Are homeostatic mechanisms aiding the reconstitution of the T-cell pool during lymphopenia in humans?

A timely recovery of T-cell numbers following haematopoietic stem-cell transplantation (HSCT) is essential for preventing complications, such as increased risk of infection and disease relapse. In analogy to the occurrence of lymphopenia-induced proliferation in mice, T-cell dynamics in humans are thought to be homeostatically regulated in a cell density-dependent manner. The idea is that T cells divide faster and/or live longer when T-cell numbers are low, thereby helping the reconstitution of the T-cell pool. T-cell reconstitution after HSCT is, however, known to occur notoriously slowly. In fact, the evidence for the existence of homeostatic mechanisms in humans is quite ambiguous, since lymphopenia is often associated with infectious complications and immune activation, which confound the study of homeostatic regulation. This calls into question whether homeostatic mechanisms aid the reconstitution of the T-cell pool during lymphopenia in humans. Here we review the changes in T-cell dynamics in different situations of T-cell deficiency in humans, including the early development of the immune system after birth, healthy ageing, HIV infection, thymectomy and hematopoietic stem cell transplantation (HSCT). We discuss to what extent these changes in T-cell dynamics are a side-effect of increased immune activation during lymphopenia, and to what extent they truly reflect homeostatic mechanisms.

Turnover of Murine Cytomegalovirus-Expanded CD8+ T Cells Is Similar to That of Memory Phenotype T Cells and Independent of the Magnitude of the Response.

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.

Transcriptomic Correlates of Immunologic Activation in Head and Neck and Cervical Cancer.

Targeting the immune system has emerged as an effective therapeutic strategy for the treatment of various tumor types, including Head and Neck Squamous Cell Carcinoma (HNSCC) and Non-small-Cell Lung Cancer (NSCLC), and checkpoint inhibitors have shown to improve patient survival in these tumor types. Unfortunately, not all cancers respond to these agents, making it necessary to identify responsive tumors. Several biomarkers of response have been described and clinically tested. As of yet what seems to be clear is that a pre-activation state of the immune system is necessary for these agents to be efficient. In this study, using established transcriptomic signatures, we identified a group of gene combination associated with favorable outcome in HNSCC linked to a higher presence of immune effector cells. CD2, CD3D, CD3E, and CXCR6 combined gene expression is associated with improved outcome of HNSCC patients and an increase of infiltrating immune effector cells. This new signature also identifies a subset of cervical squamous cell carcinoma (CSCC) patients with favorable prognosis, who show an increased presence of immune effector cells in the tumor, which outcome shows similarities with the HP-positive HNSCC cohort of patients. In addition, CD2, CD3D, CD3E, and CXCR6 signature is able to predict the best favorable prognosis in terms of overall survival of CSSC patients. Of note, these findings were not reproduced in other squamous cell carcinomas like esophageal SCC or lung SCC. Prospective confirmatory studies should be employed to validate these findings.

Transcriptomic Profiles of CD47 in Breast Tumors Predict Outcome and Are Associated with Immune Activation.

Targeting the innate immune system has attracted attention with the development of anti- CD47 antibodies. Anti-CD47 antibodies block the inhibition of the phagocytic activity of macrophages caused by the up-regulation of CD47 on tumor cells. In this study, public genomic data was used to identify genes highly expressed in breast tumors with elevated CD47 expression and analyzed the association between the presence of tumor immune infiltrates and the expression of the selected genes. We found that 142 genes positively correlated with CD47, of which 83 predicted favorable and 32 detrimental relapse-free survival (RFS). From those associated with favorable RFS, we selected the genes with immunologic biological functions and defined a CD47-immune signature composed of PTPRC, HLA-E, TGFBR2, PTGER4, ETS1, and OPTN. In the basal-like and HER2+ breast cancer subtypes, the expression of the CD47-immune signature predicted favorable outcome, correlated with the presence of tumor immune infiltrates, and with gene expression signatures of T cell activation. Moreover, CD47 up-regulated genes associated with favorable survival correlated with pro-tumoral macrophages. In summary, we described a CD47-immune gene signature composed of 6 genes associated with favorable prognosis, T cell activation, and pro-tumoral macrophages in breast cancer tumors expressing high levels of CD47.

Mapping of Genomic Vulnerabilities in the Post-Translational Ubiquitination, SUMOylation and Neddylation Machinery in Breast Cancer.

The dysregulation of post-translational modifications (PTM) transversally impacts cancer hallmarks and constitutes an appealing vulnerability for drug development. In breast cancer there is growing preclinical evidence of the role of ubiquitin and ubiquitin-like SUMO and Nedd8 peptide conjugation to the proteome in tumorigenesis and drug resistance, particularly through their interplay with estrogen receptor signaling and DNA repair. Herein we explored genomic alterations in these processes using RNA-seq and mutation data from TCGA and METABRIC datasets, and analyzed them using a bioinformatic pipeline in search of those with prognostic and predictive capability which could qualify as subjects of drug research. Amplification of UBE2T, UBE2C, and BIRC5 conferred a worse prognosis in luminal A/B and basal-like tumors, luminal A/B tumors, and luminal A tumors, respectively. Higher UBE2T expression levels were predictive of a lower rate of pathological complete response in triple negative breast cancer patients following neoadjuvant chemotherapy, whereas UBE2C and BIRC5 expression was higher in luminal A patients with tumor relapse within 5 years of endocrine therapy or chemotherapy. The transcriptomic signatures of USP9X and USP7 gene mutations also conferred worse prognosis in luminal A, HER2-enriched, and basal-like tumors, and in luminal A tumors, respectively. In conclusion, we identified and characterized the clinical value of a group of genomic alterations in ubiquitination, SUMOylation, and neddylation enzymes, with potential for drug development in breast cancer.

Cell-density independent increased lymphocyte production and loss rates post-autologous HSCT.

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.

In silico transcriptomic mapping of integrins and immune activation in Basal-like and HER2+ breast cancer.

PURPOSE: Integrins, transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions, have been linked to several cancer-associated features. A less explored function of integrins in cancer is their role in leukocyte homing and activation. Understanding their relationship with immune cell infiltrates and immune checkpoints is an area of interest in cancer research. METHODS: The expression of 33 different integrins was evaluated in relation with breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to assess the association of the identified integrin genes with immune cell infiltration, and the TCGA and METABRIC studies to assess correlations between integrin gene expression and genomic signatures of immune activation. RESULTS: We identified 7 genes coding for integrin α and ß subunits, i.e., ITGA4, ITGB2, ITGAX, ITGB7, ITGAM, ITGAL and ITGA8, which predict a favorable prognosis in Basal-like and HER2+ breast cancers. Their expression positively correlated with the presence of immune cell infiltrates within the tumor (dendritic cells, CD4+ T-cells, neutrophils, CD8+ T-cells and B-cells), with markers of T-cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast, we found that genes coding for integrins that predicted a detrimental outcome (IBSP, ITGB3BP, ITGB6, ITGB1 and ITGAV) were not associated with any of these parameters. CONCLUSIONS: We identified an integrin signature composed of 7 genes with potential to recognize immune infiltrated and activated Basal-like and HER2+ breast cancers with a favorable prognosis.

Adoptive Cell Therapy in Breast Cancer: A Current Perspective of Next-Generation Medicine.

Immunotherapy has become a cornerstone in the treatment of cancer and changed the way clinicians and researchers approach tumor vulnerabilities. Durable responses are commonly observed with immune checkpoint inhibitors in highly immunogenic tumors, while the infusion of T cells genetically engineered to express chimeric antigen receptors (CARs) has shown impressive efficacy in certain types of blood cancer. Nevertheless, harnessing our own immunity has not proved successful for most breast cancer patients. In the era of genomic medicine, cellular immunotherapies may provide a more personalized and dynamic tool against tumors displaying heterogeneous mutational landscapes and antigenic pools. This approach encompasses multiple strategies including the adoptive transfer of tumor-infiltrating lymphocytes, dendritic cells, natural killer cells, and engineered immune components such as CAR constructs and engineered T cell receptors. Although far from permeating the clinical setting, technical advances have been overwhelming in recent years, with continuous improvement in traditional challenges such as toxicity, adoptive cell persistence, and intratumoral trafficking. Also, there is an avid search for neoantigens that can be targeted by these strategies, either alone or in combination. In this work, we aim to provide a clinically-oriented overview of preclinical and clinical data regarding the use of cellular immunotherapies in breast cancer.

Breast Cancer Heterogeneity and Response to Novel Therapeutics.

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.

Genomic Mapping Identifies Mutations in RYR2 and AHNAK as Associated with Favorable Outcome in Basal-Like Breast Tumors Expressing PD1/PD-L1.

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.

In vivo deuterium labelling in mice supports a dynamic model for memory T-cell maintenance in the bone marrow.

The maintenance and dynamics of memory T-cells in the bone marrow are a matter of ongoing debate. It has been suggested that memory T-cells in the bone marrow are maintained as long-lived, quiescent cells. We have recently shown that memory T-cells isolated from goat bone marrow undergo self-renewal and recirculate via the blood and lymph. Using the well-established memory T-cell markers CD44 and CD62L we here show very similar results in mice. This provides further support for the concept that memory T-cells are continuously self-renewing and recirculating between blood, bone marrow, spleen and lymph nodes.

Genomic Signatures of Immune Activation Predict Outcome in Advanced Stages of Ovarian Cancer and Basal-Like Breast Tumors.

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.

A Transcriptomic Immunologic Signature Predicts Favorable Outcome in Neoadjuvant Chemotherapy Treated Triple Negative Breast Tumors.

Limited therapeutic options exist for the treatment of patients with triple negative breast cancer (TNBC). Neoadjuvant chemotherapy is currently the standard of care treatment in the early stages of the 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 the outcome to neoadjuvant chemotherapy. Publicly available transcriptomic data of more than 2,000 patients were evaluated. ROC plots were generated to assess the response to therapy. Cox proportional hazards regression was computed. Kaplan-Meier plots were drawn to visualize the survival differences. Higher expression of IDO1, CXCL9, CXCL10, HLA-DRA, HLA-E, STAT1, and GZMB were associated with a higher proportion without relapse in the first 5 y after chemotherapy in TNBC. The expression of these genes was associated with a high presence of CD8 T cells in responder patients using the EPIC bioinformatic tool. The strongest effect was observed for STAT1 (p = 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, FDR = 1%). However, no influence on pathological complete response (pCR) was observed. Similarly, 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 the 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 the long term outcome for immune activated tumors.

Age-related distribution and dynamics of T-cells in blood and lymphoid tissues of goats.

Neonatal mammals have increased disease susceptibility and sub-optimal vaccine responses. This raises problems in both humans and farm animals. The high prevalence of paratuberculosis in goats and the lack of an effective vaccine against it have a strong impact on the dairy sector, and calls for vaccines optimized for the neonatal immune system. We characterized the composition of the T-cell pool in neonatal kids and adult goats and quantified their turnover rates using in vivo deuterium labelling. From birth to adulthood, CD4+ T-cells were the predominant subset in the thymus and lymph nodes, while spleen and bone marrow contained mainly CD8+ lymphocytes. In blood, CD4+ T-cells were the predominant subset during the neonatal period, while CD8+ T-cells predominated in adults. We observed that thymic mass and cellularity increased during the first 5 months after birth, but decreased later in life. Deuterium labelling revealed that T-cell turnover rates in neonatal kids are considerably higher than in adult animals.

Short Lifespans of Memory T-cells in Bone Marrow, Blood, and Lymph Nodes Suggest That T-cell Memory Is Maintained by Continuous Self-Renewal of Recirculating Cells.

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.

Neuroimmunological communication via CGRP promotes the development of a regulatory phenotype in TLR4-stimulated macrophages.

Environmental signals shape the phenotype and function of activated macrophages. Here, we show that the neuropeptide calcitonin gene-related peptide (CGRP), which is released from sensory nerves, modulates the phenotype of TLR4-activated murine macrophages by enhancing expression of the regulatory macrophage markers IL-10, sphingosine kinase 1 (SPHK1), and LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes). In contrast, CGRP inhibits production of cytokines characteristic of inflammatory macrophages and does not affect expression of wound-healing macrophage markers upon TLR4 engagement. In IL-4-stimulated macrophages, CGRP increased LIGHT expression, but failed to induce IL-10 and SPHK1. The stimulatory effect of CGRP on IL-10 production required activation of protein kinase A and was linked to prolonged phosphorylation of CREB and sustained nuclear accumulation of CRTC2 and CRTC3 (where CRTC is CREB-regulated transcriptional cofactor). CGRP enhanced expression of regulatory macrophage markers during the early, but not late, phase of LPS-stimulation and this effect was independent of autocrine type-I IFN activity. In contrast, autocrine type-I IFN activity and treatment of macrophages with IFN-ß promoted late-phase IL-10 production, but had only minor influence on LIGHT and SPHK1 expression. Together, the results identify neuroimmunological communication through CGRP as a novel costimulatory pathway promoting the development of a regulatory phenotype of TLR4-stimulated macrophages. CGRP appears to act through a mechanism that involves sustained activation of CREB-dependent gene transcription.