NOD-like receptors: versatile cytosolic sentinels.

Nucleotide binding oligomerization domain (NOD)-like receptors are cytoplasmic pattern-recognition receptors that together with RIG-I-like receptor (retinoic acid-inducible gene 1), Toll-like receptor (TLR), and C-type lectin families make up the innate pathogen pattern recognition system. There are 22 members of NLRs in humans, 34 in mice, and even a larger number in some invertebrates like sea urchins, which contain more than 200 receptors. Although initially described to respond to intracellular pathogens, NLRs have been shown to play important roles in distinct biological processes ranging from regulation of antigen presentation, sensing metabolic changes in the cell, modulation of inflammation, embryo development, cell death, and differentiation of the adaptive immune response. The diversity among NLR receptors is derived from ligand specificity conferred by the leucine-rich repeats and an NH2-terminal effector domain that triggers the activation of different biological pathways. Here, we describe NLR genes associated with different biological processes and the molecular mechanisms underlying their function. Furthermore, we discuss mutations in NLR genes that have been associated with human diseases.

Phase II clinical trial of adoptive cell therapy for patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and low-dose interleukin-2.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown significant clinical benefit, but is limited by toxicities due to a requirement for post-infusion interleukin-2 (IL-2), for which high dose is standard. To assess a modified TIL protocol using lower dose IL-2, we performed a single institution phase II protocol in unresectable, metastatic melanoma. The primary endpoint was response rate. Secondary endpoints were safety and assessment of immune correlates following TIL infusion. Twelve metastatic melanoma patients were treated with non-myeloablative lymphodepleting chemotherapy, TIL, and low-dose subcutaneous IL-2 (125,000 IU/kg/day, maximum 9-10 doses over 2 weeks). All but one patient had previously progressed after treatment with immune checkpoint inhibitors. No unexpected adverse events were observed, and patients received an average of 6.8 doses of IL-2. By RECIST v1.1, two patients experienced a partial response, one patient had an unconfirmed partial response, and six had stable disease. Biomarker assessment confirmed an increase in IL-15 levels following lymphodepleting chemotherapy as expected and a lack of peripheral regulatory T-cell expansion following protocol treatment. Interrogation of the TIL infusion product and monitoring of the peripheral blood following infusion suggested engraftment of TIL. In one responding patient, a population of T cells expressing a T-cell receptor Vß chain that was dominant in the infusion product was present at a high percentage in peripheral blood more than 2 years after TIL infusion. This study shows that this protocol of low-dose IL-2 following adoptive cell transfer of TIL is feasible and clinically active. (ClinicalTrials.gov identifier NCT01883323.).

Physical activity in solid organ transplant recipients: Participation, predictors, barriers, and facilitators.

BACKGROUND: Our objectives were to describe the physical activity (PA) levels, predictors, barriers, and facilitators to PA in solid organ transplant (SOT) recipients. METHODS: A web-based questionnaire was sent to members of the Canadian Transplant Association including the Physical Activity Scale for the Elderly (PASE), and questions regarding barriers and facilitators of PA. RESULTS: One hundred and thirteen SOT recipients completed the survey. The median PASE score was 164.5 (24.6-482.7). Re-transplantation was the only statistically significant predictor of levels of PA. The most common facilitators of PA included a feeling of health from activity (94%), motivation (88%), social support (76%), knowledge and confidence about exercise (74%) and physician recommendation (59%). Influential barriers were cost of fitness centers (42%), side effects post-transplant or from medications (41%), insufficient exercise guidelines (37%), and feelings of less strength post-transplant (37%). CONCLUSION: There is a large variation in PA levels among SOT recipients. Multiple factors may explain the variance in PA levels in SOT recipients. Identification of facilitators and barriers to PA can inform the development of health and educational promotion strategies to improve participation among SOT recipients with low activity levels.

Identification of the inflammasome Nlrp1b as the candidate gene conferring diabetes risk at the Idd4.1 locus in the nonobese diabetic mouse.

Type 1 diabetes in the NOD mouse model has been linked to >30 insulin-dependent diabetes (Idd) susceptibility loci. Idd4 on chromosome 11 consists of two subloci, Idd4.1 and Idd4.2. Using congenic analysis of alleles in NOD and NOD-resistant (NOR) mice, we previously defined Idd4.1 as an interval containing >50 genes that controlled expression of genes in the type 1 IFN pathway. In this study, we report refined mapping of Idd4.1 to a 1.1-Mb chromosomal region and provide genomic sequence analysis and mechanistic evidence supporting its role in innate immune regulation of islet-directed autoimmunity. Genetic variation at Idd4.1 was mediated by radiation-sensitive hematopoietic cells, and type 1 diabetes protection conferred by the NOR allele was abrogated in mice treated with exogenous type 1 IFN-ß. Next generation sequence analysis of the full Idd4.1 genomic interval in NOD and NOR strains supported Nlrp1b as a strong candidate gene for Idd4.1. Nlrp1b belongs to the Nod-like receptor (NLR) gene family and contributes to inflammasome assembly, caspase-1 recruitment, and release of IL-1ß. The Nlrp1b of NOR was expressed as an alternative spliced isoform that skips exon 9, resulting in a premature stop codon predicted to encode a truncated protein. Functional analysis of the truncated NOR Nlrp1b protein demonstrated that it was unable to recruit caspase-1 and process IL-1ß. Our data suggest that Idd4.1-dependent protection from islet autoimmunity is mediated by differences in type 1 IFN- and IL-1ß-dependent immune responses resulting from genetic variation in Nlrp1b.

Variation in the Cd3 zeta (Cd247) gene correlates with altered T cell activation and is associated with autoimmune diabetes.

Tuning of TCR-mediated activation was demonstrated to be critical for lineage fate in T cell development, as well as in the control of autoimmunity. In this study, we identify a novel diabetes susceptibility gene, Idd28, in the NOD mouse and provide evidence that Cd3zeta (Cd247) constitutes a prime candidate gene for this locus. Moreover, we show that the allele of the Cd3zeta gene expressed in NOD and DBA/2 mouse strains confers lower levels of T cell activation compared with the allele expressed by C57BL/6 (B6), BALB/c, and C3H/HeJ mice. These results support a model in which the development of autoimmune diabetes is dependent on a TCR signal mediated by a less-efficient NOD allele of the Cd3zeta gene.

Transcriptomic Analysis and High-dimensional Phenotypic Mapping of Mononuclear Phagocytes in Mesenteric Lymph Nodes Reveal Differences Between Ulcerative Colitis and Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease [CD] and ulcerative colitis [UC] are distinct forms of inflammatory bowel disease. Heterogeneity of HLA-DR+SIRPα + mononuclear phagocytes [MNPs], including macrophages [MΦ], monocyte-derived [Mono] cells, and dendritic cells [DCs], was reported in gut tissue but not yet investigated in mesenteric lymph nodes [MLNs] of IBD patients. We here compared the phenotype, function, and molecular profile of HLA-DR+SIRPα + MNPs in CD and UC MLNs. METHODS: Cell distribution, morphology, immune function, and transcriptomic [bulk RNAseq] and high-dimensional protein expression profiles [CyTOF] of HLA-DR+SIRPα + MNPs were examined in MLNs of UC [n = 14], CD [n = 35], and non-IBD [n = 12] patients. RESULTS: Elevated frequencies of CD14+CD64+CD163+ [Mono/MΦ-like] MNPs displaying monocyte/MΦ morphology and phagocytic function were a distinct feature of UC MLNs. In CD, the proportion of CD14-CD64-CD163- [DC-like] cells was augmented relative to Mono/MΦ-like cells; DC-like cells drove naïve T cell proliferation, Th1 polarisation, and Th17 TCM plasticity. Gene expression profile corroborated the nature of DC-like cells, best represented by BTLA, SERPINF, IGJ and, of Mono/MΦ-like cells, defined by CD163, MARCO, MAFB, CD300E, S100A9 expression. CyTOF analysis showed that CD123+ plasmacytoid cells predominated over conventional DCs in DC-like cells. Four CD163+ clusters were revealed in Mono/MΦ-like cells, two of which were enriched in MARCO-CD68dimHLA-DRdim monocyte-like cells and MARCOhiCD68hiHLA-DRhi Mɸ, whose proportion increased in UC relative to CD. CONCLUSIONS: Defining the landscape of MNPs in MLNs provided evidence for expansion of CD163+ Mono/MΦ-like cells in UC only, highlighting a distinction between UC and CD, and thus the potential contribution of monocyte-like cells in driving colitis.

Association of Ipilimumab With Safety and Antitumor Activity in Women With Metastatic or Recurrent Human Papillomavirus-Related Cervical Carcinoma.

Importance: Based on evidence of human papillomavirus (HPV)-induced immune evasion, immunotherapy may be an attractive strategy in cervical cancer. Ipilimumab is a fully humanized monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4), which acts to downregulate the T-cell immune response. Objective: To assess the safety and antitumor activity of ipilimumab in recurrent cervical cancer. Design, Setting, and Participants: A multicenter trial was designed for patients with metastatic cervical cancer (squamous cell carcinoma or adenocarcinoma) with measurable disease and progression after at least 1 line of platinum chemotherapy. A run-in safety cohort using ipilimumab, 3 mg/kg, every 21 days for 4 cycles in 6 patients was followed by a phase II cohort of ipilimumab, 10 mg/kg, every 21 days for 4 cycles and then 4 cycles of maintenance therapy every 12 weeks for patients demonstrating radiologic response or stabilization. Immune correlative studies were performed on peripheral blood before and after therapy on archival tissue and fresh tumor obtained prior to registration and 7 days after cycle 2. The study was conducted from December 3, 2012, to September 15, 2014. The data were analyzed from April 2016 to June 2016 and in July 2017. Main Outcomes and Measures: The primary end points were safety and objective response rate. Immune analyses were performed on blood and tumor tissue. Results: A total of 42 women (median age, 49 years; range, 23-78 years) were enrolled (29 [69%] squamous cell cervical cancer and 13 [31%] adenocarcinoma; 37 [93%] of 40 patients with tissue available for analysis had HPV-positive confirmation; there was no archival tissue for 2 women). Grade 3 toxic effects included diarrhea in 4 patients, 3 of whom had colitis. Of 34 patients evaluated for best response (Response Evaluation Criteria in Solid Tumors, version 1.1), 1 patient had partial response and 10 had stable disease. The median progression-free survival and overall survival were 2.5 months (95% CI, 2.1-3.2 months) and 8.5 months (95% CI, 3.6-not reached; 1 patient was still alive), respectively. Intratumoral pretreatment CD3, CD4, CD8, FoxP3, indoleamine 2,3-dioxygenase, and programmed cell death ligand 1 (PD-L1) expression was not predictive of benefit and did not significantly change with treatment. Multicolor flow cytometry on peripheral lymphocytes revealed a treatment-dependent increase of inducible T-cell costimulator, human leukocyte antigen-antigen D related, and PD-1 during initial treatment, which returned to baseline during maintenance. Conclusions and Relevance: Ipilimumab was tolerable in this population but did not show significant single-agent activity. Immune changes were induced by anti-CTLA-4 therapy but did not correlate with clinical activity. Changes in these markers may guide further treatment strategies.

Defective induction of CTLA-4 in the NOD mouse is controlled by the NOD allele of Idd3/IL-2 and a novel locus (Ctex) telomeric on chromosome 1.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), or CD152, is a negative regulator of T-cell activation and has been shown to be associated with autoimmune diseases. Previous work has demonstrated a defect in the expression of this molecule in nonobese diabetic (NOD) mice upon anti-CD3 stimulation in vitro. Using a genetic approach we here demonstrate that a novel locus (Ctex) telomeric on chromosome 1 together with the Idd3 (Il-2) gene confers optimal CTLA-4 expression upon CD3 activation of T-cells. Based on these data, we provide a model for how gene interaction between Idd3 (IL-2), Ctex, and Idd5.1 (Ctla-4) could confer susceptibility to autoimmune diabetes in the NOD mouse. Additionally, we showed that the Ctex and the Idd3 regions do not influence inducible T-cell costimulator (ICOS) protein expression in NOD mice. Instead, as previously shown, higher ICOS levels in NOD mice appear to be controlled by gene(s) in the Idd5.1 region, possibly a polymorphism in the Icos gene itself.

Diabetes protection and restoration of thymocyte apoptosis in NOD Idd6 congenic strains.

Type 1 diabetes in the nonobese diabetic (NOD) mouse is a multifactorial and polygenic disease. The NOD-derived genetic factors that contribute to type 1 diabetes are named Idd (insulin-dependent diabetes) loci. To date, the biological functions of the majority of the Idd loci remain unknown. We have previously reported that resistance of NOD immature thymocytes to depletion by dexamethazone (Dxm) maps to the Idd6 locus. Herein, we refine this phenotype using a time-course experiment of apoptosis induction upon Dxm treatment. We confirm that the Idd6 region controls apoptosis resistance in immature thymocytes. Moreover, we establish reciprocal Idd6 congenic NOD and B6 strains to formally demonstrate that the Idd6 congenic region mediates restoration of the apoptosis resistance phenotype. Analysis of the Idd6 congenic strains indicates that a 3-cM chromosomal region located within the distal part of the Idd6 region controls apoptosis resistance in NOD immature thymocytes. Together, these data support the hypothesis that resistance to Dxm-induced apoptosis in NOD immature thymocytes is controlled by a genetic factor within the region that also contributes to type 1 diabetes pathogenesis. We propose that the diabetogenic effect of the Idd6 locus is exerted at the level of the thymic selection process.

CTLA-4 (CD152) and its involvement in autoimmune disease.

Autoimmune diseases (AID) are inherited as complex genetic diseases. Different Autoimmune diseases have been found to cluster in families and are believed to share some common etiological factors. With the exception of major histocompatibility complex (MHC) genes contributing susceptibility to these diseases have been difficult to identify. CD152 has emerged as one such candidate unifying several autoimmune diseases. We here review the evidence that CD152 constitutes a general susceptibility factor for multiple autoimmune diseases and discuss how CD152 and other co-stimulatory pathways may contribute to autoimmune pathogenesis.

Potentiation of immunological tolerance induction in adult mice by co-administration of pooled normal IgG and oral tolerogens: a potential therapeutic approach for autoimmune diseases.

Oral tolerance can be defined as the inability of an adult animal to produce specific antibodies or cellular immune responses upon conventional immunization, after oral antigenic administration. Recently, the oral administration of antigens has gained renewed interest because of the possibility of inducing tolerance in nonimmunized adult animals and, consequently, opening up the theoretical possibility of preventing or treating diseases caused by malfunction of the immune system. This strategy has been proven to be useful in the prevention of allergic and autoimmune diseases in rodents, as well as in the amelioration of certain autoimmune diseases in humans. Although there is experimental and clinical evidence for the usefulness of oral tolerance in medical practice, the mechanisms responsible for this phenomenon are still poorly understood, and the results obtained are not always satisfactory. Herein, we show that the thymus is required for the induction and maintenance of oral tolerance, providing evidence that it is not a pure form of clonal deletion-based peripheral tolerance. Oral tolerance could therefore depend on the formation and release to the periphery of regulatory T cells, such as gammadelta or alphabeta T cells, by the thymus. This finding may have profound implications for the treatment of autoimmune diseases, since most of them are associated with thymic hypofunction. On the other hand, due to so far unknown mechanisms, the intraperitoneal co-administration of normal IgG to mice orally treated with tolerogen leads to a sustained and intense immunological tolerance, both in euthymic and thymectomized mice, including those of the lupus erythematosus-prone NZB x NZW lineage. This approach for inducing and maintaining tolerance in thymus-deficient conditions is discussed and put forth herein as a new evidence-based proposition for the therapy of autoimmune diseases.

The NOD allele of the Idd5 locus on chromosome 1 mediates a non-cell-autonomous defect in negative selection of T cells.

Recent data have suggested that non-obese diabetic (NOD) mice display a defect in negative thymic selection. Using mixed bone marrow chimeras, we demonstrate that the NOD allele of the diabetes susceptibility region 5 (Idd5) locus on chromosome 1, confers defective negative selection in response to endogenous superantigens (SAg) Mtv8 and Mtv9. We generated mixed bone marrow (BM) chimeras in which the donor cells of NOD and C3H or NOD.Idd5(b10) and C3H coexist and are similarly exposed to the Mtv8 and Mtv9 SAg. Under these conditions, SAg-mediated deletion of Vbeta11+ T cells is less efficient in chimeric mice reconstituted with NOD+C3H BM, compared with chimeras reconstituted with NOD.Idd5(b10)+C3H BM. Interestingly, the observed discrepancy was not T cell autonomous but was found to be mediated by a BM derived cellular subset, and under control of a gene(s) in the Idd5 region.

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11.

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 x NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.