T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control.

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8+ T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells, therefore, exit the tumor, which limits the pool of CD8+ T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy.

Functional interrogation of lymphocyte subsets in alopecia areata using single-cell RNA sequencing.

Alopecia areata (AA) is among the most prevalent autoimmune diseases, but the development of innovative therapeutic strategies has lagged due to an incomplete understanding of the immunological underpinnings of disease. Here, we performed single-cell RNA sequencing (scRNAseq) of skin-infiltrating immune cells from the graft-induced C3H/HeJ mouse model of AA, coupled with antibody-based depletion to interrogate the functional role of specific cell types in AA in vivo. Since AA is predominantly T cell-mediated, we focused on dissecting lymphocyte function in AA. Both our scRNAseq and functional studies established CD8+ T cells as the primary disease-driving cell type in AA. Only the depletion of CD8+ T cells, but not CD4+ T cells, NK, B, or γδ T cells, was sufficient to prevent and reverse AA. Selective depletion of regulatory T cells (Treg) showed that Treg are protective against AA in C3H/HeJ mice, suggesting that failure of Treg-mediated immunosuppression is not a major disease mechanism in AA. Focused analyses of CD8+ T cells revealed five subsets, whose heterogeneity is defined by an "effectorness gradient" of interrelated transcriptional states that culminate in increased effector function and tissue residency. scRNAseq of human AA skin showed that CD8+ T cells in human AA follow a similar trajectory, underscoring that shared mechanisms drive disease in both murine and human AA. Our study represents a comprehensive, systematic interrogation of lymphocyte heterogeneity in AA and uncovers a novel framework for AA-associated CD8+ T cells with implications for the design of future therapeutics.

An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes.

There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

Safety and efficacy of ultrasonography as an adjunct to fluoroscopy for renal access in percutaneous nephrolithotomy (PCNL).

OBJECTIVE: • To evaluate the safety and efficacy of ultrasonography (US)-guided renal access in percutaneous nephrolithotomy (PCNL), as compared with conventional fluoroscopy-guided renal access in a prospective randomized trial. PATIENTS AND METHODS: • From January 2008 to October 2009, 224 patients with renal calculi undergoing PCNL were randomized into two groups. • Group 1 (112 patients) underwent PCNL using only fluoroscopy-guided renal access; while in group 2 (112 patients), US guidance for puncture was used in addition to fluoroscopy. • The inclusion criteria were: normal renal functions, American Society of Anesthesiology scores 1 or 2, absence of congenital abnormalities, aged 15-70 years, and anticipated single-tract procedure. The patients in both groups were matched for age, sex, and stone characteristics. • The Student t-test was used for statistical analysis with an allowable error of 5%. RESULTS: • The mean time to successful puncture was 3.2 min and 1.8 min in group 1 and group 2, respectively (P < 0.01). • The mean duration of radiation exposure to successful puncture was 28.6 s in group 1 and 14.4 s in group 2 (P < 0.01). • The mean numbers of attempts for successful puncture in the desired calyx was 3.3 in group 1 as compared with 1.5 in group 2 (P < 0.01). • The meantime taken for tract formation in group 1 was 7.4 min with radiation exposure of 82 s, while in group 2 it took 4.8 min with radiation exposure of 58 s (P < 0.01). • Successful access was achieved in all patients. All patients were stone-free at the end of the operation. The hospital stay (2-3 days) was same in both groups. There was no incidence of significant bleeding requiring transfusion during or after surgery. All the patients were followed-up for a ≥ 6 months. CONCLUSION: • US-guided puncture in PCNL helps in increasing accuracy of puncture and decreasing radiation exposure for the surgical team and the patients.