Helicobacter pylori infection reduces disease severity in an experimental model of multiple sclerosis

Recent research has demonstrated that infection with the bacterial pathogen Helicobacter pylori is less common amongst patients with multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). We aimed to compare the prevalence of H. pylori amongst MS patients and healthy controls, and also investigated the impact of this infection on an animal model for MS, experimental autoimmune encephalomyelitis (EAE). The H. pylori status of 71 MS patients and 42 healthy controls was determined by serology. Groups of C57BL/6 mice were infected with H. pylori, or given diluent alone as a placebo, prior to inducing EAE. Clinical scores were assessed for all mice, and spleens and spinal cord tissue were harvested. CD4+ T cell subsets were quantified by flow cytometry, and T cell proliferation assays were performed. In MS patients the seroprevalence of H. pylori was half that of healthy controls (p = 0.018). Over three independent experiments, prior H. pylori infection had a moderate effect in reducing the severity of EAE (p = 0.012). In line with this, the antigen-specific T cell proliferative responses of infected animals were significantly reduced (p = 0.001), and there was a fourfold reduction in the number of CD4+ cells in the CNS. CD4+ populations in both the CNS and the spleens of infected mice also contained greatly reduced proportions of IFNγ+, IL-17+, T-bet+, and RORγt+ cells, but the proportions of Foxp3+ cells were equivalent. There were no differences in the frequency of splenic CD4+cells expressing markers of apoptosis between infected and uninfected animals. H. pylori was less prevalent amongst MS patients. In mice, the infection exerted some protection against EAE, inhibiting both Th1 and Th17 responses. This could not be explained by the presence of increased numbers of Foxp3+ regulatory T cells, or T cell apoptosis. This is the first direct experimental evidence showing that H. pylori may provide protection against inflammatory demyelination in the CNS

Impact of Human FcγR Gene Polymorphisms on IgG-Triggered Cytokine Release: Critical Importance of Cell Assay Format

Monoclonal antibody (mAb) immunotherapy has transformed the treatment of allergy, autoimmunity, and cancer. The interaction of mAb with Fc gamma receptors (FcγR) is often critical for efficacy. The genes encoding the low-affinity FcγR have single nucleotide polymorphisms (SNPs) and copy number variation that can impact IgG Fc:FcγR interactions. Leukocyte-based in vitro assays remain one of the industry standards for determining mAb efficacy and predicting adverse responses in patients. Here we addressed the impact of FcγR genetics on immune cell responses in these assays and investigated the importance of assay format. FcγR genotyping of 271 healthy donors was performed using a Multiplex Ligation-Dependent Probe Amplification assay. Freeze-thawed/pre-cultured peripheral blood mononuclear cells (PBMCs) and whole blood samples from donors were stimulated with reagents spanning different mAb functional classes to evaluate the association of FcγR genotypes with T-cell proliferation and cytokine release. Using freeze-thawed/pre-cultured PBMCs, agonistic T-cell-targeting mAb induced T-cell proliferation and the highest levels of cytokine release, with lower but measurable responses from mAb which directly require FcγR-mediated cellular effects for function. Effects were consistent for individual donors over time, however, no significant associations with FcγR genotypes were observed using this assay format. In contrast, significantly elevated IFN-γ release was associated with the FCGR2A-131H/H genotype compared to FCGR2A-131R/R in whole blood stimulated with Campath (p ≤ 0.01) and IgG1 Fc hexamer (p ≤ 0.05). Donors homozygous for both the high affinity FCGR2A-131H and FCGR3A-158V alleles mounted stronger IFN-γ responses to Campath (p ≤ 0.05) and IgG1 Fc Hexamer (p ≤ 0.05) compared to donors homozygous for the low affinity alleles. Analysis revealed significant reductions in the proportion of CD14hi monocytes, CD56dim NK cells (p ≤ 0.05) and FcγRIIIa expression (p ≤ 0.05), in donor-matched freeze-thawed PBMC compared to whole blood samples, likely explaining the difference in association between FcγR genotype and mAb-mediated cytokine release in the different assay formats. These findings highlight the significant impact of FCGR2A and FCGR3A SNPs on mAb function and the importance of using fresh whole blood assays when evaluating their association with mAb-mediated cytokine release in vitro. This knowledge can better inform on the utility of in vitro assays for the prediction of mAb therapy outcome in patients

Evaluation of high-throughput genomic assays for the Fc gamma receptor locus

Cancer immunotherapy has been revolutionised by the use of monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics

The Helicobacter pylori infection mediated regulatory T-cell response and in vivo supression of allergy and autoimmunity

Helicobacter pylori is a common human bacterial infection which usually causes asymptomatic gastritis, but 10-15% of individuals develop peptic ulcer disease (PUD) or gastric cancer. H. pylori stimulates a strong yet ineffective immune response in the gastric mucosa, which allows the infection to persist for many decades. H. pylori modulates the host response to facilitate immune evasion via multiple mechanisms, however regulatory T cells (T regs) are thought to play an important role. Recent epidemiological studies have shown a reduced risk of allergy and autoimmune diseases amongst those with the infection. It was hypothesized that H. pylori-induced local T reg responses protect against PUD and pre-malignant pathology, whilst the systemic T reg response plays a role in suppressing allergy and autoimmunity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Remodeling the tumor myeloid landscape to enhance antitumor antibody immunotherapies

Among the diverse tumor resident immune cell types, tumor-associated macrophages (TAMs) are often the most abundant, possess an anti-inflammatory phenotype, orchestrate tumor immune evasion and are frequently associated with poor prognosis. However, TAMs can also be harnessed to destroy antibody-opsonized tumor cells through the process of antibody-dependent cellular phagocytosis (ADCP). Clinically important tumor-targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing macrophages to eliminate tumor cells via ADCP. For IgG mAb, this is mediated by antibody-binding activating Fc gamma receptors (FcγR), with resultant phagocytic activity impacted by the level of co-engagement with the single inhibitory FcγRIIb. Approaches to enhance ADCP in the tumor microenvironment include the repolarization of TAMs to proinflammatory phenotypes or the direct augmentation of ADCP by targeting so-called ‘phagocytosis checkpoints’. Here we review the most promising new strategies targeting the cell surface molecules present on TAMs, which include the inhibition of ‘don’t eat me signals’ or targeting immunostimulatory pathways with agonistic mAb and small molecules to augment tumor-targeting mAb immunotherapies and overcome therapeutic resistance

Activation of GABA(A) receptors inhibits T cell proliferation

Background: the major sites for fast synaptic inhibition in the central nervous system (CNS) are ion channels activated by γ-aminobutyric acid (GABA). These receptors are referred as GABA(A) receptors (GABA(A)R). Recent evidence indicates a role of GABA(A)R in modulating the immune response. This work aimed to discern the role of GABA and GABA(A)Rs in human and mouse T cell activity.Methods: mouse splenocytes or human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 antibodies and the proliferation of both CD8+ and CD4+ T cells assessed through flow cytometry. Subsequently, the effects on T cell proliferation of either GABA(A)R modulation by diazepam that is also capable of activating mitochondrial based translocator protein (TSPO), alprazolam and allopregnanolone or inhibition by bicucculine methiodide (BMI) and (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) were assessed.Results: positive modulation of GABA(A)Rs either by benzodiazepines or the neurosteroid allopregnanolone inhibits both mouse and human T cell proliferation. GABAergic inhibition of T cell proliferation by benzodiazepines could be rescued by GABA(A)R blocking. Our data suggest that benzodiazepines influence T cell proliferation through both TSPO and GABA(A)Rs activation.Conclusions: we conclude that activation of GABA(A)Rs provides immunosuppression by inhibiting T cell proliferation

Genomic dissection of the Fcγ receptor region in the context of monoclonal antibody therapy

Development of the anti-CD20 antibody, rituximab, heralded the start of monoclonal antibody (mAb) therapy as an effective means of treating cancer. Despite its undoubted impact, clinical responses remain variable and cures are rarely achieved. Evidence from pre-clinical models and human trials indicates that mAbs primarily act through engaging low-affinity Fc gamma receptor (FcγR)-expressing effector immune cells. The low-affinity FcγR genes, FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B, are located within a highly homologous 200 kb region at 1q23, which is the result of an ancestral segmental duplication event (Figure 1). The locus also contains numerous single nucleotide polymorphisms (SNPs), many of which can affect receptor affinity and/or function and are associated with differential responses following mAb immunotherapy. Moreover, the region contains extensive copy number variation (CNV) that also can affect the expression and function of these receptors, but its impact on mAb immunotherapy remains unknown. To investigate the full impact of SNPs and CNV in the FcγR locus, we have optimised a number of sensitive and specific assays which are amenable to formalin fixed paraffin embedded (FFPE) material in order to apply them to clinical trial samples in a high-throughput manner. Initially we assessed the accuracy of established TaqMan and novel allele-specific (KASP) genotyping assays for FCGR2A-131H/R (rs1801274), FCGR3A-158F/V (rs396991) and FCGR2B-232I/T (rs1050501) SNPs by analysing 2085 DNA samples derived from peripheral blood lymphocytes (PBL) from a large, multi-centre cohort. Our data showed that although clear discrimination was possible at the FCGR2A-131H/R SNP, we needed additional selective Sanger sequencing to discriminate the FF/FV and IT/TT genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs, respectively. This difficulty in genotype discrimination in the cases of FCGR3A and FCGR2B is likely due to sequence homology with other genes and CNV in the gene regions which complicate assay design and interpretation of certain genotypes. Secondly, we applied a combined KASP genotyping and Sanger sequencing approach to matched PBL DNA and FFPE-extracted DNA from follicular lymphoma (FL) patients [n=14] and showed that while FFPE material was more likely to fail genotyping, successfully genotyped cases were concordant with the matched genomic DNA samples. FFPE samples which failed to amplify PCR products of at least 100 bp using the BIOMED-2 multiplex PCR protocol were more likely to fail genotyping assays. Finally, we assessed the ability of a multiplex ligation-dependent probe amplification (MLPA) assay to concurrently determine SNP genotype and CNV in the low-affinity FCGR locus in a cohort of 155 normal donors and DNA from seven matched PBL-/FFPE-derived FL cases. We employed a paralog ratio test (PRT) assay for FCGR3A and FCGR3B CNV confirmation. In our normal donors, MLPA and PRT results were concordant. 16% of normal donors harboured a deletion [n=15] or duplication [n=10] affecting the FCGR2C locus (A summary of regions of CNV is shown in Figure 1). CNV of FCGR3B was associated with variation at FCGR2C and no CNV was observed in FCGR2A and FCGR2B. CNV affecting FCGR3A was observed in 5% of donors with deletions and duplications in 4 and 5 donors, respectively. In the FFPE-derived DNA samples, we observed elevated variability in data quality that was most noticeable in probes targeting HSPA6, FCGR2C exon 4 and HSPA7. Poor quality data correlated with samples that failed to amplify at least the 100 bp PCR product using the BIOMED-2 multiplex PCR protocol. As such, the preclusion of HSPA6, FCGR2C exon 4 and HSPA7 probes and FFPE samples that failed to amplify any BIOMED-2 PCR product from the analysis permitted the production of high-quality MLPA data. Finally, we designed, and are currently optimising, a targeted re-sequencing platform (Haloplex, Agilent) to interrogate informative regions of the FcγR region, which includes those with unique sequence identify for CNV analysis, and those that include known SNPs. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of antibody therapy. This work will ultimately provide a detailed architecture of the region and establish the importance of FcγR genetics in predicting response to antibody therapeutics

STING activation reverses lymphoma-mediated resistance to antibody immunotherapy

Tumors routinely attract and co-opt macrophages to promote their growth, angiogenesis and metastasis. Macrophages are also the key effector cell for monoclonal antibody (mAb) therapies. Here we report that the tumor microenvironment creates an immunosuppressive signature on tumor-associated macrophages (TAM) which favors expression of inhibitory rather than activating Fcγ receptors (FcγR), thereby limiting the efficacy of mAb immunotherapy. We assessed a panel of TLR and STING agonists (a) for their ability to reprogram macrophages to a state optimal for mAb immunotherapy. Both STINGa and TLRa induced cytokine release, modulated FcγR expression and augmented mAb-mediated tumor cell phagocytosis in vitro. However, only STINGa reversed the suppressive FcγR profile in vivo, providing strong adjuvant effects to anti-CD20 mAb in murine models of lymphoma. Potent adjuvants like STINGa which can improve FcγR activatory:inhibitory (A:I) ratios on TAM are appealing candidates to reprogram TAM and curb tumor-mediated immunosuppression, thereby empowering mAb efficacy.</p

Cd31 is required on cd4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4+ T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4+ T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4+ T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4+ T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the Increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4+ T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis