Same yet different - how lymph node heterogeneity affects immune responses.

Lymph nodes are secondary lymphoid organs in which immune responses of the adaptive immune system are initiated and regulated. Distributed throughout the body and embedded in the lymphatic system, local lymph nodes are continuously informed about the state of the organs owing to a constant drainage of lymph. The tissue-derived lymph carries products of cell metabolism, proteins, carbohydrates, lipids, pathogens and circulating immune cells. Notably, there is a growing body of evidence that individual lymph nodes differ from each other in their capacity to generate immune responses. Here, we review the structure and function of the lymphatic system and then focus on the factors that lead to functional heterogeneity among different lymph nodes. We will discuss how lymph node heterogeneity impacts on cellular and humoral immune responses and the implications for vaccination, tumour development and tumour control by immunotherapy.

Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming.

T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.

Lymph node medulla regulates the spatiotemporal unfolding of resident dendritic cell networks.

Unlike macrophage networks composed of long-lived tissue-resident cells within specific niches, conventional dendritic cells (cDCs) that generate a 3D network in lymph nodes (LNs) are short lived and continuously replaced by DC precursors (preDCs) from the bone marrow (BM). Here, we examined whether specific anatomical niches exist within which preDCs differentiate toward immature cDCs. In situ photoconversion and Prtn3-based fate-tracking revealed that the LN medullary cords are preferential entry sites for preDCs, serving as specific differentiation niches. Repopulation and fate-tracking approaches demonstrated that the cDC1 network unfolded from the medulla along the vascular tree toward the paracortex. During inflammation, collective maturation and migration of resident cDC1s to the paracortex created discontinuity in the medullary cDC1 network and temporarily impaired responsiveness. The decrease in local cDC1 density resulted in higher Flt3L availability in the medullary niche, which accelerated cDC1 development to restore the network. Thus, the spatiotemporal development of the cDC1 network is locally regulated in dedicated LN niches via sensing of cDC1 densities.

Cytotoxic CNS-associated T cells drive axon degeneration by targeting perturbed oligodendrocytes in PLP1 mutant mice.

Myelin defects lead to neurological dysfunction in various diseases and in normal aging. Chronic neuroinflammation often contributes to axon-myelin damage in these conditions and can be initiated and/or sustained by perturbed myelinating glia. We have previously shown that distinct PLP1 mutations result in neurodegeneration that is largely driven by adaptive immune cells. Here we characterize CD8+ CNS-associated T cells in myelin mutants using single-cell transcriptomics and identify population heterogeneity and disease-associated changes. We demonstrate that early sphingosine-1-phosphate receptor modulation attenuates T cell recruitment and neural damage, while later targeting of CNS-associated T cell populations is inefficient. Applying bone marrow chimerism and utilizing random X chromosome inactivation, we provide evidence that axonal damage is driven by cytotoxic, antigen specific CD8+ T cells that target mutant myelinating oligodendrocytes. These findings offer insights into neural-immune interactions and are of translational relevance for neurological conditions associated with myelin defects and neuroinflammation.

NFATc1 induction by an intronic enhancer restricts NKT γδ cell formation.

In thymus, the ablation of T cell receptor (TCR)-activated transcription factor NFATc1 or its inducible isoforms during the double-negative (DN) stages of thymocyte development leads to a marked increase in γδ thymocytes whereas the development of αß thymocytes remains mostly unaffected. These γδ thymocytes are characterized by the upregulation of the promyelocytic leukemia zinc-finger factor (PLZF), the "master regulator" of natural killer T (NKT) cell development, and the acquisition of an NKT γδ cell phenotype with higher cell survival rates. The suppressive function of NFATc1 in NKT γδ cell formation critically depends on the remote enhancer E2, which is essential for the inducible expression of NFATc1 directed by its distal promoter P1. Thus, the enhancer deciphers a strong γδ TCR signal into the expression of inducible NFATc1 isoforms resulting in high levels of NFATc1 protein that are essential to control the numbers of NKT γδ cells.

Lymphatic migration of unconventional T cells promotes site-specific immunity in distinct lymph nodes.

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

Type 1 conventional dendritic cells maintain and guide the differentiation of precursors of exhausted T cells in distinct cellular niches.

Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology.

A multifunctional mouse model to study the role of Samd3.

The capacity to develop immunological memory is a hallmark of the adaptive immune system. To investigate the role of Samd3 for cellular immune responses and memory development, we generated a conditional knock-out mouse including a fluorescent reporter and a huDTR cassette for conditional depletion of Samd3-expressing cells. Samd3 expression was observed in NK cells and CD8 T cells, which are known for their specific function against intracellular pathogens like viruses. After acute viral infections, Samd3 expression was enriched within memory precursor cells and the frequency of Samd3-expressing cells increased during the progression into the memory phase. Similarly, during chronic viral infections, Samd3 expression was predominantly detected within precursors of exhausted CD8 T cells that are critical for viral control. At the functional level however, Samd3-deficient CD8 T cells were not compromised in the context of acute infection with Vaccinia virus or chronic infection with Lymphocytic choriomeningitis virus. Taken together, we describe a novel multifunctional mouse model to study the role of Samd3 and Samd3-expressing cells. We found that Samd3 is specifically expressed in NK cells, memory CD8 T cells, and precursor exhausted T cells during viral infections, while the molecular function of this enigmatic gene remains further unresolved.

Neutrophils self-limit swarming to contain bacterial growth in vivo.

Neutrophils communicate with each other to form swarms in infected organs. Coordination of this population response is critical for the elimination of bacteria and fungi. Using transgenic mice, we found that neutrophils have evolved an intrinsic mechanism to self-limit swarming and avoid uncontrolled aggregation during inflammation. G protein-coupled receptor (GPCR) desensitization acts as a negative feedback control to stop migration of neutrophils when they sense high concentrations of self-secreted attractants that initially amplify swarming. Interference with this process allows neutrophils to scan larger tissue areas for microbes. Unexpectedly, this does not benefit bacterial clearance as containment of proliferating bacteria by neutrophil clusters becomes impeded. Our data reveal how autosignaling stops self-organized swarming behavior and how the finely tuned balance of neutrophil chemotaxis and arrest counteracts bacterial escape.

Accumulation of cytotoxic T cells in the aged CNS leads to axon degeneration and contributes to cognitive and motor decline.

Aging is a major risk factor for the development of nervous system functional decline, even in the absence of diseases or trauma. The axon-myelin units and synaptic terminals are some of the neural structures most vulnerable to aging-related deterioration1-6, but the underlying mechanisms are poorly understood. In the peripheral nervous system, macrophages-important representatives of the innate immune system-are prominent drivers of structural and functional decline of myelinated fibers and motor endplates during aging7. Similarly, in the aging central nervous system (CNS), microglial cells promote damage of myelinated axons and synapses8-20. Here we examine the role of cytotoxic CD8+ T lymphocytes, a type of adaptive immune cells previously identified as amplifiers of axonal perturbation in various models of genetically mediated CNS diseases21 but understudied in the aging CNS22-25. We show that accumulation of CD8+ T cells drives axon degeneration in the normal aging mouse CNS and contributes to age-related cognitive and motor decline. We characterize CD8+ T-cell population heterogeneity in the adult and aged mouse brain by single-cell transcriptomics and identify aging-related changes. Mechanistically, we provide evidence that CD8+ T cells drive axon degeneration in a T-cell receptor- and granzyme B-dependent manner. Cytotoxic neural damage is further aggravated by systemic inflammation in aged but not adult mice. We also find increased densities of T cells in white matter autopsy material from older humans. Our results suggest that targeting CD8+ CNS-associated T cells in older adults might mitigate aging-related decline of brain structure and function.

In Situ Maturation and Tissue Adaptation of Type 2 Innate Lymphoid Cell Progenitors.

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1+ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1+ ST2- ILCPs to Il18r- ST2+ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

CCL17 exerts a neuroimmune modulatory function and is expressed in hippocampal neurons.

Chemokines are important signaling molecules in the immune and nervous system. Using a fluorescence reporter mouse model, we demonstrate that the chemokine CCL17, a ligand of the chemokine receptor CCR4, is produced in the murine brain, particularly in a subset of hippocampal CA1 neurons. We found that basal expression of Ccl17 in hippocampal neurons was strongly enhanced by peripheral challenge with lipopolysaccharide (LPS). LPS-mediated induction of Ccl17 in the hippocampus was dependent on local tumor necrosis factor (TNF) signaling, whereas upregulation of Ccl22 required granulocyte-macrophage colony-stimulating factor (GM-CSF). CCL17 deficiency resulted in a diminished microglia density under homeostatic and inflammatory conditions. Further, microglia from naïve Ccl17-deficient mice possessed a reduced cellular volume and a more polarized process tree as assessed by computer-assisted imaging analysis. Regarding the overall branching, cell surface area, and total tree length, the morphology of microglia from naïve Ccl17-deficient mice resembled that of microglia from wild-type mice after LPS stimulation. In line, electrophysiological recordings indicated that CCL17 downmodulates basal synaptic transmission at CA3-CA1 Schaffer collaterals in acute slices from naïve but not LPS-treated animals. Taken together, our data identify CCL17 as a homeostatic and inducible neuromodulatory chemokine affecting the presence and morphology of microglia and synaptic transmission in the hippocampus.