European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development.

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development.

IL-17A is a pertinent therapeutic target for moderate-to-severe hidradenitis suppurativa: Combined results from a pre-clinical and phase II proof-of-concept study.

Hidradenitis Suppurativa (HS) is a chronic, recurrent, inflammatory, follicular skin disease whose pathology is complex and not fully understood. The objective of this study was to elucidate the role of IL-17A in moderate-to-severe HS. Transcriptomic and histological analyses were conducted on ex vivo HS (n = 19; lesional and non-lesional) and healthy control (n = 8) skin biopsies. Further, a Phase II exploratory, randomized, double-blind, placebo-controlled study was carried out in moderate-to-severe HS patients. Patients were treated with either CJM112 300 mg (n = 33), a fully human anti-IL-17A IgG1/κ monoclonal antibody, or placebo (n = 33). The main outcome of the translational analyses was to identify IL-17A-producing cells and indications of IL-17A activity in HS lesional skin. The primary objective of the clinical study was to determine the efficacy of CJM112 in moderate-to-severe HS patients by HS-Physician Global Assessment (HS-PGA) responder rate at Week 16. Transcriptomic and histopathologic analyses revealed the presence of heterogeneous cell types in HS lesional skin; IL-17A gene signatures were increased in HS lesional vs non-lesional or healthy skin. High expression of IL-17A was localized to T cells, neutrophils, and mast cells, confirming the transcriptional data. Clinically, the proportion of Week 16 HS-PGA responders was significantly higher (p = 0.03) in the CJM112 group vs placebo (32.3% vs 12.5%). This study elucidated the role of the IL-17A pathway in HS pathogenesis and clinically validated the IL-17A pathway in moderate-to-severe HS patients in a proof-of-concept study using the anti-IL-17A-specific antibody CJM112.

Small-molecule factor B inhibitor for the treatment of complement-mediated diseases.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1ß; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1ß pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1ß antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1ß in lung granuloma, and increased IL-1ß release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 knockout mice showed decreased and miR-223 knockout mice increased granuloma formation compared to wild-type mice. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1ß antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1ß pathway activation in sarcoidosis and suggest both as valid therapeutic targets.

Blockade of CD40-CD154 pathway interactions suppresses ectopic lymphoid structures and inhibits pathology in the NOD/ShiLtJ mouse model of Sjögren's syndrome.

OBJECTIVE: To examine the role of CD40-CD154 costimulation and effects of therapeutic pathway blockade in the non-obese diabetic (NOD/ShiLtJ) model of Sjögren's syndrome (SS). METHODS: We assessed leucocyte infiltration in salivary glands (SGs) from NOD/ShiLtJ mice by immunohistochemistry and examined transcriptomics data of SG tissue from these animals for evidence of a CD40 pathway gene signature. Additionally, we dosed MR1 (anti-CD154 antibody) in NOD mice after the onset of SS-like disease and examined the effects of MR1 treatment on sialadenitis, autoantibody production, SG leucocyte infiltration, gene expression downstream of CD40 and acquaporin 5 (AQP5) expression. RESULTS: We could detect evidence of CD40 expression and pathway activation in SG tissue from NOD mice. Additionally, therapeutic treatment with MR1 suppressed CD40 pathway genes and sialadenitis, inhibited ectopic lymphoid structure formation and autoantibody production, as well as decreased the frequency of antibody-secreting cells in SGs but had minimal effects on AQP5 expression in NOD/ShiLtJ SGs. CONCLUSION: CD40-CD154 interactions play an important role in key pathological processes in a mouse model of SS, suggesting that blockade of this costimulatory pathway in the clinic may have beneficial therapeutic effects in patients suffering from this autoimmune exocrinopathy.

Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily.

Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/ß sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8 + T cell clonal expansion (CD8 EXP ), independent of HLA mismatch or IS type. Subcloning of TCRα/ß cDNAs from CD8 EXP into Jurkat76 cells (TCR -/- ) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8 EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8 EXP , while CD8 EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples. Overall, our data define the clonal CD8 + T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.

Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/ß sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/ß cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Preclinical characterization of the Toll-like receptor 7/8 antagonist MHV370 for lupus therapy.

Genetic and in vivo evidence suggests that aberrant recognition of RNA-containing autoantigens by Toll-like receptors (TLRs) 7 and 8 drives autoimmune diseases. Here we report on the preclinical characterization of MHV370, a selective oral TLR7/8 inhibitor. In vitro, MHV370 inhibits TLR7/8-dependent production of cytokines in human and mouse cells, notably interferon-α, a clinically validated driver of autoimmune diseases. Moreover, MHV370 abrogates B cell, plasmacytoid dendritic cell, monocyte, and neutrophil responses downstream of TLR7/8. In vivo, prophylactic or therapeutic administration of MHV370 blocks secretion of TLR7 responses, including cytokine secretion, B cell activation, and gene expression of, e.g., interferon-stimulated genes. In the NZB/W F1 mouse model of lupus, MHV370 halts disease. Unlike hydroxychloroquine, MHV370 potently blocks interferon responses triggered by specific immune complexes from systemic lupus erythematosus patient sera, suggesting differentiation from clinical standard of care. These data support advancement of MHV370 to an ongoing phase 2 clinical trial.

Disease Association of Anti‒Carboxyethyl Lysine Autoantibodies in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurring suppurating lesions of the intertriginous areas, resulting in a substantial impact on patients' QOL. HS pathogenesis remains poorly understood. An autoimmune component has been proposed, but disease-specific autoantibodies, autoantigens, or autoreactive T cells have yet to be described. In this study, we identify a high prevalence of IgM, IgG, and IgA antibodies directed against Nε-carboxyethyl lysine (CEL), a methylglyoxal-induced advanced glycation end-product, in the sera of patients with HS. Titers of anti-CEL IgG and IgA antibodies were highly elevated in HS compared with those in healthy controls and individuals with other inflammatory skin diseases. Strikingly, the majority of anti-CEL IgG was of the IgG2 subclass and correlated independently with both disease severity and duration. Both CEL and anti-CEL‒producing plasmablasts could be isolated directly from HS skin lesions, further confirming the disease relevance of this autoimmune response. Our data point to an aberration of the methylglyoxal pathway in HS and support an autoimmune axis in the pathogenesis of this debilitating disease.

Nonhematopoietic IRAK1 drives arthritis via neutrophil chemoattractants.

IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.

CARD10 cleavage by MALT1 restricts lung carcinoma growth in vivo.

CARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity.

Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A4 Hydrolase.

The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720.

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis.

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis.

Lipidomics Profiling of Hidradenitis Suppurativa Skin Lesions Reveals Lipoxygenase Pathway Dysregulation and Accumulation of Proinflammatory Leukotriene B4.

Hidradenitis suppurativa (HS) is a chronic, recurring inflammatory dermatosis characterized by abscesses, deep-seated nodules, sinus tracts, and fibrosis in skin lesions around hair follicles of the axillary, inguinal, and anogenital regions. Whereas the exact pathogenesis remains poorly defined, clear evidence suggests that HS is a multifactorial inflammatory disease characterized by innate and adaptive immune components. Bioactive lipids are important regulators of cutaneous homeostasis, inflammation, and resolution of inflammation. Alterations in the lipid mediator profile can lead to malfunction and cutaneous inflammation. We used targeted lipidomics to analyze selected omega-3 and omega-6 polyunsaturated fatty acids in skin of patients with HS and of healthy volunteers. Lesional HS skin displayed enrichment of 5-lipoxygenase (LO)‒derived metabolites, especially leukotriene B4. In addition, 15-LO‒derived metabolites were underrepresented in HS lesions. Changes in the lipid mediator profile were accompanied by transcriptomic dysregulation of the 5-LO and 15-LO pathways. Hyperactivation of the 5-LO pathway in lesional macrophages identified these cells as potential sources of leukotriene B4, which may cause neutrophil influx and activation. Furthermore, leukotriene B4-induced mediators and pathways were elevated in HS lesions, suggesting a contribution of this proinflammatory lipid meditator to the pathophysiology of HS.

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.

Requirement of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Protease Activity for Fcγ Receptor-Induced Arthritis, but Not Fcγ Receptor-Mediated Platelet Elimination, in Mice.

OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.

Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure.

The immunomodulator FTY720 inhibits lymph node (LN) and thymic egress, thereby constraining T cell circulation and reducing peripheral T cell numbers. Here, we analyzed in mouse models the as yet scarcely characterized impact of long-term (up to 6 months) FTY720 exposure on T cell homeostasis and possible consequences for alloreactivity. In green fluorescent protein (GFP) hemopoietic chimeras, the turnover of (initially GFP(-)) peripheral T cell pools was markedly delayed under FTY720, while normal homeostatic differences between CD4 and CD8 T cell sub-populations were retained or amplified further. Homeostatic proliferation was enhanced, and within shrinking T cell pools, the proportions of effector memory phenotype CD4 T cells (CD4T(PEM)) increased in spleens and LNs and of central memory phenotype CD8 T cells (CD8T(PCM)) in LNs. By contrast, the fractions of CD8T(PEM) and CD4T(PCM) remained stably small under FTY720. The enrichment for CD4T(PEM) and CD8T(PCM) correlated with larger proportions of IFNgamma-producing T cells upon nonspecific but not allospecific stimulation. Splenic CD4 T cells from FTY720-treated mice proliferated more strongly upon transfer to semi-allogeneic hosts. However, heart allograft survival was not compromised in FTY720 pre-treated recipients. It correlated with reduced intra-graft CD8 T cells, and the longest surviving transplants contained the highest numbers of CD4 T cells. Thus, continuous FTY720 exposure reveals differential homeostatic responses by memory phenotype CD4 and CD8 T cell sub-populations, and it may enhance alloreactive CD4 T cell proliferation and tissue infiltration without accelerating allograft rejection.