In-depth analysis of human virus-specific CD8+ T cells delineates unique phenotypic signatures for T cell specificity prediction.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Single-cell analysis reveals clonally expanded tumor-associated CD57+ CD8 T cells are enriched in the periphery of patients with metastatic urothelial cancer responding to PD-L1 blockade.

BACKGROUND: A growing body of evidence suggests that T-cell responses against neoantigens are critical regulators of response to immune checkpoint blockade. We previously showed that circulating neoantigen-specific CD8 T cells in patients with lung cancer responding to anti-Programmed death-ligand 1 (PD-L1) (atezolizumab) exhibit a unique phenotype with high expression of CD57, CD244, and KLRG1. Here, we extended our analysis on neoantigen-specific CD8 T cells to patients with metastatic urothelial cancer (mUC) and further profiled total CD8 T cells to identify blood-based predictive biomarkers of response to atezolizumab. METHODS: We identified tumor neoantigens from 20 patients with mUC and profiled their peripheral CD8 T cells using highly multiplexed combinatorial tetramer staining. Another set of patients with mUC treated with atezolizumab (n=30) or chemotherapy (n=40) were selected to profile peripheral CD8 T cells by mass cytometry. Using single-cell transcriptional analysis (single-cell RNA sequencing (scRNA-seq)), together with CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) and paired T-cell receptor (TCR) sequencing, we further characterized peripheral CD8 T cells in a subset of patients (n=16). RESULTS: High frequency of CD57 was observed in neoantigen-specific CD8 T cells in patients with mUC responding to atezolizumab. Extending these findings to bulk CD8 T cells, we found higher frequency of CD57 expressing CD8 T cells before treatment in patients responding to atezolizumab (n=20, p<0.01) but not to chemotherapy. These findings were corroborated in a validation cohort (n=30, p<0.01) and notably were independent of known biomarkers of response. scRNA-seq analysis identified a clonally expanded cluster enriched within CD57+ CD8 T cells in responding patients characterized by higher expression of genes associated with activation, cytotoxicity, and tissue-resident memory markers. Furthermore, compared with CD57- CD8 T cells, TCRs of CD57+ CD8 T cells showed increased overlap with the TCR repertoire of tumor-infiltrating T cells. CONCLUSIONS: Collectively, we show high frequencies of CD57 among neoantigen-specific and bulk CD8 T cells in patients responding to atezolizumab. The TCR repertoire overlap between peripheral CD57+ CD8 T cells and tumor-infiltrating lymphocytes suggest that accumulation of peripheral CD57+ CD8 T cells is reflective of an ongoing antitumor T-cell response. Our findings provide evidence and rationale for using circulating CD8 T cells expressing CD57 as a readily accessible blood-based biomarker for selecting patients with mUC for atezolizumab therapy.

Preoperative autologous blood donation in placenta previa patients.

OBJECTIVE: The aim of this study was to evaluate the usability of preoperative autologous blood donation (PAD) in pregnant women with placenta previa. STUDY DESIGN: We retrospectively reviewed 142 pregnancies with placenta previa from completed 32 weeks of gestation who underwent a caesarean delivery in University clinical centre Ljubljana, over a five-year period. RESULT: Although more than two thirds of pregnant women met the criteria for PAD, it was justified for approximately 13.6% of them. The decrease in haemoglobin level after PAD was only 4.5 ± 6.7 g/l on average and did not induce anaemia. CONCLUSION: Although our study shows that PAD is not reasonable for the majority of all pregnant women with placenta previa who met the criteria for PAD from our study, we believe that with the implementation of Patient Blood Management it still has its prospects of clinical application. However, further prospective studies are needed to find risk factors for increased surgical bleeding to make a proper patient selection for PAD.

Visceral leishmaniasis diagnosis and reporting delays as an obstacle to timely response actions in Nepal and India.

BACKGROUND: To eliminate visceral leishmaniasis (VL) in India and Nepal, challenges of VL diagnosis, treatment and reporting need to be identified. Recent data indicate that VL is underreported and patients face delays when seeking treatment. Moreover, VL surveillance data might not reach health authorities on time. This study quantifies delays for VL diagnosis and treatment, and analyses the duration of VL reporting from district to central health authorities in India and Nepal. METHODS: A cross-sectional study conducted in 12 districts of Terai region, Nepal, and 9 districts of Bihar State, India, in 2012. Patients were interviewed in hospitals or at home using a structured questionnaire, health managers were interviewed at their work place using a semi-structured questionnaire and in-depth interviews were conducted with central level health managers. Reporting formats were evaluated. Data was analyzed using two-tailed Mann-Whitney U or Fisher's exact test. RESULTS: 92 VL patients having experienced 103 VL episodes and 49 district health managers were interviewed. Patients waited in Nepal 30 days (CI 18-42) before seeking health care, 3.75 times longer than in Bihar (8d; CI 4-12). Conversely, the lag time from seeking health care to receiving a VL diagnosis was 3.6x longer in Bihar (90d; CI 68-113) compared to Nepal (25d; CI 13-38). The time span between diagnosis and treatment was short in both countries. VL reporting time was in Nepal 19 days for sentinel sites and 76 days for "District Public Health Offices (DPHOs)". In Bihar it was 28 days for "District Malaria Offices". In Nepal, 73% of health managers entered data into computers compared to 16% in Bihar. In both countries reporting was mainly paper based and standardized formats were rarely used. CONCLUSIONS: To decrease the delay between onset of symptoms and getting a proper diagnosis and treatment the approaches in the two countries vary: In Nepal health education for seeking early treatment are needed while in Bihar the use of private and non-formal practitioners has to be discouraged. Reinforcement of VL sentinel reporting in Bihar, reorganization of DPHOs in Nepal, introduction of standardized reporting formats and electronic reporting should be conducted in both countries.

Apoptosis resistance, mitotic catastrophe, and loss of ploidy control in Burkitt lymphoma.

UNLABELLED: Resistance to cell death is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphoma (BL). When analyzing therapy resistance in Burkitt lymphoma (BL), we discovered a link between apoptosis resistance and ploidy control. We therefore studied systematically a panel of 15 BL lines for apoptosis induction upon treatment with microtubule inhibitors and compared three types of microtubule toxins, i.e., paclitaxel, nocodazole and vincristine. We found an inverse relationship between apoptosis sensitivity and ploidy control. Thus, cells resistant to paclitaxel- or nocodazole-induced apoptosis underwent mitotic catastrophe and developed polyploidy (>4N). Mechanistically, apoptosis resistance was linked to failure of caspase activation, which was most pronounced in cells lacking the pro-apoptotic multidomain Bcl-2 homologs Bax and Bak. Pharmacological caspase inhibition promoted polyploidy upon exposure to paclitaxel and nocodazole supporting the relationship between resistance to apoptosis and polyploidization. Of note, vincristine induced persistent mitotic arrest but no loss of ploidy control. Considering targets to facilitate Bax/Bak-independent cell death and to avoid drug-induced mitotic catastrophe and consecutive mitotic catastrophe should be of great importance to overcome therapy resistance and therapy-related events that result in ploidy changes and tumor progression. KEY MESSAGE: Inverse relation of apoptosis and polyploidy induction by paclitaxel or nocodazole in BL. Resistant cells undergo mitotic catastrophe and develop polyploidy. Lack of Bax/Bak confers resistance and leads to induction of polyploidy in BL. Intact apoptosis response protects from polyploidy as a result of mitotic catastrophe.

p14ARF induces apoptosis via an entirely caspase-3-dependent mitochondrial amplification loop.

The p14(ARF) tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF-7 breast carcinoma cells, expression of the tumor suppressor gene p14(ARF) fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase-3 proficient MCF-7 cells upon expression of p14(ARF) . This occurred in the absence of S-phase progression or mitotic entry. In contrast, syngeneic, caspase-3-deficient MCF-7 cells remained entirely resistant to p14(ARF) -induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14(ARF) -induced cell death and promotes cell death via a caspase-3-dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan-caspase inhibitors and the caspase-3/7 inhibitor zDEVD-fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase-3 proficiency indicating that caspase-3 either acts "up-stream" of the mitochondria in a "non-canonical" pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14(ARF) -induced stress signaling.

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins.

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.

Oncogene-targeting T cells reject large tumors while oncogene inactivation selects escape variants in mouse models of cancer.

The genetic instability of cancer cells frequently causes drug resistance. We established mouse cancer models, which allowed targeting of an oncogene by drug-mediated inactivation or monospecific CD8(+) effector T (T(E)) cells. Drug treatment of genetically unstable large tumors was effective but selected resistant clones in the long term. In contrast, T(E) cells completely rejected large tumors (≥500 mm(3)), if the target antigen was cancer-driving and expressed in sufficient amounts. Although drug-mediated oncogene inactivation selectively killed the cancer cells and left the tumor vasculature intact, which likely facilitated survival and growth of resistant clones, T(E) cell treatment led to blood vessel destruction and probably "bystander" elimination of escape variants, which did not require antigen cross-presentation by stromal cells.

Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3.

Induction of cell death by p14(ARF) is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21(CDKN1). Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21(CDKN1) or p53-reconstituted DU145 prostate cancer cells, we show that p14(ARF)-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14(ARF) in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14(ARF)-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14(ARF)-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21(CDKN1) strongly enhances p14(ARF)-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14(ARF)-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14(ARF)-triggered apoptosis is enhanced by loss of p21(CDKN1)-mediated cell cycle checkpoint control.