CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Tumor reactivity of CD8+ T cells favors acquisition of dysfunctional states in human melanoma.

A recent study by Oliveira et al. provides, in unprecedented detail, novel insights into the relationship between tumorreactivity and functional states of CD8 T cells in metastatic melanoma.

Ptpn2 and KLRG1 regulate the generation and function of tissue-resident memory CD8+ T cells in skin.

Tissue-resident memory T cells (TRM cells) are key elements of tissue immunity. Here, we investigated the role of the regulator of T cell receptor and cytokine signaling, Ptpn2, in the formation and function of TRM cells in skin. Ptpn2-deficient CD8+ T cells displayed a marked defect in generating CD69+ CD103+ TRM cells in response to herpes simplex virus type 1 (HSV-1) skin infection. This was accompanied by a reduction in the proportion of KLRG1- memory precursor cells and a transcriptional bias toward terminal differentiation. Of note, forced expression of KLRG1 was sufficient to impede TRM cell formation. Normalizing memory precursor frequencies by transferring equal numbers of KLRG1- cells restored TRM generation, demonstrating that Ptpn2 impacted skin seeding with precursors rather than downstream TRM cell differentiation. Importantly, Ptpn2-deficient TRM cells augmented skin autoimmunity but also afforded superior protection from HSV-1 infection. Our results emphasize that KLRG1 repression is required for optimal TRM cell formation in skin and reveal an important role of Ptpn2 in regulating TRM cell functionality.

A high-salt diet compromises antibacterial neutrophil responses through hormonal perturbation.

The Western diet is rich in salt, which poses various health risks. A high-salt diet (HSD) can stimulate immunity through the nuclear factor of activated T cells 5 (Nfat5)-signaling pathway, especially in the skin, where sodium is stored. The kidney medulla also accumulates sodium to build an osmotic gradient for water conservation. Here, we studied the effect of an HSD on the immune defense against uropathogenic E. coli-induced pyelonephritis, the most common kidney infection. Unexpectedly, pyelonephritis was aggravated in mice on an HSD by two mechanisms. First, on an HSD, sodium must be excreted; therefore, the kidney used urea instead to build the osmotic gradient. However, in contrast to sodium, urea suppressed the antibacterial functionality of neutrophils, the principal immune effectors against pyelonephritis. Second, the body excretes sodium by lowering mineralocorticoid production via suppressing aldosterone synthase. This caused an accumulation of aldosterone precursors with glucocorticoid functionality, which abolished the diurnal adrenocorticotropic hormone-driven glucocorticoid rhythm and compromised neutrophil development and antibacterial functionality systemically. Consistently, under an HSD, systemic Listeria monocytogenes infection was also aggravated in a glucocorticoid-dependent manner. Glucocorticoids directly induced Nfat5 expression, but pharmacological normalization of renal Nfat5 expression failed to restore the antibacterial defense. Last, healthy humans consuming an HSD for 1 week showed hyperglucocorticoidism and impaired antibacterial neutrophil function. In summary, an HSD suppresses intrarenal neutrophils Nfat5-independently by altering the local microenvironment and systemically by glucocorticoid-mediated immunosuppression. These findings argue against high-salt consumption during bacterial infections.

PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

Accumulation of CD103+ CD8+ T cells in a cutaneous melanoma micrometastasis.

OBJECTIVE: The immune system can halt cancer progression by suppressing outgrowth of clinically occult micrometastases in a state of cancer-immune equilibrium. Cutaneous melanoma provides a unique opportunity to study the immune contexture of such lesions, as miniscule skin metastases are accessible to clinical inspection and diagnostic biopsy. METHODS: Here, we analysed by multiplex immunofluorescence microscopy samples from a melanoma patient presenting with an overt and an occult in-transit metastasis (ITM), the latter of which appeared as a small erythematous papule. RESULTS: Microarchitecture and immune composition in the two lesions were vastly different. CD4+ and CD8+ T cells accumulated around the margin of the overt SOX10+ Melan A+ ITM but were largely excluded from the tumor centre. By contrast, the occult micrometastasis contained only few SOX10+ Melan A- melanoma cells which were scattered within a dense infiltrate of T cells, including a prominent population of CD103+ CD8+ T cells resembling tissue-resident memory T (TRM) cells. Notably, almost every single melanoma cell in the micrometastasis was in close proximity to these TRM-like cells. CONCLUSION: Such results support the emerging concept that CD103+ CD8+ TRM cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.

Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells.

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation, and function. Here, we examined the impact of the microbiota on CD8+ T cell memory. Antigen-activated CD8+ T cells transferred into germ-free mice failed to transition into long-lived memory cells and had transcriptional impairments in core genes associated with oxidative metabolism. The microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8+ T cells, and SCFAs were required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that butyrate uncoupled the tricarboxylic acid cycle from glycolytic input in CD8+ T cells, which allowed preferential fueling of oxidative phosphorylation through sustained glutamine utilization and fatty acid catabolism. Our findings reveal a role for the microbiota in promoting CD8+ T cell long-term survival as memory cells and suggest that microbial metabolites guide the metabolic rewiring of activated CD8+ T cells to enable this transition.

Author Correction: Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

Panel j was inadvertently labelled as panel k in the caption to Fig. 4. Similarly, 'Fig. 4k' should have been 'Fig. 4j' in the sentence beginning 'TNF-α-deficient gBT-I cells were…'. In addition, the surname of author Umaimainthan Palendira was misspelled 'Palendria'. These errors have been corrected online.

Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

The immune system can suppress tumour development both by eliminating malignant cells and by preventing the outgrowth and spread of cancer cells that resist eradication1. Clinical and experimental data suggest that the latter mode of control-termed cancer-immune equilibrium1-can be maintained for prolonged periods of time, possibly up to several decades2-4. Although cancers most frequently originate in epithelial layers, the nature and spatiotemporal dynamics of immune responses that maintain cancer-immune equilibrium in these tissue compartments remain unclear. Here, using a mouse model of transplantable cutaneous melanoma5, we show that tissue-resident memory CD8+ T cells (TRM cells) promote a durable melanoma-immune equilibrium that is confined to the epidermal layer of the skin. A proportion of mice (~40%) transplanted with melanoma cells remained free of macroscopic skin lesions long after epicutaneous inoculation, and generation of tumour-specific epidermal CD69+ CD103+ TRM cells correlated with this spontaneous disease control. By contrast, mice deficient in TRM formation were more susceptible to tumour development. Despite being tumour-free at the macroscopic level, mice frequently harboured melanoma cells in the epidermal layer of the skin long after inoculation, and intravital imaging revealed that these cells were dynamically surveyed by TRM cells. Consistent with their role in melanoma surveillance, tumour-specific TRM cells that were generated before melanoma inoculation conferred profound protection from tumour development independently of recirculating T cells. Finally, depletion of TRM cells triggered tumour outgrowth in a proportion (~20%) of mice with occult melanomas, demonstrating that TRM cells can actively suppress cancer progression. Our results show that TRM cells have a fundamental role in the surveillance of subclinical melanomas in the skin by maintaining cancer-immune equilibrium. As such, they provide strong impetus for exploring these cells as targets of future anticancer immunotherapies.

CRISPR/Cas9: A tool for immunological research.

The CRISPR/Cas9-system was originally identified as part of the adaptive immune system in bacteria and has since been adapted for the genetic manipulation of eukaryotic cells. The technique is of particular value for biomedical sciences, as it enables the genetic manipulation of cell lines and primary cells as well as whole organisms with unprecedented ease and efficiency. Furthermore, the CRISPR/Cas9-technology has the potential for future therapeutic applications in the clinic. Here, we discuss the use of CRISPR/Cas9 for the genetic modification of haematopoietic cells and the generation of mouse models for immunological research. Additionally, we explain how the technique can be applied as a screening-tool to identify genes involved in different immunological processes. Moreover, we will talk about recent extensions of using the CRISPR/Cas9 technology, such as a transcriptional activator or repressor. Finally, we discuss the first clinical trials that use CRISPR/Cas9 and discuss potential future applications.

Effective Priming of Herpes Simplex Virus-Specific CD8+ T Cells In Vivo Does Not Require Infected Dendritic Cells.

Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection.IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.

Sustained accumulation of antigen-presenting cells after infection promotes local T-cell immunity.

Antigen-presenting cells (APC), such as dendritic cells (DC) and macrophages, are critical for T-cell-mediated immunity. Although it is established that memory T cells accumulate and persist in peripheral tissues after the resolution of infection, whether this is also the case for APC remains unclear. Here, we report that CCR2-dependent cells infiltrate skin during acute infection with herpes simplex virus (HSV)-1 and subsequently give rise to localized populations of DCs and macrophages. These APC are found at elevated numbers at sites of resolved infection or inflammation compared with unaffected regions of skin. Importantly, this local accumulation of APC is sustained for prolonged periods of time and has important functional consequences, as it promotes interferon-γ responses by virus-specific CD4+ T cells upon localized challenge infection with HSV-1. Thus, our results highlight how infection history determines long-term changes in immune cell composition in skin and how different types of immune cells accumulate, persist and co-operate to provide optimal immunity at this critical barrier site.

Cutting Edge: The RIG-I Ligand 3pRNA Potently Improves CTL Cross-Priming and Facilitates Antiviral Vaccination.

Protective immunity against intracellular pathogens involves the induction of robust CTL responses. Vaccination with protein Ags establishes such responses only when combined with immune-stimulatory adjuvants. In this study, we compared different adjuvants and identified triphosphate RNA (3pRNA) as especially effective at inducing CTL responses. 3pRNA sensing required IPS-1/MAVS signaling and induced type I IFN in plasmacytoid dendritic cells and macrophages, with the latter being more important for the adjuvant effect. Type I IFN acted on CD11c(+) cells, especially on CD8α(+) Batf3-dependent dendritic cells. Vaccination with OVA in combination with 3pRNA protected mice from a subsequent OVA-encoding adenovirus infection in a CD8(+) cell-dependent manner and more efficiently than other adjuvants. In summary, 3pRNA is a superior adjuvant for CTL activation and might be useful to facilitate antiviral immunization strategies.

Selective Dependence of Kidney Dendritic Cells on CX3CR1--Implications for Glomerulonephritis Therapy.

As central regulators of the adaptive immune response, dendritic cells (DCs) are found in virtually all lymphatic and non-lymphatic organs. A compact network of DCs also spans the kidneys. DCs play a central role in maintenance of organ homeostasis as well as in induction of immune responses against invading pathogens. They can mediate protective or destructive functions in a context-dependent manner.We recently identified CX(3)CR1 as a kidney-specific "homing receptor" for DCs. There was a strong reduction of DCs in the kidneys of CX(3)CR1-deficient mice compared to controls. This reduction was not observed in other organs except the small intestine. As a possible underlying reason we found a strong expression of the CX(3)CR1 ligand fractalkine in the kidneys. Due to this CX(3)CR1-dependent reduction of DCs, especially in the renal cortex, a glomerulonephritis (GN) model was ameliorated in CX(3)CR1-deficient mice. In contrast, the immune defense against the most common renal infection, bacterial pyelonephritis (PN), was not significantly influenced by CX(3)CR1-deficiency. This was explained by the much smaller CX(3)CR1-dependency of medullary DCs, which recruit effector cells into the kidney during PN. Additionally, once neutrophils had been recruited by mechanisms distinct from CX(3)CR1, they carried out some of the functions of DCs.Taken together, we suggest CX(3)CR1 as a therapeutic target for GN treatment, as the absence of CX(3)CR1 selectively influences DCs in the kidney without rendering mice more susceptible towards bacterial kidney infections.

Exclusive CX3CR1 dependence of kidney DCs impacts glomerulonephritis progression.

DCs and macrophages both express the chemokine receptor CX3CR1. Here we demonstrate that its ligand, CX3CL1, is highly expressed in the murine kidney and intestine. CX3CR1 deficiency markedly reduced DC numbers in the healthy and inflamed kidney cortex, and to a lesser degree in the kidney medulla and intestine, but not in other organs. CX3CR1 also promoted influx of DC precursors in crescentic glomerulonephritis, a DC-dependent aggressive type of nephritis. Disease severity was strongly attenuated in CX3CR1-deficient mice. Primarily CX3CR1-dependent DCs in the kidney cortex processed antigen for the intrarenal stimulation of T helper cells, a function important for glomerulonephritis progression. In contrast, medullary DCs played a specialized role in inducing innate immunity against bacterial pyelonephritis by recruiting neutrophils through rapid chemokine production. CX3CR1 deficiency had little effect on the immune defense against pyelonephritis, as medullary DCs were less CX3CR1 dependent than cortical DCs and because recruited neutrophils produced chemokines to compensate for the DC paucity. These findings demonstrate that cortical and medullary DCs play specialized roles in their respective kidney compartments. We identify CX3CR1 as a potential therapeutic target in glomerulonephritis that may involve fewer adverse side effects, such as impaired anti-infectious defense or compromised DC functions in other organs.

Ligand-specific binding and activation of the human adenosine A(2B) receptor.

Adenosine A(2B) receptors, which play a role in inflammation and cancer, are of considerable interest as novel drug targets. To gain deeper insights into ligand binding and receptor activation, we exchanged amino acids predicted to be close to the binding pocket. The alanine mutants were stably expressed in CHO cells and characterized by radioligand binding and cAMP assays using three structural classes of ligands: xanthine (antagonist), adenosine, and aminopyridine derivatives (agonists). Asn282(7.45) and His280(7.43) were found to stabilize the binding site by intramolecular hydrogen bond formation as in the related A(2A) receptor subtype. Trp247(6.48), Val250(6.51), and particularly Ser279(7.42) were shown to be important for binding of nucleosidic agonists. Leu81(3.28), Asn186(5.42), and Val250(6.51) were discovered to be crucial for binding of the xanthine-derived antagonist PSB-603. Leu81(3.28), which is not conserved among adenosine receptor subtypes, may be important for the high selectivity of PSB-603. The N186(5.42)A mutant resulted in an increased potency for agonists. The interactions of the non-nucleosidic agonist BAY60-6583 were different from those of the nucleosides: while BAY60-6583 appeared not to interact with Ser279(7.42), its interactions with Trp247(6.48) and Val250(6.51) were significantly weaker compared to those of NECA. Moreover, our results discount the hypothesis of Trp247(6.48) serving as a "toogle switch" because BAY60-6583 was able to activate the corresponding mutant. This study reveals distinct interactions of structurally diverse ligands with the human A(2B) receptor and differences between closely related receptor subtypes (A(2B) and A(2A)). It will contribute to the understanding of G protein-coupled receptor function and advance A(2B) receptor ligand design.

Isolation of functional dendritic cells from murine kidneys for immunological characterization.

AIM: The kidney is a complex organ, requiring the contributions of multiple cell types to perform its various functions. Within this system the dendritic cell has been demonstrated to play a key role in maintaining the immunological balance of the kidney. In this methods paper we aim to identify the best method for isolating murine renal dendritic cells. METHODS: The efficiency of isolating dendritic cells from enzymatically digested renal parenchyma by density centrifugation, MACS and FACS was compared. RESULTS: Density centrifugation enriched dendritic cells by only approximately two fold. However, MACS and FACS resulted in a much higher purity (80% versus 95% respectively). CONCLUSIONS: Although FACS gave the highest purity, MACS is the optimal method for isolating dendritic cells given cost and time factors. Isolation of a homogeneous population of renal dendritic cells will enable the molecular and functional dissection of these cells in both homeostasis and disease models.