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Background: Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury., Methods: Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina., Results: Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer., Conclusions: Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration., (© 2024. The Author(s).)

Tissue-resident CD8 + T cells (T RM ) continuously scan peptide-MHC (pMHC) complexes in their organ of residence to intercept microbial invaders. Recent data showed that T RM lodged in exocrine glands scan tissue in the absence of any chemoattractant or adhesion receptor signaling, thus bypassing the requirement for canonical migration-promoting factors. The signals eliciting this noncanonical motility and its relevance for organ surveillance have remained unknown. Using mouse models of viral infections, we report that exocrine gland T RM autonomously generated front-to-back F-actin flow for locomotion, accompanied by high cortical actomyosin contractility, and leading-edge bleb formation. The distinctive mode of exocrine gland T RM locomotion was triggered by sensing physical confinement and was closely correlated with nuclear deformation, which acts as a mechanosensor via an arachidonic acid and Ca 2+ signaling pathway. By contrast, naïve CD8 + T cells or T RM surveilling microbe-exposed epithelial barriers did not show mechanosensing capacity. Inhibition of nuclear mechanosensing disrupted exocrine gland T RM scanning and impaired their ability to intercept target cells. These findings indicate that confinement is sufficient to elicit autonomous T cell surveillance in glands with restricted chemokine expression and constitutes a scanning strategy that complements chemosensing-dependent migration.

Background: Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis-because of injury, disease, or aging-initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration., Methods: In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration., Results: We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood-retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1 + cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8 + T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response., Conclusions: Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The tyrosine kinase inhibitor dasatinib is approved for Philadelphia chromosome-positive leukemia, including chronic myeloid leukemia (CML). Although effective and well tolerated, patients typically exhibit a transient lymphocytosis after dasatinib uptake. To date, the underlying physiological process linking dasatinib to lymphocytosis remains unknown. Here, we used a small rodent model to examine the mechanism of dasatinib-induced lymphocytosis, focusing on lymphocyte trafficking into and out of secondary lymphoid organs. Our data indicate that lymphocyte homing to lymph nodes and spleen remained unaffected by dasatinib treatment. In contrast, dasatinib promoted lymphocyte egress from spleen with kinetics consistent with the observed lymphocytosis. Unexpectedly, dasatinib-induced lymphocyte egress occurred independently of canonical sphingosine-1-phosphate-mediated egress signals; instead, dasatinib treatment led to a decrease in spleen size, concomitant with increased splenic stromal cell contractility, as measured by myosin light chain phosphorylation. Accordingly, dasatinib-induced lymphocytosis was partially reversed by pharmacological inhibition of the contraction-promoting factor Rho-rho associated kinase. Finally, we uncovered a decrease in spleen size in patients with CML who showed lymphocytosis immediately after dasatinib treatment, and this reduction was proportional to the magnitude of lymphocytosis and dasatinib plasma levels. In summary, our work provides evidence that dasatinib-induced lymphocytosis is a consequence of drug-induced contractility of splenic stromal cells., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (T RM ) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes -OVA. In bone marrow chimeras, Rgs1 -/- and Rgs1 +/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes -OVA, however, OT-I Rgs1 +/+ T cells outnumbered the co-transferred OT-I Rgs1 - /- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1 -/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1 +/+ T RM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1 -/- T RM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8 + T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 von Werdt, Gungor, Barreto de Albuquerque, Gruber, Zysset, Kwong Chung, Corrêa-Ferreira, Berchtold, Page, Schenk, Kehrl, Merkler, Imhof, Stein, Abe, Turchinovich, Finke, Hayday, Corazza and Mueller.)

Migration and adhesion play critical roles in B cells, regulating recirculation between lymphoid organs, migration within lymphoid tissue, and interaction with CD4+ T cells. However, there is limited knowledge of how B cells integrate chemokine receptor and integrin signaling with B cell activation to generate efficient humoral responses. Here, we show that the WNK1 kinase, a regulator of migration and adhesion, is essential in B cells for T-dependent and -independent antibody responses. We demonstrate that WNK1 transduces signals from the BCR, CXCR5, and CD40, and using intravital imaging, we show that WNK1 regulates migration of naive and activated B cells, and their interactions with T cells. Unexpectedly, we show that WNK1 is required for BCR- and CD40-induced proliferation, acting through the OXSR1 and STK39 kinases, and for efficient B cell-T cell collaboration in vivo. Thus, WNK1 is critical for humoral immune responses, by regulating B cell migration, adhesion, and T cell-dependent activation., (© 2023 Hayward et al.)

High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass.

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1 + VCAM-1 + follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wissmann, Stolp, Jímenez and Stein.)

Lymph nodes (LNs) comprise two main structural elements: fibroblastic reticular cells that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. Immunological challenge causes LNs to increase more than tenfold in size within a few days. Here, we characterized the biomechanics of LN swelling on the cellular and organ scale. We identified lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing fibroblastic reticular cells of the T-zone (TRCs) and their associated conduits to stretch. After an initial phase of relaxation, TRCs sensed the resulting strain through cell matrix adhesions, which coordinated local growth and remodeling of the stromal network. While the expanded TRC network readopted its typical configuration, a massive fibrotic reaction of the organ capsule set in and countered further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multitier fashion., (© 2022. The Author(s).)

The gastrointestinal (GI) tract constitutes an essential barrier against ingested microbes, including potential pathogens. Although immune reactions are well studied in the lower GI tract, it remains unclear how adaptive immune responses are initiated during microbial challenge of the oral mucosa (OM), the primary site of microbial encounter in the upper GI tract. Here, we identify mandibular lymph nodes (mandLNs) as sentinel lymphoid organs that intercept ingested Listeria monocytogenes (Lm). Oral Lm uptake led to local activation and release of antigen-specific CD8 + T cells that constituted most of the early circulating effector T cell (T EFF ) pool. MandLN-primed T EFF disseminated to lymphoid organs, lung, and OM and contributed substantially to rapid elimination of target cells. In contrast to CD8 + T EFF generated in mesenteric LN (MLN) during intragastric infection, mandLN-primed T EFF lacked a gut-seeking phenotype, which correlated with low expression of enzymes required for gut-homing imprinting by mandLN stromal and dendritic cells. Accordingly, mandLN-primed T EFF decreased Lm burden in spleen but not MLN after intestinal infection. Our findings extend the concept of regional specialization of immune responses along the length of the GI tract, with CD8 + T EFF generated in the upper GI tract displaying homing profiles that differ from those imprinted by lymphoid tissue of the lower GI tract.

Bruton's tyrosine kinase inhibitor ibrutinib has significantly changed treatment landscape in chronic lymphocytic leukemia (CLL). Growing evidence supports ibrutinib to work beyond the effect on tumor cells by means of, for example, restoring functionality of the T-cell compartment and increasing circulating T-cell numbers. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, whether the latter physiological change is also a consequence of a forced retention in blood remains undisclosed. Since CCR7 is the main chemokine receptor taking over the homing of T-cells from peripheral compartments to lymph nodes and other SLO, we aimed to investigate the impact of ibrutinib on CCR7 functionality in T-cells. To this end, we documented receptor expression in T-cells from a large cohort of ibrutinib-treated CLL patients, and performed different in vivo and in vitro migration models. Overall, our data confirm that CCR7 expression or receptor-mediated migration in CLL T-cells is not affected by ibrutinib. Furthermore, it does not modulate CCR7-driven homing nor nodal interstitial migration. Together, our results support that ibrutinib-induced CLL T-cell accumulation in the blood stream is not derived from an impairment of CCR7-driven recirculation between the SLO and bloodstream, and therefore T-cell expansion is the most plausible cause.

CD169 + macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169 + macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTβ) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTβR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTβR revealed that both receptors contribute equally to LN CD169 + macrophage differentiation. In the spleen, the Cd169 -directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8 + T cells. Taken together, the data provide evidence that CD169 + macrophage differentiation in LN and spleen requires dual signals from LTβR and RANK with implications for the immune response., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)