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The potential of memory T cells to provide protection against reinfection is beyond question. Yet, it remains debated whether long-term T cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8⁺ T cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T cells, depending on factors such as exposure to cognate Ag. CMV infection induces not only conventional, contracting T cell responses, but also inflationary CD8⁺ T cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8⁺ T cells. Using in vivo deuterium labeling and mathematical modeling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8⁺ T cells are very similar to those of bulk memory-phenotype CD8⁺ T cells. Even CMV-specific inflationary CD8⁺ T cell responses that differ 3-fold in size were found to turn over at similar rates. Key Points MCMV-specific CD8⁺ T cells have similar lifespans as bulk memory CD8⁺ T cells. CD8⁺ T cells of inflationary and noninflationary responses have similar lifespans.

Lymphocyte numbers need to be quite tightly regulated. It is generally assumed that lymphocyte production and lifespan increase homeostatically when lymphocyte numbers are low and, vice versa, return to normal once cell numbers have normalized. This widely accepted concept is largely based on experiments in mice, but is hardly investigated in vivo in humans. Here we quantified lymphocyte production and loss rates in vivo in patients 0.5–1 year after their autologous hematopoietic stem cell transplantation (autoHSCT). We indeed found that the production rates of most T- and B-cell subsets in autoHSCT-patients were two to eight times higher than in healthy controls, but went hand in hand with a threefold to ninefold increase in cell loss rates. Both rates also did not normalize when cell numbers did. This shows that increased lymphocyte production and loss rates occur even long after autoHSCT and can persist in the face of apparently normal cell numbers.

Targeted cancer therapies against oncogenic drivers are actively being developed and tested in clinical trials. Targeting an oncogenic driver may only prove effective if the mutation is present in most tumoral cells. Therefore, highly heterogeneous tumors may be refractory to these therapies. This makes tumor heterogeneity a major challenge in cancer therapy. Although heterogeneity has traditionally been attributed to genetic diversity within cancer cell populations, it is now widely recognized that human cancers are heterogeneous in almost all distinguishable phenotypic characteristics. Understanding the genetic variability and also the non-genetic influences of tumor heterogeneity will provide novel insights into how to reverse therapeutic resistance and improve cancer therapy.

Treatment with anti-PD-L1 antibodies has shown efficacy in basal-like breast cancer. In this context, identification of pre-activated immune tumors is a main goal. Here we explore mutations in PD1 and PD-L1 high-expressing tumors to identify genomic correlates associated with outcome. To do so, RNA-seq and mutation data from 971 breast cancer patients from the TCGA dataset were used to identify most prevalent mutations in patients with high levels of PD1 and PD-L1. Transcriptomic signatures associated with the selected mutations were identified and analyzed in terms of outcome and immune cell infiltration. We identified co-occurrent mutations in RYR2 and AHNAK in 8% and 5% of basal-like tumors respectively, which conferred good prognosis in patients with high expression of PD1 and PD-L1 genes. The transcriptomic signature associated with these mutations, composed of CXCL9, GBP5, C1QA, IL2RG, CSF2RB, IDO1 and LAG3 genes, also conferred good prognosis and correlated with immune infiltrations within the tumors. The joint signature classified patients with favorable relapse-free survival (HR: 0.28; CI: 0.2–0.38; p = 1.7 × 10⁻¹⁶) and overall survival (HR: 0.18; CI: 0.09–0.34; p = 6.8 × 10⁻⁹), showing a stronger prediction capacity than previous reported signatures. In conclusion, we describe two novel mutations and their transcriptomic signature, both associated with a favorable outcome and immune infiltrates in PD1 and PD-L1 high-expressing basal-like tumors.

Lymphocyte numbers need to be quite tightly regulated. It is generally assumed that lymphocyte production and survival rates increase homeostatically when lymphocyte numbers decrease. This widely-accepted concept is largely based on experiments in mice. In humans, lymphocyte reconstitution usually occurs very slowly, which challenges the idea that density dependent homeostasis aids recovery from lymphopenia. Using in vivo deuterium labelling, we quantified lymphocyte production and survival rates in patients who underwent an autologous hematopoietic stem cell transplantation (autoHSCT). We indeed found that the production rates of most T-cell and B-cell subsets in autoHSCT-patients were 2 to 8-times higher than in healthy controls. These increased lymphocyte production rates went hand in hand with a 3 to 9-fold increase in cell loss rates, and both rates did not normalize when cell numbers did. This challenges the concept of homeostatic regulation of lymphocyte production and survival rates in humans.

There is an unmet need for new therapies in metastatic ovarian cancer and basal-like breast cancer since no curative therapies are currently available. Immunotherapy has shown to be active in several solid tumors, but particularly more in those where a pre-activated immune state does exist. In this work, we aim to identify biomarkers that could distinguish immune-activated tumors and predict response to therapies in ovarian and basal-like breast cancer, as well as their association with the level of tumor immune infiltration. We found that the combined expression of IFNG, CD30, CXCL13, and PRF1 correlated with better overall survival (OS) in advanced stage ovarian cancer. This was confirmed using an independent dataset from TCGA. Interestingly, we observed that this gene combination also predicted for better prognosis in ovarian tumors with low mutational load, which typically respond less to immunotherapy. Expression of IFNG, CD30, CXCL13, and PRF1 was associated with increased level of immune infiltrates (CD8⁺ T cells, dendritic cells, and neutrophils) within the tumor. Moreover, we found that these gene signature also correlated with an increased OS and with a higher level of tumor immune infiltrates (B cells, CD8⁺ T cells, CD4⁺ T cells, neutrophils, and dendritic cells) in basal-like breast cancer. In conclusion, our analysis identifies genes signatures with potential to recognize immune activated ovarian and basal-like breast cancers with favorable prognosis and with a remarkable predictive capacity in tumors with low mutational burden. The presented results led to a hypothesis being formulated, but prospective clinical studies are needed to support a potential clinical application.

Limited therapeutic options exist for the treatment of patients with triple negative breast cancer (TNBC) tumors beyond the administration of chemotherapy, and very recently, the approval of the anti PD-L1 inhibitor atezolizumab. Neoadjuvant chemotherapy is currently the standard of care treatment in the early stage disease, although reliable biomarkers of response have been scarcely described. In our study we explored whether immunologic signatures associated with inflamed tumors or hot tumors could predict outcome to neoadjuvant chemotherapy. Publicly available transcriptomic data of more than 2,000 patients were evaluated. ROC plots were generated to assess response to therapy. Cox proportional hazards regression was computed to explore the association between gene expression and outcomes. Kaplan-Meier plots were drawn to visualize the survival differences. Higher expression of IDO1, CXCL9, CXCL10, HLA-DRA, , ISGF-3 from the IFN gamma signature, CXCL13, HLA-E,, LAG3 and STAT1, from the expanded gene signature and GZMB from the CTL-level signature were associated with higher proportion without relapse in the first five years after chemotherapy in TNBC. The strongest effect was observed for STAT1 (p value =1.8e-05 and AUC 0.69, p=2.7e-06). The best gene-set signature to predict favorable RFS was the combination of IDO1, LAG3, STAT1 and GZMB (HR 0.28 CI 0.17-0.46 p= 9.8 E-08). However, no influence on pathological complete response (pCR) was observed. Similar, no benefit was identified in any other tumor subtype: HER2 or estrogen receptor positive. In conclusion, we describe a set of immunologic genes that predict outcome to neoadjuvant chemotherapy in TNBC, but not pCR. suggesting that this non-time to event endpoint is not a good surrogate marker to detect long term outcome for immune activated tumors.

Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived, non-circulating memory T-cells, it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study, we used in vivo deuterium labeling in goats to simultaneously quantify the average turnover rates—and thereby expected lifespans—of memory T-cells from BM, blood and lymph nodes (LN). While the fraction of Ki-67 positive cells, a snapshot marker for recent cell division, was higher in memory T-cells from blood compared to BM and LN, in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery, and are continuously recirculating between the blood, BM, and LN.