No abstract

Genetic and immunological evidence clearly points to a role for T cells in the pathogenesis of rheumatoid arthritis (RA). Selective targeting of such disease-associated T cell clones might be highly effective while having few side effects. However, such selective targeting may only be feasible if the same T cell clones dominate the immune response at different sites of inflammation. We leveraged high-throughput technology to quantitatively assess whether different T cell clones dominate the inflammatory infiltrate at various sites of inflammation in this prototypic autoimmune disease. In 13 RA patients, we performed quantitative next-generation sequencing-based human TCRβ repertoire analysis in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, and from synovial fluid (SF) and peripheral blood (PB). Identical TCRβ clones dominate inflammatory responses in ST samples taken from different locations within a single joint and when sampled in different joints. Although overall ST-SF overlap was comparable to higher ST-ST values, the overlap in dominant TCRβ clones in ST-SF comparisons was much lower than ST-ST and comparable to the low ST-PB overlap. In individual RA patients, a limited number of TCRβ clones dominate the immune response in the inflamed ST regardless of the location within a joint and which joint undergoes biopsy; in contrast, there is limited overlap of ST with SF or PB TCR repertoires. This limited breadth of the T cell response in ST of the individual RA patient indicates that development of immunotherapies that selectively modulate dominant T cell responses might be feasible.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.

BackgroundMalignant pleural mesothelioma (MPM) is a highly lethal malignancy in need for new treatment options. Although immunotherapies have been shown to boost a tumor-specific immune response, not all patients respond and prognostic biomarkers are scarce. In this study, we determined the peripheral blood T cell receptor β (TCRβ) chain repertoire of nine MPM patients before and 5 weeks after the start of dendritic cell (DC)-based immunotherapy.Materials and methodsWe separately profiled PD1+and PD1−CD4+and CD8+T cells, as well as Tregs and analyzed 70 000 TCRβ sequences per patient.ResultsStrikingly, limited TCRβ repertoire diversity and high average clone sizes in total CD3+T cells before the start of immunotherapy were associated with a better clinical response. To explore the differences in TCRβ repertoire prior-DC-therapy and post-DC-therapy, for each patient the TCRβ clones present in the total CD3+T cell fractions were classified into five categories, based on therapy-associated frequency changes: expanding, decreasing, stable, newly appearing and disappearing clones. Subsequently, the presence of these five groups of clones was analyzed in the individual sorted T cell fractions. DC-therapy primarily induced TCRβ repertoire changes in the PD1+CD4+and PD1+CD8+T cell fractions. In particular, in the PD1+CD8+T cell subpopulation we found high frequencies of expanding, decreasing and newly appearing clones. Conversion from a PD1−to a PD1+phenotype was significantly more frequent in CD8+T cells than in CD4+T cells. Hereby, the number of expanding PD1+CD8+T cell clones—and not expanding PD1+CD4+T cell clones following immunotherapy positively correlated with overall survival, progression-free survival and reduction of tumor volume.ConclusionWe conclude that the clinical response to DC-mediated immunotherapy is dependent on both the pre-existing TCRβ repertoire of total CD3+T cells and on therapy-induced changes, in particular expanding PD1+CD8+T cell clones. Therefore, TCRβ repertoire profiling in sorted T cell subsets could serve as predictive biomarker for the selection of MPM patients that benefit from immunotherapy.Trial registration numberNCT02395679.