Tissue-resident memory T cells in human kidney transplants have alloreactive potential.

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Association of HBsAg levels with differential gene expression in NK, CD8 T, and memory B cells in treated patients with chronic HBV.

Background & Aims: HBsAg secretion may impact immune responses to chronic HBV infection. Thus, therapeutic approaches to suppress HBsAg production are being investigated. Our study aims to examine the immunomodulatory effects of high and low levels of circulating HBsAg and thereby improve our understanding of anti-HBV immunity. Methods: An optimized 10x Genomics single-cell RNA sequencing workflow was applied to blood samples and liver fine-needle aspirates from 18 patients undergoing tenofovir/entecavir (NUC) treatment for chronic HBV infection. They were categorized based on their HBsAg levels: high (920-12,447 IU/ml) or low (1-100 IU/ml). Cluster frequencies, differential gene expression, and phenotypes were analyzed. Results: In the blood of HBV-infected patients on NUC, the proportion of KLRC2+ "adaptive" natural killer (NK) cells was significantly lower in the HBsAg-high group and, remarkably, both KLRC2+ NK and KLRG1+ CD8 T cells display enrichment of lymphocyte activation-associated gene sets in the HBsAg-low group. High levels of HBsAg were associated with mild immune activation in the liver. However, no suppression of liver-resident CXCR6+ NCAM1+ NK or CXCR6+ CD69+ CD8 T cells was detected, while memory B cells showed signs of activation in both the blood and liver. Conclusions: Among NUC-treated patients, we observed a minimal impact of HBsAg on leukocyte populations in the blood and liver. However, for the first time, we found that HBsAg has distinct effects, restricted to NK-, CD8 T-, and memory B-cell subsets, in the blood and liver. Our findings are highly relevant for current clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Impact and implications: This study provides unique insight into the impact of HBsAg on gene expression levels of immune cell subsets in the blood and liver, particularly in the context of NUC-treated chronic HBV infection. It holds significant relevance for current and future clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Our findings raise questions about the effectiveness of such treatment strategies and challenge the previously hypothesized immunomodulatory effects of HBsAg on immune responses against HBV.

Functional Homologous Recombination (HR) Screening Shows the Majority of BRCA1/2-Mutant Breast and Ovarian Cancer Cell Lines Are HR-Proficient.

Tumors with a pathogenic BRCA1/2 mutation are homologous recombination (HR)-deficient (HRD) and consequently sensitive to platinum-based chemotherapy and Poly-[ADP-Ribose]-Polymerase inhibitors (PARPi). We hypothesized that functional HR status better reflects real-time HR status than BRCA1/2 mutation status. Therefore, we determined the functional HR status of 53 breast cancer (BC) and 38 ovarian cancer (OC) cell lines by measuring the formation of RAD51 foci after irradiation. Discrepancies between functional HR and BRCA1/2 mutation status were investigated using exome sequencing, methylation and gene expression data from 50 HR-related genes. A pathogenic BRCA1/2 mutation was found in 10/53 (18.9%) of BC and 7/38 (18.4%) of OC cell lines. Among BRCA1/2-mutant cell lines, 14/17 (82.4%) were HR-proficient (HRP), while 1/74 (1.4%) wild-type cell lines was HRD. For most (80%) cell lines, we explained the discrepancy between functional HR and BRCA1/2 mutation status. Importantly, 12/14 (85.7%) BRCA1/2-mutant HRP cell lines were explained by mechanisms directly acting on BRCA1/2. Finally, functional HR status was strongly associated with COSMIC single base substitution signature 3, but not BRCA1/2 mutation status. Thus, the majority of BRCA1/2-mutant cell lines do not represent a suitable model for HRD. Moreover, exclusively determining BRCA1/2 mutation status may not suffice for platinum-based chemotherapy or PARPi patient selection.