic conditions and for control of listeria infection or can be effectively compensated by other mechanisms.

Molecular imaging of tumors with nanobodies and antibodies: Timing and dosage are crucial factors for improved in vivo detection.

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.

Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies. First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results. The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

In vivo near-infrared fluorescence targeting of T cells: comparison of nanobodies and conventional monoclonal antibodies.

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody-Fc-fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP-ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2-specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc-fusion protein of s + 16a (s + 16-mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near-infrared fluorescence imaging. Nanobody s + 16a(680) and s + 16mFc(680) labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102(680) required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2-positive lymph nodes but not of ART2-negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc(680) showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a(680) , since renal elimination of unbound s + 16a(680) significantly reduced background signals. Our results indicate that small single-domain nanobodies are best suited for short-term uses, such as noninvasive imaging, whereas larger nanobody-Fc-fusion proteins are better suited for long-term uses, such as therapy of inflammation and tumors.