The cytotoxic T lymphocyte antigen4 (CTLA4) gene plays a critical role in the control of T cell activation. The gene encodes a surface molecule with inhibitory effects on activated T cells. Several studies have disclosed an association between the previously known variants of the CTLA4 gene and autoimmune disorders, but no study has as yet found any definite association between vitiligo and the CTLA4 polymorphisms. A recent study identified new candidate susceptibility polymorphisms in this region, associated with differential gene splicing and thereby the relative abundance of soluble CTLA4. To assess these new polymorphisms in patients with vitiligo, we genotyped 100 vitiligo patients and 140 healthy controls from the UK, for these novel polymorphisms. No association was found in patients with isolated vitiligo, but a significant association was seen in patients with vitiligo and other autoimmune diseases. The results indicate that the polymorphisms in the CTLA4 gene region confer susceptibility to vitiligo when occurring together with other autoimmune diseases, but not in patients with isolated vitiligo. This raises the possibility that there are two distinct forms of vitiligo where only a subgroup of patients may have a disease caused by the autoimmune destruction of melanocytes.

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

A fusion protein consisting of the extracellular domain of CTLA4 and an Ig C gamma 1 chain (CTLA4-Ig) was used to examine the distribution of the ligands for CTLA4 within the murine thymus and to characterize the nature of these ligands. Two-color immunofluorescence of thymus tissue revealed binding of the fusion protein to medullary thymic epithelial cells and dendritic cells within the corticomedullary and medullary areas of the thymus. Medullary cells binding the fusion protein also expressed MHC class II products and ICAM-1. Thymus tissue sections treated with cross-linking fixatives, such as glutaraldehyde, paraformaldehyde, or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide no longer bound the CTLA4 fusion protein, indicating that binding was very sensitive to the tertiary structure of the tissue ligand. The ability of thymic tissue to bind the fusion protein was developmentally regulated. At day 14 of gestation, only scattered single cells were labeled. Clusters of labeled cells, which were detected by day 16 of gestation, increased in frequency with advancing gestational age. Consistent with the in situ labeling studies, CTLA4-Ig also labeled several thymic epithelial cell lines previously shown to have a medullary phenotype. Polymerase chain reaction analysis of mRNA extracted from these cells indicated they contained mRNA for B7, a known counter receptor for CTLA4 and CD28. Immunoprecipitation of 125I-labeled thymic epithelial cells with the CTLA4-Ig detected a M(r) 65,000 to 70,000 species under reducing conditions, consistent with previous studies of B7. These data suggest that the ligand for CTLA4 expressed by thymic epithelial cells in vitro is B7 and that the expression of this ligand in situ is largely restricted to the medullary compartment and is associated with epithelial cells and dendritic cells.

BACKGROUND

CTLA4 is an inhibitory receptor expressed on a subset of T lymphocytes. Single nucleotide polymorphisms of the CTLA4 gene have been implicated in autoimmune diseases, including autoimmune hepatitis and primary biliary cirrhosis. In reverse form, CTLA4 variations are associated with chronic infections such as chronic hepatitis B.

METHODS

CTLA4 variations -318C>T and +49A>G were analyzed in 2366 patients with chronic liver disease of various etiologies, including 323 patients with chronic hepatitis B virus (HBV) infection, 1181 patients with chronic hepatitis C virus infection, 180 patients with primary biliary cirrhosis, and 127 patients with autoimmune hepatitis, as well as 202 healthy control individuals. Genotyping was performed by melting curve analysis.

RESULTS

The -318C>T variation was underrepresented in patients with chronic HBV infection compared with healthy controls (14.6 vs. 25.7%, P=0.002) and with patients with chronic liver disease of other origin (14.6 vs. 20.7%, P=0.011). Patients with cryptogenic cirrhosis also showed a lower frequency of the -318T allele than healthy controls (12.0 vs. 25.7%, P=0.014). No association of the +49G>A variation was found with any diagnosis, including autoimmune hepatitis and primary biliary cirrhosis.

CONCLUSIONS

We describe the association of the CTLA4 -318C>T variation with chronic HBV infection and cryptogenic cirrhosis but find no association of the +49G>A variation with autoimmune liver disease.

The appearance of vitiligo and spontaneous regression of the primary lesion in melanoma patients illustrate a relationship between tumor immunity and autoimmunity. T lymphocytes play a major role both in tumor immunity and autoimmunity. CD28, Cytotoxic T lymphocyte antigen 4 (CTLA4) and inducible costimulator (ICOS) molecules are important secondary signal molecules in the T lymphocyte activation. Single nucleotide polymorphisms (SNPs) in the CD28/CTLA4/ICOS gene region were reported to be associated with several autoimmune diseases including, type-1 diabetes, SLE, autoimmune thyroid diseases and celiac disease. In this study, we investigated the association of SNPs in the CD28, CTLA4 and ICOS genes with the risk of melanoma. We also assessed the prognostic effect of the different polymorphisms in melanoma patients. Twenty-four tagging SNPs across the three genes and four additional SNPs were genotyped in a cohort of 763 German melanoma patients and 734 healthy German controls. Influence on prognosis was determined in 587 melanoma cases belonging to stage I or II of the disease. In general, no differences in genotype or allele frequencies were detected between melanoma patients and controls. However, the variant alleles for two polymorphisms in the CD28 gene were differentially distributed in cases and controls. Similarly no association of any polymorphism with prognosis, except for the rs3181098 polymorphism in the CD28 gene, was observed. In addition, individuals with AA genotype for rs11571323 polymorphism in the ICOS gene showed reduced overall survival. However, keeping in view the correction for multiple hypothesis testing our results suggest that the polymorphisms in the CD28, CTLA4 and ICOS genes at least do not modulate risk of melanoma and nor do those influence the disease prognosis in the investigated population.

Coeliac disease is a common enteropathy with a strong inherited risk characterised by dietary wheat, rye and barley induced T-cell activation. Although there is replicated linkage to 2q33, results are inconsistent from association studies of the most promising candidate genes: the CD28/CTLA4/ICOS cluster. CTLA4 plays a key role in regulating T lymphocyte mediated inflammatory responses, and variants in the 3' region influence development of diabetes and thyroid disease. We genotyped CTLA4 variants (-1722 C/T, -658 T/C, -318 C/T, +49 A/G, +1822 C/T, CT60 A/G) to tag all common haplotypes (>5% frequency) and an ICOS variant (IVS+173 C/T) in 340 white UK Caucasian coeliac disease cases. Strict ascertainment criteria for coeliac cases required both villous atrophy at diagnosis and positive serology. In total, 973 healthy controls were available for SNP, and 705 for CTLA4 haplotype, based association analyses. Coeliac disease showed weak association with the CTLA4 +1822T (P=0.019) and CT60 G (P=0.047) alleles. Strong association was seen with a common CTLA4 haplotype (P=0.00067, odds ratio 1.41) of frequency 32.7% in coeliac disease and 25.5% in healthy controls. A common CTLA4 haplotype shows strong association with coeliac disease, and contains multiple alleles reported to affect immunological function. Loss of tolerance to dietary antigens in coeliac disease may be mediated in part by heritable variants in co-signalling genes regulating T-cell responses.

Treatment with fully human monoclonal antibodies against programmed death 1 (PD1) receptor has shown great promise for a number of advanced malignancies. Although inflammatory adverse events have been well described with anti-CTL antigen 4 (CTLA4) therapy, experience with the range of adverse effects of anti-PD1 remains comparatively limited. Here, we report on a patient with advanced mucosal melanoma who received four doses of MK-3475, a fully human monoclonal antibody against PD1, and experienced a durable near-complete response but developed severe hypothyroidism, rhabdomyolysis, and acute kidney injury. To our knowledge, this is the first case reported of a patient with advanced mucosal melanoma who responded to anti-PD1 therapy. With the promising antitumor effects of anti-PD1 in a wide array of tumors, we expect an increasing number of patients to be exposed to anti-PD1 therapies. Recognition of infrequent presentations of adverse events such as elevated creatine kinase levels and thyroid disorders in patients who receive anti-PD1 therapy is important.

Type 1 diabetes (T1D) is a multifactorial disease with genetic and environmental components involved. Recent studies of an animal model of T1D, the Komeda diabetes-prone rat, have demonstrated that the Casitas-B-lineage lymphoma b (cblb) gene is a major susceptibility gene in the development of diabetes and other autoimmune features of this rat. As a result of the inhibitory role of Cbl-b in T cell costimulation, dysregulation of Cbl-b may also contribute to autoimmune diseases in man. Different isoforms of Cbl-b exist; we evaluated expression levels of two known transcript variants. Constitutive expression of both isoforms was demonstrated, as well as an increased expression, after cytokine exposure, of an isoform lacking exon 16, suggesting a possible role of this variant in the pathogenesis of autoimmunity. We screened coding regions of the human CBLB gene for mutations in a panel of individuals affected with several autoimmune diseases. Eight single nucleotide polymorphisms (SNPs) were detected. One SNP in exon 12 of the CBLB gene was significantly demonstrated to be associated to T1D in a large Danish T1D family material of 480 families. Evidence for common genetic factors underlying several autoimmune diseases has come from studies of cytotoxic T lymphocyte antigen 4 (CTLA4), which encodes another negatively regulatory molecule in the immune system. Gene-gene interactions probably play substantial roles in T1D susceptibility. We performed stratification of CBLB exon 12 SNP data, according to an established CTLA4 marker, CT60, and evidence for a genetic interaction between the CTLA4 and CBLB genes, involved in the same biological pathway of T cell receptor signaling, was observed.

BACKGROUND

Chronic Chagas disease cardiomyopathy (CCC), a late consequence of Trypanosoma cruzi infection, is an inflammatory cardiomyopathy with prognosis worse than those of noninflammatory etiology (NIC). Although the T cell-rich myocarditis is known to play a pathogenetic role, the relative contribution of each of the functional T cell subsets has never been thoroughly investigated. We therefore assessed gene expression of cytokines and transcription factors involved in differentiation and effector function of each functional T cell subset (TH1/TH2/TH17/Treg) in CCC, NIC, and heart donor myocardial samples.

RESULTS

Quantitative PCR showed markedly upregulated expression of IFN-γ and transcription factor T-bet, and minor increases of GATA-3; FoxP3 and CTLA-4; IL-17 and IL-18 in CCC as compared with NIC samples. Conversely, cytokines expressed by TH2 cells (IL-4, IL-5, and IL-13) or associated with Treg (TGF-β and IL-10) were not upregulated in CCC myocardium. Expression of TH1-related genes such as T-bet, IFN-γ, and IL-18 correlated with ventricular dilation, FoxP3, and CTLA-4.

CONCLUSIONS

Results are consistent with a strong local TH1-mediated response in most samples, possibly associated with pathological myocardial remodeling, and a proportionally smaller FoxP3(+)CTLA4(+) Treg cell population, which is unable to completely curb IFN-γ production in CCC myocardium, therefore fueling inflammation.

We comprehensively screened CTLA4 for novel genetic variations in patients with MS. We studied genetic variations by association methods in a population-based sample of 122 sporadic patients with MS and 244 age-, gender- and ethnicity-matched controls, and by linkage and family-based association methods in 395 individuals from 59 American multiplex pedigrees with 141 affected individuals. Being homozygous for AT(8) (common) allele of the 3'(514) microsatellite (OR: 1.69; CI: 0.99-2.86) and for the common 5'(318)*C/E1(49)*A/3'(514*AT(8) haplotype (OR: 1.96; CI: 1.13-3.39) was associated with increased susceptibility to MS in Olmsted County. The genotype frequencies of other individual polymorphisms were not significantly different between cases and controls. A pooled analysis of association studies revealed an odds ratio of 1.28 (95% CI: 1.01-1.63; p=0.043) for 5'(-318)*C homozygotes and 1.28 (95% CI: 1.08-1.51; p=0.005) for the 3'(514)*AT(8) allele. We did not detect linkage with MS susceptibility in multiplex families. We did not find a strong association with age at onset, disease course or severity. CTLA-4 is associated with susceptibility to MS.

Accumulated evidence suggested that cytotoxic T-lymphocyte antigen 4 (CTLA4) plays an important role in the negative regulation of T-cell proliferation and activation, and thus participates in antitumor immunity and cancer surveillance. Previously we reported that the CTLA4 49A/G (rs231775) single nucleotide polymorphism (SNP) was a candidate cancer susceptibility marker for breast, lung, esophageal, and gastric cancers. In the present study, we expanded our study to two infection-related cancers, namely, hepatocellular carcinoma (HCC) and cervical cancer. We genotyped rs231775 in two independent case-control studies of 864 HCC patients and 864 control subjects, and 719 cervical cancer patients and 719 control subjects. In the multivariate logistic regression models, CTLA4 +49 A/G variant genotype was associated with increased risk (AA vs GG) by 1.43-fold (95% CI = 0.94-2.17) for HCC, and 1.66-fold (95% CI = 1.13-2.44) for cervical cancer. Taken together, the results suggest that CTLA4 rs231775 may serve as a common cancer susceptibility marker.

BACKGROUND

Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T(reg)) cells may contribute to the immunosuppressive effects of glucocorticoids (GCs).

OBJECTIVE

We readdressed the influence of GC therapy on T(reg) cells in immunocompetent human subjects and naïve mice.

METHODS

Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T(reg) cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T(reg) cells were analyzed prior and after a 14 day GC therapy based on different markers.

RESULTS

Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T(reg) cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10(4) cells/ml vs. 33±11×10(4) in control mice) and spleen (dexamethasone: 2.8±1.9×10(5)/spleen vs. 95±22×10(5)/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3(+) T(reg) cells amongst the CD4(+) T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T(reg) cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T(reg) cells in a relevant manner, although there was some variation depending on the definition of the T(reg) cells (FOXP3(+): 4.0±1.5% vs 3.4±1.5%*; AITR(+): 0.6±0.4 vs 0.5±0.3%, CD127(low): 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05).

CONCLUSIONS

Short-term GC therapy does not induce the hitherto supposed increase in circulating T(reg) cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T(reg) cell numbers.

Triple-negative breast cancer (TNBC) has been subdivided into six distinct subgroups: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR). We recently identified a subgroup of TNBC with loss of the tumor suppressor PTEN and five specific microRNAs that exhibits exceedingly poor clinical outcome and contains TP53 mutation, RB1 loss and high MYC and WNT signalling. Here, show that these PTEN-low/miRNA-low lesions cluster with BL1 TNBC. These tumors exhibited high RhoA signalling and were significantly stratified on the basis of PTEN-low/RhoA-signalling-high with hazard ratios (HRs) of 8.2 (P = 0.0009) and 4.87 (P = 0.033) in training and test cohorts, respectively. For BL2 TNBC, we identified AKT1 copy gain/high mRNA expression as surrogate for poor prognosis (HR = 3.9; P = 0.02 and HR = 6.1; P = 0.0032). In IM, programmed cell death 1 (PD1) was elevated and predictive of poor prognosis (HR = 5.3; P = 0.01 and HR = 3.5; P < 0.004). Additional alterations, albeit without prognostic power, characterized each subtype including high E2F2 and TGFβ signalling and CXCL8 expression in BL2, high IFNα and IFNγ signalling and CTLA4 expression in IM, and high EGFR signalling in MSL, and may be targeted for therapy. This study identified PTEN-low/RhoA-signalling-high, and high AKT1 and PD1 expression as potent prognostications for BL1, BL2 and IM subtypes with survival differences of over 14, 2.75 and 10.5 years, respectively. This intrinsic heterogeneity could be exploited to prioritize patients for precision medicine.

Although cancer cells can be immunogenic, tumour progression is associated with the evasion of immunosurveillance, the promotion of tumour tolerance and even the production of pro-tumorigenic factors by immune cells. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) represents a crucial immune checkpoint, the blockade of which can potentiate anti-tumour immunity. CTLA4-blocking antibodies are now an established therapeutic approach for malignant melanoma, and clinical trials with CTLA4-specific antibodies in prostate cancer have also shown clinical activity. This treatment may provide insights into the targets that the immune system recognizes to drive tumour regression, and could potentially improve both outcome and toxicity for patients with prostate cancer.

OBJECTIVE

We studied differences between patients with latent autoimmune diabetes in adults (LADA), type 2 diabetes, and classical type 1 diabetes diagnosed after age 35 years.

METHODS

Polymorphisms in HLA-DQB1, INS, PTPN22, and CTLA4 were genotyped in patients with LADA (n = 213), type 1 diabetes diagnosed at >35 years of age (T1D(>35y); n = 257) or <20 years of age (T1D(<20y); n = 158), and type 2 diabetes.

RESULTS

Although patients with LADA had an increased frequency of HLA-DQB1 and PTPN22 risk genotypes and alleles compared with type 2 diabetic subjects, the frequency was significantly lower compared with T1D(>35y) patients. Genotype frequencies, measures of insulin secretion, and metabolic traits within LADA differed according to GAD antibody (GADA) quartiles, but even the highest quartile differed from type 1 diabetes. Having two or more risk genotypes was associated with lower C-peptide concentrations in LADA.

CONCLUSIONS

LADA patients differed genetically and phenotypically from both T1D(>35y) and type 2 diabetic patients in a manner dependent on GADA levels.

BACKGROUND

Immune system affects prognosis of various malignancies. Anti-immune pathways like PD-L1 and CTLA4 are used by the tumor to overcome immune system and they serve as immunotherapy targets. The immune microenvironment of head-and-neck squamous cell carcinoma (SCCHN) has not been sufficiently studied.

METHODS

152 SCCHN were immunohistochemically studied for the expression of CD3, CD8, CD57, CD4, granzyme b, CD20, CD163, S100, PD-L1, CTLA4 and CXCR4.

RESULTS

CD3, CD8, CD57 and stromal S100 higher density is a good prognostic factor (p=0.02, 0.01, 0.02, 0.03 respectively). CTLA4 tumor expression is a poor prognostic factor (p=0.05). The rest immune cells do not affect prognosis. CD3 and CD8 density does not correlate with clinicopathological factors or p16/p53 expression, while CD57 and CD4 higher density is associated with the absence of distant metastases (p=0.03 and 0.07, respectively). Higher CD20 and S100 density is associated with lower T stage (p=0.04 and 0.03, respectively). PD-L1 expression is higher in CD3, CD8, and CD163 infiltrated tumors and in histologically more aggressive tumors. Response to neoadjuvant chemotherapy is better in highly CD3 infiltrated tumors and in tumors with less intraepithelial macrophages.

CONCLUSIONS

Rich T-lympocytic and dendritic cell response is a good prognostic factor in SCCHN, whereas tumors expressing CTLA4 show poor prognosis. PDL1 expression does not affect prognosis, but it is expressed in histologically more aggressive tumors and in T-cells rich tumors. Response to induction chemotherapy is better in tumors less infiltrated by macrophages and mostly infiltrated by T cells.

Tolerizing mechanisms within the host and tumor microenvironment inhibit T-cell effector functions that can control cancer. These mechanisms blunt adoptive immunotherapy with infused T-cells due to a complex array of signals that determine T-cell tolerance, survival, or deletion. Ligation of the negative regulatory receptors CTLA4, PD-1(PDCD1), or LAG3 on T-cells normally hinders their response to antigen through nonredundant biochemical processes that interfere with stimulatory pathways. In this study, we used an established mouse model of T-cell tolerance to define the roles of these inhibitory receptors in regulating CD8(+) T-cell tolerance during adoptive immunotherapy to treat leukemia. Blocking CTLA4 and PD-1 in vivo combined to promote survival of transferred T-cells despite powerful deletional signals that mediate Bim (BCL2L11)-dependent apoptosis. However, this dual blockade was not optimal for stimulating effector function by responding T-cells, which required the additional blockade of LAG3 to induce full expansion and allow the acquisition of robust cytolytic activity. Thus, the cooperation of multiple distinct regulatory pathways was needed for the survival and effector differentiation of adoptively transferred tumor-reactive CD8(+) T-cells. Our work defines the immune escape pathways in which simultaneous blockade could yield durable immunotherapeutic responses that can eradicate disseminated leukemia.

The mechanism of idiosyncratic drug-induced liver injury (IDILI) remains poorly understood, to a large degree because of the lack of a valid animal model. Recently, we reported an animal model in which treatment of female C57BL/6 mice with amodiaquine (AQ) resulted in mild liver injury with a delayed onset and resolution despite continued treatment. Such adaptation is a common outcome in the IDILI caused by drugs that can cause liver failure. We had hypothesized that most IDILI is immune-mediated and adaptation represents immune tolerance. In this study we found that AQ treatment of Cbl-b(-/-) and PD-1(-/-) mice, which have impaired immune tolerance, resulted in a slightly greater injury. Cotreatment of C57BL/6 with AQ and anti-CTLA4 also resulted in a greater increase in ALT than treatment with AQ alone; however, these mice also had an increase in T regulatory (Treg) cells and T helper cells expressing PD-1 and CTLA4. The increase in these cells implies the induction of immune tolerance, and the alanine aminotransferase (ALT) activity in these mice returned to normal despite continued treatment. Cotreatment of PD-1(-/-) mice with anti-CTLA4 antibody and AQ resulted in the greatest increase in ALT (200-300 U/L), and necroinflammatory responses characterized by portal infiltration of lymphocytes with interface hepatitis. The lymphocyte infiltration included T and B cells, and the CD8(+) T cells produced perforin and granzyme. In addition, the ALT activity in PD-1(-/-) mice cotreated with anti-CTLA4 antibody and AQ did not return to normal, as it had in other mice.

CONCLUSIONS

We report here the first animal model of IDILI that is similar to the IDILI that occurs in humans, and it was accomplished by inhibiting immune tolerance.

BACKGROUND

To investigate in the NeoSphere trial the contribution of the immune system to pathologic complete response in the breast (pCRB) after neoadjuvant docetaxel with trastuzumab (TH), pertuzumab (TP), or both (THP), or monoclonal antibodies alone (HP).

METHODS

Immune gene mRNA expression (n = 350, 83.8%), lymphocyte infiltration (TIL, n = 243, 58.3%), and PDL1 by immunohistochemistry (n = 305, 73.1%) were correlated with pCRB. We studied five selected genes (IFNG, PD1, PDL1, PDL2, CTLA4) and six immune metagenes corresponding to plasma cells (IGG), T cells (CD8A), antigen-presenting cells (MHC2), and to MHC1 genes (MHC1), STAT1 co-expressed genes (STAT1), and interferon-inducible genes (IF-I). Gene expression data from the NOAH trial were used for validation.

RESULTS

TIL as continuous variable and PDL1 protein expression were not significantly associated with pCRB. Expression of immune genes/metagenes had different association with pCRB after THP than after other therapies. With THP, higher expression of PD1 and STAT1, or any among PDL1, CTLA4, MHC1, and IF-I were linked with lower pCRB. In the combined TH/TP/HP treatment group, in multivariate analysis, higher expression of PD1, MHC2, and STAT1 were linked with pCRB, and higher PDL1, MHC1, or IF-I to lower pCRB. In the NOAH, a similar association of higher STAT1 with higher pCRB, and higher MHC1 and IF-I with lower pCRB was found for trastuzumab/chemotherapy but not for chemotherapy treatment only.

CONCLUSIONS

The immune system modulates response to therapies containing trastuzumab and pertuzumab. Greatest benefit from THP is observed for low expression of some immune markers (i.e. MHC1, CTLA4). The involvement of PDL1 in resistance supports testing combinations of HER2-directed antibodies and immune-checkpoint inhibitors.

OBJECTIVE

The precipitating event(s) that triggers Wegener's granulomatosis (WG) is unknown. Cytokines, costimulatory molecules, and counterregulatory molecules control the quality and intensity of immune responses. Thus, they are relevant candidates for genetic studies of immune dysregulation in WG, the pathogenesis of which may be facilitated by multiple acquired and/or inherited factors. This study was undertaken to investigate possible genetic associations of various proinflammatory cytokines and CTLA-4, a receptor for T cell inhibition, with WG.

METHODS

Using polymerase chain reaction-based DNA genotyping, we investigated the polymorphisms located in the genes encoding a variety of proinflammatory cytokines and CTLA-4 in 117 American patients with WG and 123 ethnically matched healthy controls.

RESULTS

Compared with controls, patients with WG had a significantly lower frequency of homozygosity for the shortest allele (designated allele 86) of the Ctla4 microsatellite polymorphism (AT)n located in the 3'-untranslated region (3'-UTR) of exon 3 (47.0% versus 69.9%; P = 0.0005). Significant differences between patients and controls in the allelic and genotypic frequencies of polymorphisms in the other cytokine and cytokine receptor genes studied (tumor necrosis factor alpha [TNFalpha], TNF receptor I [TNFRI], TNFRII, interleukin-1beta [IL-1beta], IL-6) were not found.

CONCLUSIONS

The Ctla4 (AT)n 86 allele has been previously demonstrated to be crucial for maintenance of normal levels of CTLA-4 expression and balance between T cell activation and inhibition. Our results in American patients confirm findings from a Scandinavian cohort in which a positive association between WG and longer alleles of (AT)n in the Ctla4 3'-UTR was demonstrated. Diminished frequencies of the most effective allele for CTLA-4 expression may represent a WG-related susceptibility mutation that accounts, in part, for increased T cell activation and clonal expansion in WG. Blockade of T cell costimulation using CTLA-4Ig might be a useful therapeutic intervention, providing an alternative or complementary approach to conventional treatment with immunosuppressive agents.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.

The immune status of the tumor microenvironment is a key indicator in determining the antitumor effectiveness of immunotherapies. Data support the role of activation and expansion of tumor-infiltrating lymphocytes (TILs) in increasing the benefit of immunotherapies in patients with solid tumors. We found that intratumoral injection of a tumor-selective oncolytic vaccinia virus encoding interleukin-7 (IL-7) and IL-12 into tumor-bearing immunocompetent mice activated the inflammatory immune status of previously poorly immunogenic tumors and resulted in complete tumor regression, even in distant tumor deposits. Mice achieving complete tumor regression resisted rechallenge with the same tumor cells, suggesting establishment of long-term tumor-specific immune memory. Combining this virotherapy with anti-programmed cell death-1 (PD-1) or anti-cytotoxic T lymphocyte antigen 4 (CTLA4) antibody further increased the antitumor activity as compared to virotherapy alone, in tumor models unresponsive to either of the checkpoint inhibitor monotherapies. These findings suggest that administration of an oncolytic vaccinia virus carrying genes encoding for IL-7 and IL-12 has antitumor activity in both directly injected and distant noninjected tumors through immune status changes rendering tumors sensitive to immune checkpoint blockade. The benefit of intratumoral IL-7 and IL-12 expression was also observed in humanized mice bearing human cancer cells. These data support further investigation in patients with non-inflamed solid tumors.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.

BACKGROUND

Manipulation of immune checkpoints such as CTLA4 or PD-1 with targeted antibodies has recently emerged as an effective anticancer strategy in multiple malignancies. Sarcomas are a heterogeneous group of diseases in need of more effective treatments. Different subtypes of soft tissue and bone sarcomas have been shown to express PD-1 ligand.

METHODS

We retrospectively analyzed a cohort of patients (pts) with relapsed metastatic/unresectable sarcomas, who were treated with nivolumab provided under a patient assistance program from the manufacturer. Pts underwent CT or PET/CT imaging at baseline and after at least four doses of nivolumab; RECIST 1.1 criteria were used for response assessment.

RESULTS

Twenty-eight pts with soft tissue (STS, N = 24) or bone sarcoma (N = 4), received IV nivolumab 3 mg/kg every 2 weeks from July 2015. Median age was 57 (24-78), male:female ratio was 14:14; the median number of nivolumab cycles was eight. Eighteen pts concomitantly received pazopanib at 400-800 mg daily. The most common side effect was grade 1-2 LFT elevations; grade 3-4 toxicity occurred in five patients (colitis, LFT elevations, pneumonitis). Twenty-four pts received at least four cycles. We observed three partial responses: one dedifferentiated chondrosarcoma, one epithelioid sarcoma and one maxillary osteosarcoma (last two patients on pazopanib); nine patients had stable disease including three leiomyosarcomas; 12 patients had progression of disease including 4 leiomyosarcoma. Clinical benefit (response + stability) was observed in 50% of the evaluable patients.

CONCLUSIONS

These data provide a rationale for further exploring the efficacy of nivolumab and other checkpoint inhibitors in soft tissue and bone sarcoma.