Tissue-resident memory T (TRM) cells provide infectious, cancer and vaccine-trained immunity across barrier sites. TRM cells are implicated in autoimmunity, successful response to immune checkpoint blockade in the tumor microenvironment and toxicities that occur after immune checkpoint blockade in peripheral tissues. Here, we identified that signaling through the immune checkpoint programmed death receptor 1 (PD-1) strongly impacts the early specification of CD8+ TRM cells in the skin. PD-1 is expressed broadly across mouse and human skin TRM cells, in the absence of persistent infection, and is retained on skin TRM cells in aged mice. PD-1 supports early TRM cell colonization, skin-specific programming and silencing of other differentiation programs and promotes TGFβ responsivity and skin engraftment. Thus, PD-1 signaling mediates skin TRM cell specification during immune initiation. These findings may inform therapeutic PD-1 agonist and antagonist use to modulate successful peripheral memory. Anandasabapathy and colleagues show that the inhibitory receptor PD-1 impacts the specification of tissue-resident memory T cells in the skin.

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.

Background Immune checkpoint inhibitors (ICIs) are limited by the high incidence of immune-related adverse events (irAEs) occurring in up to 40% of solid tumor patients on anti-PD-1 monotherapy 1 2 and 72% in anti-CTLA-4/anti-PD-1 combination.3 4 These toxicities can cause treatment cessation, hospitalization and even death.5–7 IrAEs are variable in severity, timing, onset, and remain poorly understood. Amongst the different toxicities, skin irAEs are most frequent, occur the earliest, and are correlated with a positive prognosis.4 8 However, there is a lack of preclinical models to study checkpoint toxicity. We evaluated a murine model of allergic contact dermatitis (contact hypersensitivity to 2,4-dinitrofluorobenzene) that is mediated by CD8+ T cells to gain a mechanistic understanding of skin checkpoint toxicity. Methods C57BL/6 mice (n = 5 per group) were sensitized epicutaneously on shaved flank with hapten 0.5% DNFB on day -5 and elicited on their ears with DNFB on day 0. Starting four weeks later, mice were treated with either anti-programmed cell death protein (PD-1) or isotype. At the time of the first recall challenge only, mice were given either anti-PD-1 or isotype. Mice received subsequent rechallenges with DNFB to the ears and ear swelling was measured at various time points. Mice were depleted of circulating or skin CD8+ T cells by anti-CD8 mAbs from day 29 onwards, and maintained weekly, as in this model CD8+ T cells are the main hapten responder population. Samples were collected for histochemistry and analyzed by flow cytometry. Results Our data indicate that despite the depletion of circulating T cells, anti-PD-1 recipients mount a higher initial recall response to contact agents. Higher ear swelling was observed with increased inflammation in these mice. Our data suggest anti-PD-1 can liberate local T cell responses in the absence of a contribution from blood, and may offer a model to test therapeutic interventions to alleviate peripheral immune toxicities. Conclusions Our results suggest that this murine model of contact hypersensitivity represents a potential model for skin immune checkpoint toxicities. This model of locally-mediated inflammatory recall may advance the goal of uncoupling toxicity from efficacy in patients with immune-related adverse events. Ethics Approval The animal study was approved by Weill Cornell Medicine’s IACUC; approval number D16-00186. References Naidoo J, Page DB, Li BT, et al. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies. Ann Oncol 2015;26(12):2375–91. doi: 10.1093/annonc/mdv383. Belum VR, Benhuri B, Postow MA, et al. Characterisation and management of dermatologic adverse events to agents targeting the PD-1 receptor. Eur J Cancer 2016;60:12–25. doi: 10.1016/j.ejca.2016.02.010. Postow MA, Sidlow R, Hellmann MD. Immune-Related Adverse Events Associated with Immune Checkpoint Blockade. N Engl J Med 2018;378(2):158–168. doi: 10.1056/NEJMra1703481. Martins F, Sofiya L, Sykiotis GP, et al. Adverse effects of immune-checkpoint inhibitors: epidemiology, management and surveillance. Nat Rev Clin Oncol 2019;16(9):563–580. doi: 10.1038/s41571-019-0218-0. Puzanov I, Diab A, Abdallah K, et al. Managing toxicities associated with immune checkpoint inhibitors: consensus recommendations from the society for immunotherapy of cancer (SITC) Toxicity Management Working Group. J Immunother Cancer 2017;5(1):95. doi: 10.1186/s40425-017-0300-z. Wills B, Brahmer JR, Naidoo J. Treatment of complications from immune checkpoint inhibition in patients with lung cancer. Curr Treat Options Oncol 2018;19(9):46. doi: 10.1007/s11864-018-0562-9. Michot JM, Bigenwald C, Champiat S, et al. Immune-related adverse events with immune checkpoint blockade: a comprehensive review. Eur J Cancer 2016;54:139–148. doi: 10.1016/j.ejca.2015.11.016. Phillips GS, Wu J, Hellmann MD, et al. Treatment outcomes of immune-related cutaneous adverse events. J Clin Oncol 2019:JCO1802141. doi: 10.1200/JCO.18.02141.

Here we observe that PD-1 and other immune checkpoints are part of a highly conserved tissue specific resident memory program in both mouse and human TRM. We find activated T cells upregulate high cell-surface level of PD1 following scar with vaccinia virus expressing whole OVA protein (VACV-OVAss). Between weeks 3-4 post VACV-OVAss a dramatic decline of PD1 on memory T cells was observed whereas a population of 20%-80% of TRM retained intermediate levels of PD1 between 2-6 weeks. Mass cytometry data of human skin revealed that PD-1+ T cells in human skin express markers supporting antigen experience (CD45RO), skin-homing (CLA), epidermal residence (CD69, CD103, CD49a), and chemokine receptors like CXCR3 and CXCR6. PD-1 was observed across CD49a CD45RO T cells across other human peripheral tissues such as skin, lung, and liver above blood. We also observe PD-1 expression to be induced on TRM generated by inflammation secondary to the contact sensitizer DPCP (diphenylcyclopropenone), suggesting PD-1 is broadly conserved on TRM across species, and in pre-existing and newly generated TRM by either infection or inflammation. TRM co-express molecules highly related to cytotoxic T cells and effector cytokine production including multiple granzymes, perforin, and IFNg, and we find that both immune checkpoints and activation molecules are co-regulated by a single transcription factor Runx3. Together these data demonstrate that immune activation and inhibitory checkpoints typically associated with exhaustion are co-expressed and co-regulated and conserved across protective TRM, and future studies will test the functional biology of PD-1 on TRM development, persistence and recall.

Formation and recall of protective memory T cells is likely necessary for optimal anti-tumoral immunity in the adjuvant and neoadjuvant setting. Here, we find that tumor infiltrating lymphocytes (TILs) forming in memory-conditioned mice are phenotypically different from naive mice when implanted with dermal B16 melanoma engineered to express the OVA protein. TILs formed in memory conditioned mice express CD69+ and are CD103+/- whereas TILs formed in naive mice express low CD69 and CD103, comparable to CD45-low non-hematopoietic populations, suggesting a tumor memory-specific resident composition. In memory mice treated with FTY720, T cell numbers were observed to be 1.5-2 logs lower than controls. In keeping with this, when TILs were harvested from FTY720 treated memory mice a nearly complete absence of CD8+ TILs were observed demonstrating the majority of TILs enter from blood. However, when anti-PD1 was administered to memory mice on FTY720, melanoma growth was slowed and CD69+CD103+/- T cell numbers were restored in the tumor microenvironment. We also observed CD8+CD69+CD103+/- T cells expanded locally throughout the skin after anti-PD1 treatment, even at sites never exposed to antigen during either the priming or recall phase. These data suggest anti-PD1 expands T cells locally within tissues and tumor alike, which was further supported by visible aPD-1 targeting to both TILs and skin T cells. In contrast we did not identify targeting to or expansion of TCM or TEM in dLN or spleen in this context with the caveat that at 6 weeks of memory and recall, PD1 was low on memory T cells, and we did not preselect antigen specific T cells in the LN and spleen by tetramer. These results support the targeting and expansion of CD69+CD103+/- T cells in tumor and tissue alike. Future work examining the contribution of aPD-1 to TRM formation and recall may offer insight into current immunotherapies and associated checkpoint toxicities in tissues.

In contrast to an established role for tissue resident memory T cells (TRM) formation and protection to viral infections, formation and activity during cancer formation, including skin cancers is unknown. Here, we compare tumor-infiltrating lymphocytes (TILs) composition, circulating T cells, and tumor adjacent and non-adjacent skin T cells during a slow spontaneous model of melanoma formation (42-52 days). We tested BrafV600E, CDKN2A-/-, PTEN-/- mice to model the 40-50% of melanoma patients harboring Braf mutations (>90% BrafV600E). A majority of tumor infiltrating CD4+ (70%) and CD8+ (90%) of T lymphocytes express phenotypic markers for tissue residence such as CD69+ CD103+/-. We observed PD-1 expression on multiple T cell populations in the tumor microenvironment including CD3+ FoxP3+ CD25+ Treg. A significant accumulation of CD4+CD44+CD62L+KLRG1+CCR7low T cells was observed in the tumor draining LNs of mice with tumors, but not controls. Mice with tumors demonstrated higher T cell numbers in adjacent and non-adjacent skin. In contrast to TILs, PD-1 expression levels in tamoxifen-induced tumor mice were found to be similar to isotype and levels on control mice in the skin draining lymph nodes, tumor-adjacent and tumor non-adjacent skin, and spleen, suggesting that in this model PD-1 expression is largely restricted to tumor infiltrating lymphocytes. Collectively, these data suggest that in conditions favoring slow progressive spontaneous melanoma, TILs share phenotypic properties of TRM and are amongst the highest PD-1 expressing TILs. Subsequent work will further interrogate the cutaneous immune response to spontaneous melanoma skin cancer and the role of PD-1.

Tissue resident memory T cells (TRM) are non-recirculating memory T cells residing at epithelial sites which confer tissue-wide pathogen alarm and inflammatory recall. Here we identify broad expression of immune checkpoint transcripts programmed cell death protein 1 (PD-1), CTLA-4, and TIGIT on TRM across multiple viral infections and peripheral tissue sites in mouse. We find PD-1 expression is rapidly upregulated within 72 hours of viral priming in all memory-forming subsets after vaccinia viral infection but is contracted in TCM/TEM populations by 4 weeks. In contrast to prior reports, we find PD-1 protein expression is retained on a large fraction of CD69+CD103+/-TRM when tested at 5 and 6 weeks. PD-1 is expressed in steady state human skin TRM and on TRM accumulating in skin isolated from DPCP (diphenylcyclopropenone)-treated patients, above placebo, at 120 days post treatment. Because the PD-1 cognate ligand PD-L1 (CD274) is highly enriched on skin-derived DCs in mouse and human we tested the impact of PD-L1 and PD-L2 (CD273) loss on TRM-based recall against melanoma. We find an acute and transient loss of PD-L1 and PD-L2 in ZBTB46-dependent classical DCs during TRM recall significantly contracts tumors growth and improves overall survival. These results support classical DCs as a critical regulator of TRM-based recall and a dynamic role for the PD-L1/PD-L2:PD-1 axis.

Tumor infiltrating lymphocytes (TILs) isolated from epithelial tumors were previously reported to express phenotypic markers consonant with tissue resident memory T cells (TRM). We now find TILs isolated from human melanoma metastases, a non-epithelial tumor, share markers with TRM such as CD69 and CD103. A significant accumulation CD69+CD103+ CD8 T cells was seen comparing tumor involved nodes and non-involved nodes isolated from the same individual (p < .05). A significant fraction of CD69+ CD103+ T cells were also observed in human small bowel and skin metastases. While most CD8+ human melanoma TILs express CD69+, 10-40% of CD8+ T cells co-expressed CD103, a molecule known to regulate epithelial retention of T cells. The largest fraction of CD8+ T cells expressing PD-1 co-stained for both CD69+ and CD103+ and these cells bore high cell surface levels of both PD1 and Granzyme B. To further investigate the relatedness of TILs to TRM we derived a consensus signature using the transcriptome of murine TRM generated across 3 different sites (skin, lung, and gut) and infections (HSV, influenza, LCMV). When applying this signature against the single cell transcriptomes of 1500 T cells isolated from melanoma metastases we find two thirds of human melanoma T cells share common transcripts with tissue resident memory T cells. TILs sharing high tissue resident memory T cell signature transcripts also expressed high levels of immune checkpoint molecules, and markers of T cell activation such as Granzyme B. Future work will deconvolve the overlapping transcriptome program identified between activated, exhausted, and resident memory T cells and further relate these states to the programing and differentiation of T cells within the tumor microenvironment.

To maintain tissue immune homeostasis, a coordinated balance exists between maintenance of self-tolerance and protective immunity to pathogen challenge. Immune homeostasis is associated with a common tissue specific programming that prevails when immune cells enter peripheral sites. We previously identified a highly conserved program of 227 signature transcripts in human and mouse skin-derived DCs. We now find these transcripts are highly conserved across species and demonstrate transcriptional convergence across the entire monophagocyte lineage during tissue entry and progenitor differentiation in both fetal and adult states. We find this program broadly enriched across 30 primary human tumors largely originating in peripheral tissues. Moreover, human patient melanoma metastases bearing high, medium or low 227 signature expression demonstrated high, medium and low survival outcomes (p < .01), which did not correlate to overall non-silent gene mutation. Examining the single cell transcriptome of DCs and monocytes isolated from metastatic melanoma, we find enrichment of these signatures within monophagocytes in the tumor microenvironment when compared against T, B, NK, melanoma, endothelial, or cancer associated fibroblast cells. Collectively these data suggest programmatic convergence during monophagocyte differentiation in peripheral tissues such as the skin, may shape immune programming in the tumor microenvironment.

Dendritic cells (DCs) are specialized immune sentinels that play key role in maintaining immune homeostasis by efficiently regulating the delicate balance between protective immunity and tolerance to self. Although DCs respond to maturation signals present in the surrounding milieu, multiple layers of suppression also co-exist that reduce the infringement of tolerance against self-antigens. These tolerance inducing properties of DCs are governed by their origin and a range of other factors including distribution, cytokines, growth factors, and transcriptional programing, that collectively impart suppressive functions to these cells. DCs directing tolerance secrete anti-inflammatory cytokines and induce naïve T cells or B cells to differentiate into regulatory T cells (Tregs) or B cells. In this review, we provide a detailed outlook on the molecular mechanisms that induce functional specialization to govern central or peripheral tolerance. The tolerance-inducing nature of DCs can be exploited to overcome autoimmunity and rejection in graft transplantation.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Skin migratory dendritic cells (DCs) are subsets of specialize antigen-presenting cells that initiate and direct self-tolerogenic or pathogen-responsive immunity by recognizing self and foreign antigens in the skin and skin-draining LNs. However, their activating stimuli and networks for regulation are incompletely understood. Previously we identified a role for skin migratory DCs (migDCs) at actively dampening an ongoing immune response, using a protein vaccine that can be specifically recognized by DCs. Here, we describe a novel mechanism of regulation via induction of suppressor of cytokine signaling 2 (Socs2) during NF-kB signaling in migDCs. Socs2 is highly expressed in murine skin migratory DCs (migDCs), but not classical DC (cDCs), which reside in draining lymph node and spleen. Elevated levels of phosphorylated RelB (p-RelB), the active form of RelB that resides in the nuclei, was also observed in migDCs as compared to cDCs. Using two different bone marrow derived DC culture systems ex vivo, we find Socs2 is not expressed in DCs differentiated by Flt3L but constitutively expressed in GM-CSF/IL-4 differentiated DCs, suggesting alternative pathways of regulation. DCs isolated from Socs2 deficient mice demonstrate increased RelB phosphorylation and nuclear translocation upon LPS activation. These data indicate that Socs2 negatively regulates RelB during DC activation. Further work will examine whether this inhibitory activity in Socs2 is a determinant of DC differentiation in the steady state.

Skin migratory dendritic cells express high levels of PDL1 mRNA and dampen immune priming during protein immunization. However the role of the PDL1-PDL2/PD1 axis at contracting immune priming through the skin is unknown. Here we find that all skin-derived subsets of murine and human DCs, and LN resident CD11b+ classical DCs (cDC) express intermediate to high levels of the PDL1 cell surface protein. In contrast, CD8a+ LN resident DCs previously identified as critical for immunity express low levels of PDL1. High PDL1 was also identified on DCs and monocytes isolated from human melanoma metastases. To better understand the role of the PDL1/2:PD1 axis during DC based priming of T cells, we compared DC-targeted protein immunization of mice deficient in PDL1 alone or deficient in both PDL1 and PDL2. We find loss of PDL1/L2 but not PDL1 led to heightened T cell proliferation, during 4 day peptide recall. While the percentage of antigen specific CD4+ IFNg+ T cells assayed directly ex vivo by intracellular cytokine staining was unaltered in the lymph node directly draining the vaccination site of PDL1/PDL2-/- animals, in distal lymph nodes higher percentages of antigen specific T cells producing IFN-γ, IL-2 and TNF-α were identified. Collectively these data suggest that in absence of PDL1/L2 signaling, rapidly proliferating T cells exit the draining lymph node and circulate through distal nodes. Concordantly, we observe increased levels of E and P selectin binding on CD4 and CD8 T cells isolated from PDL1/2 deficient as compared to control animals. Subsequent work will examine the role of DC based PDL1/L2 signaling on the circulation and homing properties of T cells.

The present study delves into a combined bio-nano-macromolecular approach for bone tissue engineering. This approach relies on the properties of an ideal scaffold material imbued with all the chemical premises required for fostering cellular growth and differentiation. A tannic acid based water dispersible hyperbranched polyurethane is fabricated with bio-nanohybrids of carbon dot and four different peptides (viz. SVVYGLR, PRGDSGYRGDS, IPP, and CGGKVGKACCVPTKLSPISVLYK) to impart target specific in vivo bone healing ability. This polymeric bio-nanocomposite is blended with 10 wt% of gelatin and examined as a non-invasive delivery vehicle. In vitro assessment of the developed polymeric system reveals good osteoblast adhesion, proliferation, and differentiation. Aided by this panel of peptides, the polymeric bio-nanocomposite exhibits in vivo ectopic bone formation ability. The study on in vivo mineralization and vascularization reveals the occurrence of calcification and blood vessel formation. Thus, the study demonstrates carbon dot/peptide functionalized hyperbranched polyurethane gel for bone tissue engineering application.

Polysaccharides or heteroglycan isolated from fungal species have attained wide recognition for their immune modulating functions. However, there exists a significant relationship in the structural and functional activity of this molecule. In this commentary, we focus on a well characterized heteroglycan isolated from the mycelia of Pleurotus ostreatus which have been shown to drive immune stimulation in tumor bearing mice. The heteroglycan possessed high molecular weight and was primarily composed of glucose, mannose and fucose in a 3:2:1 ratio with major β- linkages in the repeating chain. The molecule also exhibited triple helical conformation in solutions. These immune driving molecules have a significant impact in decreasing the tumor volume in model mice and they are known to play a significant role in the proliferation and activation of lymphocytes in the tumor bearing mice. The physical traits of the heteroglycan were associated with its immune stimulating ability and such compounds can therefore be utilized as adjuvants in immune based therapies.

Active polysaccharides isolated from various fungal sources have been implicated to stimulate immune response against various pathogens as well as self anomalies such as cancer. Therefore, the nuanced approach presented in our work was to blend polysaccharides derived from Pleurotus ostreatus with bio-compatible ferrite nanoparticles and thereafter investigate the enhanced immune functionality of the polysaccharide-nanoparticle composite. A Schiff base reductive amination reaction occurred between the aldehyde group of the polysaccharide and the amine group of the nanoparticles in the presence of a strong reducing agent such as sodium cyanoborohydride to form a stable amide bond between the two conjugating molecules. The multifaceted conjugate was characterized by physiochemical techniques such as electron microscopy, FTIR. VSM and DLS measurements. This particulate form of the polysaccharide showed a marked escalation in the production of free radicals such as reactive oxygen and nitrogen species in murine macrophages as compared to the soluble form. Animal based experiments demonstrated a reduction in tumor volume and augmentation in the proliferation of splenocytes in particulate or conjugated polysaccharide treated mice. Furthermore, molecular signaling studies showed a high upregulation in p-p38 and p-MEK molecules in particulate polysaccharide treated RAW264.7 cells suggesting a cellular downstream mechanistic regulation behind the immunostimulative response. (C) 2014 Elsevier B.V. All rights reserved.

A water soluble β-glucan having molecular weight ∼2×10(5)Da was isolated from hot water extract of the fruit bodies of an edible mushroom Entoloma lividoalbum (Kühner & Romagn) Kubička. This polysaccharide (ELPS) contains (1→3,6)-β-D-Glcp, (1→3)-β-D-Glcp, (1→6)-β-D-Glcp, and terminal β-D-Glcp moieties in a molar ratio of nearly 1:1:3:1. Chemical and spectroscopic analysis showed that the backbone of glucan consists of three (1→6)-β-D-glucopyranosyl and two (1→3)-β-D-glucopyranosyl residues, out of which one (1→3)-β-D-glucopyranosyl moiety was branched at O-6 with a terminal β-D-glucopyranosyl residue. This β-glucan exhibited macrophage, splenocyte, and thymocyte stimulations. It possesses promising antioxidant activities as evidenced from its hydroxyl and superoxide radical scavenging activities and reducing properties.

The therapeutic benefits of glycans have garnered much attention over the last few decades with most studies being reported in 2D cultures or in animal models. The present work is therefore aimed to assess the effects of an immunomodulatory heteroglycan in a 3D milieu. Briefly, HT29 tumor spheroids were incubated with THP-1 macrophages at 1:1 ratio in a culture medium supplemented with immune stimulants such as heteroglycans or LPS. Spheroidal distortion, migration of tumor cells from the periphery of the spheroids and 46% of higher macrophage invasiveness was noted in heteroglycan-treated co-cultures with respect to control cultures. Histological sections of the treated co-cultures revealed the presence of high apoptotic tumor cells in the spheroidal periphery. CD11c and CD68 staining further suggested the predominance of macrophages in the vicinity of the apoptotic tumor cells. Such an in vitro created tissue system may thereby confirm the effectiveness of heteroglycan in activating the immune cells to exhibit tumor cytotoxic properties.