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Checkpoint blockade therapies that reactivate tumour-associated T cells can induce durable tumour control and result in the long-term survival of patients with advanced cancers 1 . Current predictive biomarkers for therapy response include high levels of intratumour immunological activity, a high tumour mutational burden and specific characteristics of the gut microbiota 2 , 3 . Although the role of T cells in antitumour responses has thoroughly been studied, other immune cells remain insufficiently explored. Here we use clinical samples of metastatic melanomas to investigate the role of B cells in antitumour responses, and find that the co-occurrence of tumour-associated CD8 + T cells and CD20 + B cells is associated with improved survival, independently of other clinical variables. Immunofluorescence staining of CXCR5 and CXCL13 in combination with CD20 reveals the formation of tertiary lymphoid structures in these CD8 + CD20 + tumours. We derived a gene signature associated with tertiary lymphoid structures, which predicted clinical outcomes in cohorts of patients treated with immune checkpoint blockade. Furthermore, B-cell-rich tumours were accompanied by increased levels of TCF7 + naive and/or memory T cells. This was corroborated by digital spatial-profiling data, in which T cells in tumours without tertiary lymphoid structures had a dysfunctional molecular phenotype. Our results indicate that tertiary lymphoid structures have a key role in the immune microenvironment in melanoma, by conferring distinct T cell phenotypes. Therapeutic strategies to induce the formation of tertiary lymphoid structures should be explored to improve responses to cancer immunotherapy. The co-occurrence of tumour-associated CD8 + T cells and CD20 + B cells, and the formation of tertiary lymphoid structures, are linked with improved survival in cohorts of patients with metastatic melanoma.

Evidence from mouse chronic viral infection models suggests that CD8 + T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8 + T lymphocyte populations with high (PD-1 T ), intermediate (PD-1 N ) and no PD-1 expression (PD-1 – ) from non-small-cell lung cancer patients. PD-1 T T cells showed a markedly different transcriptional and metabolic profile from PD-1 N and PD-1 – lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1 T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1 T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention. Tumor-infiltrating CD8 + T cells with high expression of PD-1 in non-small-cell lung cancer are distinct from exhausted T cells in chronic virus infection, have high tumor reactivity and associate with response to PD-1-targeted immunotherapy.

Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4⁺ helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1⁺CXCL13⁺ T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46%of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5⁺ normal B cells in close proximity to CXCL13⁺ T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1⁺CXCL13⁺ T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL (P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1⁺CXCL13⁺ T cells as a treatment target in LR-CHL.

Recent studies have implicated chemokines in microglial activation and pathogenesis of neuropathic pain. C-X-C motif chemokine 13 (CXCL13) is a B lymphocyte chemoattractant that activates CXCR5. Using the spinal nerve ligation (SNL) model of neuropathic pain, we found that CXCL13 was persistently upregulated in spinal cord neurons after SNL, resulting in spinal astrocyte activation via CXCR5 in mice. shRNA-mediated inhibition of CXCL13 in the spinal cord persistently attenuated SNL-induced neuropathic pain. Interestingly, CXCL13 expression was suppressed by miR-186-5p, a microRNA that colocalized with CXCL13 and was downregulated after SNL. Spinal overexpression of miR-186-5p decreased CXCL13 expression, alleviating neuropathic pain. Furthermore, SNL induced CXCR5 expression in spinal astrocytes, and neuropathic pain was abrogated in Cxcr5-/- mice. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. Intrathecal injection of CXCL13 was sufficient to induce pain hypersensitivity and astrocyte activation via CXCR5 and ERK. Finally, intrathecal injection of CXCL13-activated astrocytes induced mechanical allodynia in naive mice. Collectively, our findings reveal a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain. Thus, miR-186-5p and CXCL13/CXCR5-mediated astrocyte signaling may be suitable therapeutic targets for neuropathic pain.

Tertiary lymphoid structures are immune cell aggregates linked with cancer outcomes, but their interactions with tumour cell aggregates are unclear. Using nasopharyngeal carcinoma as a model, here we analyse single-cell transcriptomes of 343,829 cells from 77 biopsy and blood samples and spatially-resolved transcriptomes of 31,316 spots from 15 tumours to decipher their components and interactions with tumour cell aggregates. We identify essential cell populations in tertiary lymphoid structure, including CXCL13 + cancer-associated fibroblasts, stem-like CXCL13 + CD8 + T cells, and B and T follicular helper cells. Our study shows that germinal centre reaction matures plasma cells. These plasma cells intersperse with tumour cell aggregates, promoting apoptosis of EBV-related malignant cells and enhancing immunotherapy response. CXCL13 + cancer-associated fibroblasts promote B cell adhesion and antibody production, activating CXCL13 + CD8 + T cells that become exhausted in tumour cell aggregates. Tertiary lymphoid structure-related cell signatures correlate with prognosis and PD-1 blockade response, offering insights for therapeutic strategies in cancers. The interactions between tertiary lymphoid structures (TLS) and tumor cell aggregates remain to be investigated. Here, single-cell and spatial transcriptomics reveal the cellular composition, formation, and function of TLS during tumour progression and immunotherapy response in nasopharyngeal carcinoma.

Fat-associated lymphoid clusters are lymphoid tissues that support B-1 cells. Caamaño and colleagues show that inflammation that elicits the cytokine TNF and activates natural killer cells contributes to the formation of these clusters in visceral fat. Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

The authors identify in patients with rheumatoid arthritis a pathogenic subset of CD4+ T cells that augments B cell responses within inflamed tissues. Peripheral helper T cells in rheumatoid arthritis Michael Brenner and colleagues identify a subset of pathogenically activated PD-1 hi CD4-positive T cells in patients with rheumatoid arthritis, and show that it promotes B-cell responses in tertiary lymphoid structures. The cells, which the authors designate as 'peripheral helper' T cells, differ from follicular helper cells in that they lack CXCR5, have altered BCL6 expression, and express chemokine receptors that direct migration to inflamed sites. CD4 + T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4 + T cells within affected tissues may be identified by expression of markers of recent activation 1 . Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population 2 . This approach revealed a markedly expanded population of PD-1 hi CXCR5 − CD4 + T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1 hi CXCR5 − ‘peripheral helper’ T (T PH ) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1 hi CXCR5 + T follicular helper cells, T PH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3 , 4 ). However, global transcriptomics highlight differences between T PH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in T PH cells. T PH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

In human inflammatory sites, PD-1 hi CXCR5 − CD4 + T cells are involved in the formation of ectopic lymphoid-like structures (ELSs) by the secretion of chemokine CXCL13, but how the transcription of CXCL13 is regulated in CD4 + T cells is still unclear. Here we show that Sox4 is a key transcription factor for CXCL13 production in human CD4 + T cells under inflammatory conditions. In vitro TGF-β + , IL-2-neutralizing culture conditions give rise to PD-1 hi CXCR5 − CD4 + T cells that preferentially express CXCL13, and transcriptome analysis and lentiviral overexpression indicate Sox4 association with the CXCL13 transcription. In vivo, Sox4 is significantly upregulated in synovial CD4 + T cells, when compared with blood CD4 + T cells, from patients with rheumatoid arthritis (RA), and further correlates with ELS formation in RA synovium. Overall, our studies suggest that Sox4 contributes to CXCL13 production and ELS formation at inflammatory sites in humans. At inflammatory sites, ectopic lymphoid-like structures (ELS) can be induced through the function of chemokine CXCL13 produced by CD4 + T cells. Here the authors show that a transcription factor, Sox4, induces the expression of CXCL13 in CD4 T cells in vitro, and is associated with ELS formation in patients with rheumatoid arthritis.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell–B cell interactions 1 , 2 . Expansion of T follicular helper (T FH ) and T peripheral helper (T PH ) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE 3 , 4 . Human T FH and T PH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs.  5 , 6 ), yet regulation of T cell CXCL13 production and the relationship between CXCL13 + T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4 + T cell phenotypes in patients with SLE, with expansion of PD-1 + /ICOS + CXCL13 + T cells and reduction of CD96 hi IL-22 + T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4 + T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13 + T PH /T FH cell differentiation and promote an IL-22 + phenotype. Type I interferon, a pathogenic driver of SLE 7 , opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13 + T PH /T FH cells on a polarization axis opposite from T helper 22 (T H 22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states. Insufficient AHR activation has been suggested in SLE, and augmenting AHR activation therapeutically may prevent CXCL13 + T PH /T FH differentiation and the subsequent recruitment of B cells and formation of lymphoid aggregates in inflamed tissues.

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13 + follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients. Morphogens such as chemokines form gradients to direct graded responses and modulate cell behaviors. Here the authors show, using imaging and computer simulation, that the chemokine CXCL13 originated from follicular reticular cells in the lymph nodes forms both soluble and immobilized gradients to regulate B cell recruitment and migration.