High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity.

OBJECTIVE: A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC. DESIGN: Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence. RESULTS: We discovered that a previously unappreciated innate lymphocyte population (Lin-CD7+CD127-CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes. CONCLUSION: This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

Revisiting immune escape in colorectal cancer in the era of immunotherapy.

In colorectal cancer (CRC), T-cell checkpoint blockade is only effective in patients diagnosed with mismatch repair-deficient (MMR-d) cancers. However, defects in Human Leukocyte Antigen (HLA) class I expression were reported to occur in most MMR-d CRCs, which would preclude antigen presentation in these tumours, considered essential for the clinical activity of this immunotherapeutic modality. We revisited this paradox by characterising HLA class I expression in two independent cohorts of CRC. We determined that loss of HLA class I expression occurred in the majority (73-78%) of MMR-d cases. This phenotype was rare in CRC liver metastases, irrespective of MMR status, whereas weak, inducible expression of HLA class I molecules was frequent in liver lesions. We propose that HLA class I is an important determinant of metastatic homing in CRCs. This observation is paramount to understand CRC carcinogenesis and for the application of immunotherapies in the metastatic setting.

Genome-wide association meta-analysis of human longevity identifies a novel locus conferring survival beyond 90 years of age.

The genetic contribution to the variation in human lifespan is ∼ 25%. Despite the large number of identified disease-susceptibility loci, it is not known which loci influence population mortality. We performed a genome-wide association meta-analysis of 7729 long-lived individuals of European descent (≥ 85 years) and 16 121 younger controls (<65 years) followed by replication in an additional set of 13 060 long-lived individuals and 61 156 controls. In addition, we performed a subset analysis in cases aged ≥ 90 years. We observed genome-wide significant association with longevity, as reflected by survival to ages beyond 90 years, at a novel locus, rs2149954, on chromosome 5q33.3 (OR = 1.10, P = 1.74 × 10(-8)). We also confirmed association of rs4420638 on chromosome 19q13.32 (OR = 0.72, P = 3.40 × 10(-36)), representing the TOMM40/APOE/APOC1 locus. In a prospective meta-analysis (n = 34 103), the minor allele of rs2149954 (T) on chromosome 5q33.3 associates with increased survival (HR = 0.95, P = 0.003). This allele has previously been reported to associate with low blood pressure in middle age. Interestingly, the minor allele (T) associates with decreased cardiovascular mortality risk, independent of blood pressure. We report on the first GWAS-identified longevity locus on chromosome 5q33.3 influencing survival in the general European population. The minor allele of this locus associates with low blood pressure in middle age, although the contribution of this allele to survival may be less dependent on blood pressure. Hence, the pleiotropic mechanisms by which this intragenic variation contributes to lifespan regulation have to be elucidated.

The effect of forced exercise on knee joints in Dio2(-/-) mice: type II iodothyronine deiodinase-deficient mice are less prone to develop OA-like cartilage damage upon excessive mechanical stress.

OBJECTIVE: To further explore deiodinase iodothyronine type 2 (DIO2) as a therapeutic target in osteoarthritis (OA) by studying the effects of forced mechanical loading on in vivo joint cartilage tissue homeostasis and the modulating effect herein of Dio2 deficiency. METHODS: Wild-type and C57BL/6-Dio2(-/-) -mice were subjected to a forced running regime for 1 h per day for 3 weeks. Severity of OA was assessed by histological scoring for cartilage damage and synovitis. Genome-wide gene expression was determined in knee cartilage by microarray analysis (Illumina MouseWG-6 v2). STRING-db analyses were applied to determine enrichment for specific pathways and to visualise protein-protein interactions. RESULTS: In total, 158 probes representing 147 unique genes showed significantly differential expression with a fold-change ≥1.5 upon forced exercise. Among these are genes known for their association with OA (eg, Mef2c, Egfr, Ctgf, Prg4 and Ctnnb1), supporting the use of forced running as an OA model in mice. Dio2-deficient mice showed significantly less cartilage damage and signs of synovitis. Gene expression response upon exercise between wild-type and knockout mice was significantly different for 29 genes. CONCLUSIONS: Mice subjected to a running regime have significant increased cartilage damage and synovitis scores. Lack of Dio2 protected against cartilage damage in this model and was reflected in a specific gene expression profile, and either mark a favourable effect in the Dio2 knockout (eg, Gnas) or an unfavourable effect in wild-type cartilage homeostasis (eg, Hmbg2 and Calr). These data further support DIO2 activity as a therapeutic target in OA.

A gain of function mutation in TNFRSF11B encoding osteoprotegerin causes osteoarthritis with chondrocalcinosis.

OBJECTIVE: To identify pathogenic mutations that reveal underlying biological mechanisms driving osteoarthritis (OA). METHODS: Exome sequencing was applied to two distant family members with dominantly inherited early onset primary OA at multiple joint sites with chondrocalcinosis (familial generalised osteoarthritis, FOA). Confirmation of mutations occurred by genotyping and linkage analyses across the extended family. The functional effect of the mutation was investigated by means of a cell-based assay. To explore generalisability, mRNA expression analysis of the relevant genes in the discovered pathway was explored in preserved and osteoarthritic articular cartilage of independent patients undergoing joint replacement surgery. RESULTS: We identified a heterozygous, probably damaging, read-through mutation (c.1205A=>T; p.Stop402Leu) in TNFRSF11B encoding osteoprotegerin that is likely causal to the OA phenotype in the extended family. In a bone resorption assay, the mutant form of osteoprotegerin showed enhanced capacity to inhibit osteoclastogenesis and bone resorption. Expression analyses in preserved and affected articular cartilage of independent OA patients showed that upregulation of TNFRSF11B is a general phenomenon in the pathophysiological process. CONCLUSIONS: Albeit that the role of the molecular pathway of osteoprotegerin has been studied in OA, we are the first to demonstrate that enhanced osteoprotegerin function could be a directly underlying cause. We advocate that agents counteracting the function of osteoprotegerin could comply with new therapeutic interventions of OA.

Underlying molecular mechanisms of DIO2 susceptibility in symptomatic osteoarthritis.

OBJECTIVES: To investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches. METHODS: Epigenetically regulated expression of DIO2 was explored by assessing methylation of positional CpG-dinucleotides and the respective DIO2 expression in OA-affected and macroscopically preserved articular cartilage from end-stage OA patients. In a human in vitro chondrogenesis model, we measured the effects when thyroid signalling during culturing was either enhanced (excess T3 or lentiviral induced DIO2 overexpression) or decreased (iopanoic acid). RESULTS: OA-related changes in methylation at a specific CpG dinucleotide upstream of DIO2 caused significant upregulation of its expression (ß=4.96; p=0.0016). This effect was enhanced and appeared driven specifically by DIO2 rs225014 risk allele carriers (ß=5.58, p=0.0006). During in vitro chondrogenesis, DIO2 overexpression resulted in a significant reduced capacity of chondrocytes to deposit extracellular matrix (ECM) components, concurrent with significant induction of ECM degrading enzymes (ADAMTS5, MMP13) and markers of mineralisation (ALPL, COL1A1). Given their concurrent and significant upregulation of expression, this process is likely mediated via HIF-2α/RUNX2 signalling. In contrast, we showed that inhibiting deiodinases during in vitro chondrogenesis contributed to prolonged cartilage homeostasis as reflected by significant increased deposition of ECM components and attenuated upregulation of matrix degrading enzymes. CONCLUSIONS: Our findings show how genetic variation at DIO2 could confer risk to OA and raised the possibility that counteracting thyroid signalling may be a novel therapeutic approach.

IL7R gene expression network associates with human healthy ageing.

BACKGROUND: The level of expression of the interleukin 7 receptor (IL7R) gene in blood has recently been found to be associated with familial longevity and healthy ageing. IL7R is crucial for T cell development and important for immune competence. To further investigate the IL7R pathway in ageing, we identified the closest interacting genes to construct an IL7R gene network that consisted of IL7R and six interacting genes: IL2RG, IL7, TSLP, CRLF2, JAK1 and JAK3. This network was explored for association with chronological age, familial longevity and immune-related diseases (type 2 diabetes, chronic obstructive pulmonary disease and rheumatoid arthritis) in 87 nonagenarians, 337 of their middle-aged offspring and 321 middle-aged controls from the Leiden Longevity Study (LLS). RESULTS: We observed that expression levels within the IL7R gene network were significantly different between the nonagenarians and middle-aged controls (P = 4.6 × 10(-4)), being driven by significantly lower levels of expression in the elderly of IL7, IL2RG and IL7R. After adjustment for multiple testing and white blood cell composition and in comparison with similarly aged controls, middle-aged offspring of nonagenarian siblings exhibit a lower expression level of IL7R only (P = 0.006). Higher IL7R gene expression in the combined group of middle-aged offspring and controls is associated with a higher prevalence of immune-related disease (P = 0.001). On the one hand, our results indicate that lower IL7R expression levels, as exhibited by the members of long-lived families that can be considered as 'healthy agers', are beneficial in middle age. This is augmented by the observation that higher IL7R gene expression associates with immune-related disease. On the other hand, IL7R gene expression in blood is lower in older individuals, indicating that low IL7R gene expression might associate with reduced health. Interestingly, this contradictory result is supported by the observation that a higher IL7R gene expression level is associated with better prospective survival, both in the nonagenarians (Hazard ratio (HR) = 0.63, P = 0.037) and the middle-aged individuals (HR = 0.33, P = 1.9 × 10(-4)). CONCLUSIONS: Overall, we conclude that the IL7R network reflected by gene expression levels in blood may be involved in the rate of ageing and health status of elderly individuals.

Multimodal profiling of chordoma immunity reveals distinct immune contextures.

BACKGROUND: Chordomas are rare cancers from the axial skeleton which present a challenging clinical management with limited treatment options due to their anatomical location. In recent years, a few clinical trials demonstrated that chordomas can respond to immunotherapy. However, an in-depth portrayal of chordoma immunity and its association with clinical parameters is still lacking. METHODS: We present a comprehensive characterization of immunological features of 76 chordomas through application of a multimodal approach. Transcriptomic profiling of 20 chordomas was performed to inform on the activity of immune-related genes through the immunologic constant of rejection (ICR) signature. Multidimensional immunophenotyping through imaging mass cytometry was applied to provide insights in the different immune contextures of 32 chordomas. T cell infiltration was further evaluated in all 76 patients by means of multispectral immunofluorescence and then associated with clinical parameters through univariate and multivariate Cox proportional hazard models as well as Kaplan-Meier estimates. Moreover, distinct expression patterns of human leukocyte antigen (HLA) class I were assessed by immunohistochemical staining in all 76 patients. Finally, clonal enrichment of the T cell receptor (TCR) was sought through profiling of the variable region of TCRB locus of 24 patients. RESULTS: Chordomas generally presented an immune "hot" microenvironment in comparison to other sarcomas, as indicated by the ICR transcriptional signature. We identified two distinct groups of chordomas based on T cell infiltration which were independent from clinical parameters. The highly infiltrated group was further characterized by high dendritic cell infiltration and the presence of multicellular immune aggregates in tumors, whereas low T cell infiltration was associated with lower overall cell densities of immune and stromal cells. Interestingly, patients with higher T cell infiltration displayed a more pronounced clonal enrichment of the TCR repertoire compared with those with low T cell counts. Furthermore, we observed that the majority of chordomas maintained HLA class I expression. CONCLUSION: Our findings shed light on the natural immunity against chordomas through the identification of distinct immune contextures. Understanding their immune landscape could guide the development and application of immunotherapies in a tailored manner, ultimately leading to an improved clinical outcome for patients with chordoma.

Aging as accelerated accumulation of somatic variants: whole-genome sequencing of centenarian and middle-aged monozygotic twin pairs.

It has been postulated that aging is the consequence of an accelerated accumulation of somatic DNA mutations and that subsequent errors in the primary structure of proteins ultimately reach levels sufficient to affect organismal functions. The technical limitations of detecting somatic changes and the lack of insight about the minimum level of erroneous proteins to cause an error catastrophe hampered any firm conclusions on these theories. In this study, we sequenced the whole genome of DNA in whole blood of two pairs of monozygotic (MZ) twins, 40 and 100 years old, by two independent next-generation sequencing (NGS) platforms (Illumina and Complete Genomics). Potentially discordant single-base substitutions supported by both platforms were validated extensively by Sanger, Roche 454, and Ion Torrent sequencing. We demonstrate that the genomes of the two twin pairs are germ-line identical between co-twins, and that the genomes of the 100-year-old MZ twins are discerned by eight confirmed somatic single-base substitutions, five of which are within introns. Putative somatic variation between the 40-year-old twins was not confirmed in the validation phase. We conclude from this systematic effort that by using two independent NGS platforms, somatic single nucleotide substitutions can be detected, and that a century of life did not result in a large number of detectable somatic mutations in blood. The low number of somatic variants observed by using two NGS platforms might provide a framework for detecting disease-related somatic variants in phenotypically discordant MZ twins.

Genome-wide association study (GWAS)-identified disease risk alleles do not compromise human longevity.

A set of currently known alleles increasing the risk for coronary artery disease, cancer, and type 2 diabetes as identified by genome-wide association studies was tested for compatibility with human longevity. Here, we show that nonagenarian siblings from long-lived families and singletons older than 85 y of age from the general population carry the same number of disease risk alleles as young controls. Longevity in this study population is not compromised by the cumulative effect of this set of risk alleles for common disease.

CD103 and CD39 coexpression identifies neoantigen-specific cytotoxic T cells in colorectal cancers with low mutation burden.

BACKGROUND: Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden. EXPERIMENTAL DESIGN: Whole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+ T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+ cytotoxic T cells in tumors. RESULTS: Neoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+ (double positive, DP) CD8+ T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+ T cells could be attributed to CD4+ T cells. CD8+ T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression. CONCLUSION: Coexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+ T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients.

Neoantigen Targetability in Progressive Advanced Melanoma.

PURPOSE: The availability of (neo)antigens and the infiltration of tumors by (neo)antigen-specific T cells are crucial factors in cancer immunotherapy. In this study, we aimed to investigate the targetability of (neo)antigens in advanced progessive melanoma and explore the potential for continued T-cell-based immunotherapy. EXPERIMENTAL DESIGN: We examined a cohort of eight patients with melanoma who had sequential metastases resected at early and later time points. Antigen-presenting capacity was assessed using IHC and flow cytometry. T-cell infiltration was quantified through multiplex immunofluorescence. Whole-exome and RNA sequencing were conducted to identify neoantigens and assess the expression of neoantigens and tumor-associated antigens. Mass spectrometry was used to evaluate antigen presentation. Tumor recognition by autologous T cells was assessed by coculture assays with cell lines derived from the metastatic lesions. RESULTS: We observed similar T-cell infiltration in paired early and later metastatic (LM) lesions. Although elements of the antigen-presenting machinery were affected in some LM lesions, both the early and later metastasis-derived cell lines were recognized by autologous T cells. At the genomic level, the (neo)antigen landscape was dynamic, but the (neo)antigen load was stable between paired lesions. CONCLUSIONS: Our findings indicate that subsequently isolated tumors from patients with late-stage melanoma retain sufficient antigen-presenting capacity, T-cell infiltration, and a stable (neo)antigen load, allowing recognition of tumor cells by T cells. This indicates a continuous availability of T-cell targets in metastases occurring at different time points and supports further exploration of (neo)antigen-specific T-cell-based therapeutic approaches for advanced melanoma.

Increased type II deiodinase protein in OA-affected cartilage and allelic imbalance of OA risk polymorphism rs225014 at DIO2 in human OA joint tissues.

OBJECTIVE: Genetic variation at the type II deiodinase (D2) gene (DIO2) was previously identified as osteoarthritis (OA) risk factor. To investigate mechanisms possibly underlying this association, we assessed D2 protein in healthy and OA-affected cartilage and investigated allelic balance of the OA risk polymorphism rs225014 at DIO2 in human OA joints. METHODS: Immunohistochemical staining of healthy and OA-affected cartilage was performed for D2. We then assessed allelic balance of DIO2 mRNA within OA-affected cartilage both at and away from the lesion, ligaments and subchondral bone. Allelic balance was measured by the amount of alleles 'C' and 'T' of the intragenic OA risk polymorphism rs225014 in heterozygous carriers. RESULTS: A markedly higher amount of D2 positive cells and staining intensity was observed in OA cartilage. A significant, 1.3-fold higher presence was observed for the OA-associated rs225014 'C' allele relative to the 'T' allele of DIO2, which was significant in 28 of 31 donors. CONCLUSION: In OA cartilage, D2 protein presence is increased. The allelic imbalance of the DIO2 mRNA transcript, with the OA risk allele 'C' of rs225014 more abundant than the wild-type 'T' allele in heterozygote carriers provides a possible mechanism by which genetic variation at DIO2 confers OA risk.

Large replication study and meta-analyses of DVWA as an osteoarthritis susceptibility locus in European and Asian populations.

Recently, through a genome wide association study in Japanese knee osteoarthritis (OA) cases, a previously unknown gene, DVWA, was identified. The non-synonymous single nucleotide polymorphism (SNP) rs7639618 was subsequently found to be consistent and most significantly associated in Japanese and Han Chinese knee OA studies and functional relevant. Here, the association of the DVWA polymorphisms (rs7639618, rs11718863 and rs9864422) was genotyped in 1120 knee OA cases, 1482 hip OA cases and 2147 controls, all of white European descent from the Netherlands, the UK, Spain and Greece. Random effect DerSimonian and Laird meta-analyses were performed to assess the association in the different strata. To assess a more global effect, the original Japanese and Chinese data were included with the European. The meta-analyses provided evidence for global association of rs7639618 with knee OA with an odds ratio (OR) of 1.29, 95% confidence interval (CI) of 1.15-1.45 and a P-value of 2.70 x 10(-5). This effect, however, showed moderate heterogeneity, and rs7639618 was not independently associated with knee OA in Europeans, with an OR of 1.16, 95% CI of 0.99-1.35 and a P-value of 0.063. Furthermore, no association was observed with hip OA in Europeans, with a P-value of 0.851. Our results suggest that there may be global relevance for the DVWA SNP rs7639618 among knee OA cases, however, the apparent lower effect size in combination with the higher risk allele frequency in the European samples highlights again the ethnic differences in effects of discovered OA susceptibility genes.

Meta-analyses of genes modulating intracellular T3 bio-availability reveal a possible role for the DIO3 gene in osteoarthritis susceptibility.

OBJECTIVE: To study whether common genetic variants of the genes involved in the complex regulatory mechanism determining the intracellular bio-availability of T3 influence osteoarthritis onset. METHODS: In total 17 genetic variants within the genes encoding WD40-repeat/SOCS-box protein 1, ubiquitin specific protease 33, thyroid hormone receptor α, deiodinase, iodothyronine, type III (DIO3) and Indian hedgehog were measured and associated with osteoarthritis in a meta-analyses in European populations from the UK, The Netherlands, Greece and Spain containing a total of 3252 osteoarthritis cases and 2132 controls. RESULTS: The minor allele of the DIO3 variant rs945006 showed suggestive evidence for protective association in the overall meta-analyses, which was supported by individual osteoarthritis studies and osteoarthritis subtypes. The association appeared most significant in cases with knee and/or hip with an allelic OR of 0.81 (95% CI 0.70 to 0.930) with a nominal p value of 0.004 and a permutation-based corrected p value for multiple testing of 0.039. CONCLUSION: The findings suggest that the DIO3 gene modulates osteoarthritis disease risk; however, additional studies are necessary to replicate our findings. To elucidate the molecular mechanisms focus should be on the local adaptation to T3 availability either during the endochondral ossification process or during ageing of the articular cartilage.

A meta-analysis of European and Asian cohorts reveals a global role of a functional SNP in the 5' UTR of GDF5 with osteoarthritis susceptibility.

We have performed a meta-analysis combining data for more than 11,000 individuals. It provides compelling evidence for a positive association between a functional single-nucleotide polymorphism (SNP) in the 5'-UTR of GDF5 (+104T/C; rs143383) and osteoarthritis (OA) in European and Asian populations. This SNP has recently been reported to be associated with OA in Japanese and Han Chinese populations. Attempts to replicate this association in European samples have been inconclusive, as no association was found in the case-control cohorts from the UK, Spain and Greece when studied individually. However, the pooled data of UK and Spain found an association of the T-allele with an odds ratio (OR) of 1.10. Although the European studies had adequate power to replicate the original findings from the Japanese cohort (OR = 1.79), these results suggest that the role of the GDF5 polymorphism may not be as strong in Europeans. To clarify whether the European studies were hampered by insufficient power, we combined new data from the UK and the Netherlands with the three published studies of Europe and Asia. The results provide strong evidence of a positive association of the GDF5 SNP with knee OA for Europeans as well as for Asians. The combined association for both ethnic groups is highly significant for the allele frequency model (P = 0.0004, OR = 1.21) and the dominant model (P < 0.0001, OR = 1.48). These findings represent the first highly significant evidence for a risk factor for the development of OA which affects two highly diverse ethnic groups.

Identification of DIO2 as a new susceptibility locus for symptomatic osteoarthritis.

Osteoarthritis [MIM 165720] is a common late-onset articular joint disease for which no pharmaceutical intervention is available to attenuate the cartilage degeneration. To identify a new osteoarthritis susceptibility locus, a genome-wide linkage scan and combined linkage association analysis were applied to 179 affected siblings and four trios with generalized osteoarthritis (The GARP study). We tested, for confirmation by association, 1478 subjects who required joint replacement and 734 controls in a UK population. Additional replication was tested in 1582 population-based females from the Rotterdam study that contained 94 cases with defined hip osteoarthritis and in 267 Japanese females with symptomatic hip osteoarthritis and 465 controls. Suggested evidence for linkage in the GARP study was observed on chromosome 14q32.11 (log of odds = 3.03, P = 1.9 x 10(-4)). Genotyping tagging single-nucleotide polymorphisms covering three important candidate genes revealed a common coding variant (rs225014; Thr92Ala) in the iodothyronine-deiodinase enzyme type 2 (D2) gene (DIO2 [MIM 601413]) which significantly explained the linkage signal (P = 0.006). Confirmation and replication by association in the additional osteoarthritis studies indicated a common DIO2 haplotype, exclusively containing the minor allele of rs225014 and common allele of rs12885300, with a combined recessive odds ratio of 1.79, 95% confidence interval (CI) 1.37-2.34 with P = 2.02 x 10(-5) in female cases with advanced/symptomatic hip osteoarthritis. The gene product of this DIO2 converts intracellular pro-hormone-3,3',5,5'-tetraiodothyronine (T4) into the active thyroid hormone 3,3',5-triiodothyronine (T3) thereby regulating intracellular levels of active T3 in target tissues such as the growth plate. Our results indicate a new susceptibility gene (DIO2) conferring risk to osteoarthritis.

A 40-Marker Panel for High Dimensional Characterization of Cancer Immune Microenvironments by Imaging Mass Cytometry.

Multiplex immunophenotyping technologies are indispensable for a deeper understanding of biological systems. Until recently, high-dimensional cellular analyses implied the loss of tissue context as they were mostly performed in single-cell suspensions. The advent of imaging mass cytometry introduced the possibility to simultaneously detect a multitude of cellular markers in tissue sections. This technique can be applied to various tissue sources including snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. However, a number of methodological challenges must be overcome when developing large antibody panels in order to preserve signal intensity and specificity of antigen detection. We report the development of a 40-marker panel for imaging mass cytometry on FFPE tissues with a particular focus on the study of cancer immune microenvironments. It comprises a variety of immune cell markers including lineage and activation markers as well as surrogates of cancer cell states and tissue-specific markers (e.g., stroma, epithelium, vessels) for cellular contextualization within the tissue. Importantly, we developed an optimized workflow for maximum antibody performance by separating antibodies into two distinct incubation steps, at different temperatures and incubation times, shown to significantly improve immunodetection. Furthermore, we provide insight into the antibody validation process and discuss why some antibodies and/or cellular markers are not compatible with the technique. This work is aimed at supporting the implementation of imaging mass cytometry in other laboratories by describing methodological procedures in detail. Furthermore, the panel described here is an excellent immune monitoring tool that can be readily applied in the context of cancer research.

Neoantigen-specific immunity in low mutation burden colorectal cancers of the consensus molecular subtype 4.

BACKGROUND: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers. METHODS: Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material. RESULTS: Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39+CD103+ T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-ß pathway activation and worse clinical outcome. CONCLUSIONS: We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-ß pathway to reinforce T cell reactivity in this patient group.

Annotating Transcriptional Effects of Genetic Variants in Disease-Relevant Tissue: Transcriptome-Wide Allelic Imbalance in Osteoarthritic Cartilage.

OBJECTIVE: Multiple single-nucleotide polymorphisms (SNPs) conferring susceptibility to osteoarthritis (OA) mark imbalanced expression of positional genes in articular cartilage, reflected by unequally expressed alleles among heterozygotes (allelic imbalance [AI]). We undertook this study to explore the articular cartilage transcriptome from OA patients for AI events to identify putative disease-driving genetic variation. METHODS: AI was assessed in 42 preserved and 5 lesioned OA cartilage samples (from the Research Arthritis and Articular Cartilage study) for which RNA sequencing data were available. The count fraction of the alternative alleles among the alternative and reference alleles together (φ) was determined for heterozygous individuals. A meta-analysis was performed to generate a meta-φ and P value for each SNP with a false discovery rate (FDR) correction for multiple comparisons. To further validate AI events, we explored them as a function of multiple additional OA features. RESULTS: We observed a total of 2,070 SNPs that consistently marked AI of 1,031 unique genes in articular cartilage. Of these genes, 49 were found to be significantly differentially expressed (fold change <0.5 or >2, FDR <0.05) between preserved and paired lesioned cartilage, and 18 had previously been reported to confer susceptibility to OA and/or related phenotypes. Moreover, we identified notable highly significant AI SNPs in the CRLF1, WWP2, and RPS3 genes that were related to multiple OA features. CONCLUSION: We present a framework and resulting data set for researchers in the OA research field to probe for disease-relevant genetic variation that affects gene expression in pivotal disease-affected tissue. This likely includes putative novel compelling OA risk genes such as CRLF1, WWP2, and RPS3.