Triple Negative Breast Cancer (TNBC) is a very aggressive tumor subtype, which still lacks specific markers for an effective targeted therapy. Despite the common feature of negativity for the three most relevant receptors (ER, PgR and HER2), TNBC is a very heterogeneous disease where different subgroups can be recognized, and both gene and microRNA profiling studies have recently been carried out to dissect the different molecular entities. Moreover, several microRNAs playing a crucial role in triple negative breast cancer biology have been identified, providing the experimental basis for a possible therapeutic application. Indeed, the causal involvement of microRNAs in breast cancer and the possible use of these small noncoding RNA molecules as biomarkers has been extensively studied with promising results. Their application as therapeutic tools might represent an innovative approach, especially for a tumor subgroup still lacking an efficient and specific therapy such as TNBC. In this review, we summarize our knowledge on the most important microRNAs described in TNBC.

T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8⁺ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8⁺ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8⁺ TCRs—classic features of protective immunity—are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8⁺ T cell immunity.

Engagement of the inhibitory T cell receptor programmed cell death protein 1 (PD-1) associates with dysfunctional states of pathogen- or tumor-specific T cells. Accordingly, systemic antibody-mediated blockade of PD-1 has become a central target for immunotherapies but is also associated with severe toxicities due to loss of peripheral tolerance. Therefore, selective ablation of PD-1 expression on adoptively transferred T cells through direct genetic knockout (KO) is currently being explored as an alternative therapeutic approach. However, since PD-1 might also be required for the regulation of physiological T cell function and differentiation, the suitability of PD-1 as an engineering target is controversial. In this study, we systematically investigated the maintenance of T cell functionality after CRISPR/Cas9-mediated PD-1 KO in vivo during and after acute and chronic antigen encounter. Under all tested conditions, PD-1 ablation preserved the persistence, differentiation, and memory formation of adoptively transferred receptor transgenic T cells. Functional PD-1 KO T cells expressing chimeric antigen receptors (CARs) targeting CD19 could be robustly detected for over 390 d in a syngeneic immunocompetent mouse model, in which constant antigen exposure was provided by continuous B cell renewal, representing the longest in vivo follow-up of CAR-T cells described to date. PD-1 KO CAR-T cells showed no evidence for malignant transformation during the entire observation period. Our data demonstrate that genetic ablation of PD-1 does not impair functionality and longevity of adoptively transferred T cells per se and therefore may be pursued more generally in engineered T cell-based immunotherapy to overcome a central immunosuppressive axis.

The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen–specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma—combined with transgenic reexpression of identified TCRs by CRISPR-Cas9–mediated orthotopic TCR replacement—revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.