B. Van den Eynde

Rheumatoid arthritis synovial fibroblasts produce a soluble form of the interleukin-7 receptor in response to pro-inflammatory cytokines.

We previously demonstrated that baseline synovial overexpression of the interleukin‐7 receptor α‐chain (IL‐7R) is associated with poor response to tumour necrosis factor (TNF) blockade in rheumatoid arthritis (RA). We found that IL‐7R gene expression is induced in fibroblast‐like synovial cells (FLS) by the addition of TNF‐α, IL‐1β and combinations of TNF‐α+ IL‐1β or TNF‐α+ IL‐17, thereby suggesting that these cytokines play a role in the resistance to TNF blockade in RA. Because FLS and CD4 T cells also produce a soluble form of IL‐7R (sIL‐7R), resulting from an alternative splicing of the full‐length transcript, we wondered whether expression of sIL‐7R is similarly regulated by pro‐inflammatory cytokines. We also investigated whether sIL‐7R is detectable in the serum of RA patients and associated with response to TNF blockade. RA FLS were cultured in the presence of pro‐inflammatory cytokines and sIL‐7R concentrations were measured in culture supernatants. Similarly, sIL‐7R titres were measured in sera obtained from healthy individuals, early untreated RA patients with active disease and disease‐modifying anti‐rheumatic drug (DMARD)‐resistant RA patients prior to initiation of TNF‐blockade. Baseline serum sIL‐7R titres were correlated with validated clinical measurements of disease activity. We found that exposure of RA FLS to pro‐inflammatory cytokines (TNF‐α, IL‐1β and combinations of TNF‐α and IL‐1β or TNF‐α and IL‐17) induces sIL‐7R secretion. Activated CD4 T cells also produce sIL‐7R. sIL‐7R serum levels are higher in RA patients as compared to controls. In DMARD‐resistant patients, high sIL‐7R serum concentrations are strongly associated with poor response to TNF‐blockade. In conclusion, sIL‐7R is induced by pro‐inflammatory cytokines in RA FLS. sIL‐7R could qualify as a new biomarker of response to therapy in RA.

Rituximab treatment induces the expression of genes involved in healing processes in the rheumatoid arthritis synovium

OBJECTIVE Rituximab displays therapeutic benefits in the treatment of patients with rheumatoid arthritis (RA) resistant to tumor necrosis factor (TNF) blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. We undertook this study to investigate the global molecular effects of rituximab in synovial biopsy samples obtained from anti-TNF-resistant RA patients before and after administration of the drug. METHODS Paired synovial biopsy samples were obtained from the affected knee of anti-TNF-resistant RA patients before (time 0) and 12 weeks after (time 12) initiation of rituximab therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction experiments were performed to confirm the differential expression of selected transcripts. RESULTS According to Students paired t-tests, 549 of 54,675 investigated probe sets were differentially expressed between time 0 and time 12. Pathway analysis revealed that genes down-regulated between time 0 and time 12 were significantly enriched in immunoglobulin genes and genes involved in chemotaxis, leukocyte activation, and immune responses (Gene Ontology annotations). In contrast, genes up-regulated between time 0 and time 12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (Gene Set Enrichment Analysis). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy. CONCLUSION Rituximab displays unique effects on global gene expression profiles in the synovial tissue of RA patients. These observations open new perspectives in the understanding of the biologic effects of the drug and in the selection of patients likely to benefit from this therapy.

Cytolytic response of human T cells against allogeneic small cell lung carcinoma treated with interferon gamma.

SummaryHuman lymphocytes stimulated in vitro by allogeneic small cell lung carcinoma cell lines did not show any significant cytolytic activity against the stimulator tumor cells. However, a high level of lysis was observed when both stimulator and target small cell lung carcinoma cells were pretreated with inferferon γ, which increased considerably the expression of major histocompatibility class I molecules by these cells. The demonstration that small cell lung carcinoma cells can be lysed by cytolytic T lymphocytes, suggests that it will be feasible to study the autologous T cell response of patients against this tumor.

605 Tumor antigens recognized by cytolytic T lymphocytes

In human tumors, several antigens recognized by autologous CTL have been identified. A first class results from the activation of genes such as MAGE-1, MAGE-3, BAGE and GAGE, which are not expressed in normal tissues with the exception of testis. MAGE-derived peptides binding to HLA-A1, Cwl6 and A2 have been identified. The MAGE family comprises genes that are expressed in tumors of several histological types. A second type of antigens identified in melanoma consists of differentiation antigens derived from proteins such as tyrosinase and Melan-A that are specific for melanocytes and melanomas. Recently, we have identified a melanoma antigen which results from a point mutation in an intron. The antigenic peptide is encoded by the end of an exon and the initial part of intron. Another antigen recognized on a large fraction of HLA-A2 melanomas involves an antigenic peptide encoded by an intron. The identification of new antigens will extend the range of patients eligible for specific immunotherapy, allowing also to immunize against several antigens borne by the same tumor. This may be a critical condition for therapeutic success.

The immune microenvironment of HPV-negative oral squamous cell carcinoma from never-smokers and never-drinkers patients suggests higher clinical benefit of IDO1 and PD1/PD-L1 blockade.

Background Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. Patients and methods Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (three validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was carried out. Formalin-fixed paraffin-embedded samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1, and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. Results A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND than in SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared with SD. Conclusion The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.

995 Genes coding for tumor rejection antigens

We have isolated a number of genes that code for antigens recognized on human melanomas by autologous cytolytic T cells (CTL). A gene named MAGE-1 codes for two different antigenic peptides that are recognized by CTL on MHC molecules HLA-A1 and HLA-Cw16 respectively. This gene belongs to a family of 12 closely related genes. No expression of these genes was found on a large panel of normal tissues except for testis. The genes of the MAGE family are all located on the q terminal region of the X chromosome. The putative proteins produced by these genes present almost identical hydrophobicity patterns, suggesting that they exert the same function, but this function remains unknown. Gene MAGE-4 carries at least eight alternative first exons preceded by different promoters. The MAGE gene family may therefore ensure that the same function is placed under the control of nineteen different promoters, allowing for very specific spatial and temporal regulation. Gene MAGE-3 codes for a second antigen presented by HLA-A1. The relevant antigenic peptide is encoded by the MAGE-3 sequence that is homologous to the MAGE-1 sequence that also codes for an antigen presented by HLA-A1. Recently, another peptide that is encoded by MAGE-3 and binds to HLA-A2 has been found to be recognized by CTL. Two additional genes that code for tumor antigens and are expressed only in tumors and in testis have been isolated. These genes, named BAGE and GAGE, are unrelated to each other and to the MAGE family. MAGE, BAGE and GAGE are expressed in a significant proportion of tumors of different histological types, such as melanomas head and neck carcinomas, non small cell lung carcinomas and bladder tumors. They are not expressed in certain types of tumors such as leukemias. Genes coding for differentiation products, such as tyrosinase and Melan A in melanomas, also code for antigens recognized by autologous CTL.

Tumor Rejection Antigens and Immune Surveillance

The existence of specific tumor rejection antigens was first demonstrated with chemically induced mouse sarcomas: each tumor was found to express a different antigen [1]. Similar findings were made with ultraviolet-induced tumors [2]. Later, the generality of the existence of tumor rejection antigens was questioned when spontaneous mouse tumors were found to be completely incapable of eliciting an immune rejection response [3]. However, further experiments demonstrated that even these tumors express weak transplantation antigens that are potential targets for immune rejection by the syngeneic host [4].