Histone-Specific CD4+ T Cell Plasticity in Active and Quiescent Systemic Lupus Erythematosus.

OBJECTIVE: The aim of this study was to assess whether circulating histone-specific T cells represent tools for precision medicine in systemic lupus erythematosus (SLE). METHODS: Seroprevalence of autoantibodies and HLA-DR beta (DRB) 1 profile were assessed among 185 patients with SLE and combined with bioinformatics and literature evidence to identify HLA-peptide autoepitope couples for ex vivo detection of antigen-specific T cells through flow cytometry. T cell differentiation and polarization was investigated in patients with SLE, patients with Takayasu arteritis, and healthy controls carrying HLA-DRB1*03:01 and/or HLA-DRB1*11:01. SLE Disease Activity Index 2000 and Lupus Low Disease Activity State were used to estimate disease activity and remission. RESULTS: Histone-specific CD4+ T cells were selectively detected in patients with SLE. Among patients with a history of anti-DNA antibodies, 77% had detectable histone-specific T cells, whereas 50% had lymphocytes releasing cytokines or upregulating activation markers after in vitro challenge with histone peptide antigens. Histone-specific regulatory and effector T helper (Th) 1-, Th2-, and atypical Th1/Th17 (Th1*)-polarized cells were significantly more abundant in patients with SLE with quiescent disease. In contrast, total Th1-, Th2-, and Th1*-polarized and regulatory T cells were similarly represented between patients and controls or patients with SLE with active versus quiescent disease. Histone-specific effector memory T cells accumulated in the blood of patients with quiescent SLE, whereas total effector memory T cell counts did not change. Immunosuppressants were associated with expanded CD4+ histone-specific naive T (TN) and terminally differentiated T cells. CONCLUSION: Histone-specific T cells are selectively detected in patients with SLE, and their concentration in the blood varies with disease activity, suggesting that they represent innovative tools for patient stratification and therapy.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells.

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity. Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs). Results: Activated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature. Conclusion: These data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Dynamics of polyclonal immuno-reconstitution after allogeneic transplant with post-transplant cyclophosphamide and letermovir.

Cytomegalovirus (CMV) reactivations are strong stimulators of immune-reconstitution (IR) in hematopoietic stem cell transplantation (HSCT) recipients. Herein, we analyzed 317 CMV-seropositive consecutive patients (n = 109 letermovir, LTV; n = 208 no-LTV), undergoing HSCT with post-transplant cyclophosphamide (PTCy) and calcineurin inhibitor- (CNI) free graft-versus-host-disease (GvHD) prophylaxis. At day+90, median CD19+/mm3 was higher in LTV-cohort: 5.5 [0;439] versus 2 [0;294], p = 0.008; median CD3+/mm3 counts were lower in LTV-cohort, with no differences in CD4+, CD8+ and NK-cells. At day+180 median CD3+, CD4+ and CD8+/mm3 values were comparable between groups. Higher CD19+/mm3 counts were observed in LTV-cohort: 62 [0; 2983] versus 42 [0; 863]. Significantly higher median NK/mm3 values were seen in LTV-cohort: 225.5 [0;763] versus 163.5 [0;1181], p = 0.0003. The impact of LTV on B-cell IR at 3 months and NK-cell levels at 6 months was retained in multivariate analysis (p < 0.01), whereas the effect on T-cells was not confirmed. Moreover, we confirmed a significant reduction of clinically-relevant CMV, and moderate-to- severe chronic GvHD in LTV-cohort. Overall, in our study the use of LTV was associated with a slight improvement of B-cell and NK-cells reconstitution, with only minor impact on T-cell subsets, giving new insights on polyclonal IR for HSCT recipients in the LTV era.

Case Report: Favorable outcome of allogeneic hematopoietic stem cell transplantation in SARSCoV2 positive recipient, risk-benefit balance between infection and leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in SARS-CoV-2 positive candidates is usually delayed until the clinical resolution of the infection's symptoms and a negative nasopharyngeal molecular test. However, prolonged SARS-CoV-2 positivity has been frequently observed in haematological malignancies, thus representing a challenge for the timing of transplant procedures. Here, we report on the case of a 34-year-old patient with recent pauci-symptomatic COVID-19 undergoing transplant for high-risk acute B-lymphoblastic leukemia before achieving viral clearance. Shortly before their scheduled allogeneic HSCT from a matched unrelated donor, the patient developed mild Omicron BA.5 infection receiving nirmatrelvir/ritonavir with fever resolution within 72 hours. Twenty-three days after COVID-19 diagnosis, because of increasing minimal residual disease values in the context of high-risk refractory leukemia and clinical resolution of SARS-2-CoV infection with reduction of viral load at surveillance nasopharyngeal swabs, it was decided not to delay further allo-HSCT. During myelo-ablative conditioning, the nasopharyngeal SARS-CoV-2 viral load increased while the patient remained asymptomatic. Consequently, two days before the transplant, intra-muscular tixagevimab/cilgavimab 300/300 mg and a 3-day course of intravenous remdesivir were administered. During the pre-engraftment phase, veno-occlusive disease (VOD) occurred at day +13, requiring defibrotide treatment to obtain a slow but complete recovery. The post-engraftment phase was characterized by mild COVID-19 at day +23 (cough, rhino-conjunctivitis, fever) that spontaneously resolved, achieving viral clearance at day +28. At day +32, she experienced grade I acute graft-versus host disease (a-GVHD, skin grade II) treated with steroids and photo-apheresis, without further complications during follow-up until day +180. Addressing the issue of allo-HSCT timing in patients recovering from SARS-CoV-2 infection with high-risk malignant diseases is challenging because of 1] the high risk of COVID-19 clinical progression, 2] the impact of transplant delay on leukemia prognosis and 3] the occurrence of endothelial complications such as VOD, a-GVHD, and transplant associated thrombotic micro-angiopathy. Our report describes the favourable outcome of allo-HSCT in a recipient with active SARS-CoV2 infection and high-risk leukemia thanks to timely anti-SARS-CoV-2 preventive therapies and prompt management of transplant-related complications.

Human herpesvirus 6-specific T-cell immunity in allogeneic hematopoietic stem cell transplant recipients.

Human herpesvirus 6 (HHV-6) can reactivate after allogeneic hematopoietic stem cell transplant (allo-HSCT) and may lead to severe symptoms. HHV-6-specific immune responses after HSCT are largely unexplored. We conducted a prospective observational study on 208 consecutive adult patients who received allo-HSCT to investigate HHV-6 reactivations and specific immune responses. Interferon gamma-producing HHV-6-specific T cells were quantified using enzyme-linked immunospot assay (ELISpot). HHV-6 reactivation occurred in 63% of patients, at a median of 25 days from allo-HSCT. Only 40% of these presented a clinically relevant infection, defined by the presence of classical HHV-6 end-organ diseases (EODs), based on European Conference on Infections in Leukaemia (ECIL) guidelines, and other possible HHV6-related EODs. Using multivariate analysis, we identified risk factors for HHV-6 reactivation: previous allo-HSCT, posttransplant cyclophosphamide (PT-Cy), and time-dependent steroids introduction. The use of PT-Cy and steroids were associated with clinically relevant infections, whereas higher CD3+ cell counts seemed to be protective. Interestingly, circulating HHV-6-specific T cells were significantly higher in patients with reactivated virus. Moreover, HHV-6-specific T-cell responses, quantified at >4 days after the first viremia detection, predicted clinically relevant infections (P < .0001), with higher specificity (93%) and sensitivity (79%) than polyclonal CD3+ cells per µL. Overall survival and transplant-related mortality were not affected by time-dependent HHV-6 reactivation, whereas a significant association was observed between clinically relevant infections and acute graft-versus-host disease. These results shed light on the role of HHV-6 in allo-HSCT and may affect HHV-6 monitoring and treatment.

Cytomegalovirus-specific T cells restricted for shared and donor human leukocyte antigens differentially impact on cytomegalovirus reactivation risk after allogeneic hematopoietic stem cell transplantation.

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations.

Profiling Antibody Response Patterns in COVID-19: Spike S1-Reactive IgA Signature in the Evolution of SARS-CoV-2 Infection.

This contribution explores in a new statistical perspective the antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 141 coronavirus disease 2019 (COVID-19) patients exhibiting a broad range of clinical manifestations. This cohort accurately reflects the characteristics of the first wave of the SARS-CoV-2 pandemic in Italy. We determined the IgM, IgA, and IgG levels towards SARS-CoV-2 S1, S2, and NP antigens, evaluating their neutralizing activity and relationship with clinical signatures. Moreover, we longitudinally followed 72 patients up to 9 months postsymptoms onset to study the persistence of the levels of antibodies. Our results showed that the majority of COVID-19 patients developed an early virus-specific antibody response. The magnitude and the neutralizing properties of the response were heterogeneous regardless of the severity of the disease. Antibody levels dropped over time, even though spike reactive IgG and IgA were still detectable up to 9 months. Early baseline antibody levels were key drivers of the subsequent antibody production and the long-lasting protection against SARS-CoV-2. Importantly, we identified anti-S1 IgA as a good surrogate marker to predict the clinical course of COVID-19. Characterizing the antibody response after SARS-CoV-2 infection is relevant for the early clinical management of patients as soon as they are diagnosed and for implementing the current vaccination strategies.

Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells.

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

TCR Redirected T Cells for Cancer Treatment: Achievements, Hurdles, and Goals.

Adoptive T cell therapy (ACT) is a rapidly evolving therapeutic approach designed to harness T cell specificity and function to fight diseases. Based on the evidence that T lymphocytes can mediate a potent anti-tumor response, initially ACT solely relied on the isolation, in vitro expansion, and infusion of tumor-infiltrating or circulating tumor-specific T cells. Although effective in a subset of cases, in the first ACT clinical trials several patients experienced disease progression, in some cases after temporary disease control. This evidence prompted researchers to improve ACT products by taking advantage of the continuously evolving gene engineering field and by improving manufacturing protocols, to enable the generation of effective and long-term persisting tumor-specific T cell products. Despite recent advances, several challenges, including prioritization of antigen targets, identification, and optimization of tumor-specific T cell receptors, in the development of tools enabling T cells to counteract the immunosuppressive tumor microenvironment, still need to be faced. This review aims at summarizing the major achievements, hurdles and possible solutions designed to improve the ACT efficacy and safety profile in the context of liquid and solid tumors.

Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade.

PURPOSE: Hyperprogression (HP), a paradoxical boost in tumor growth, was described in a subset of patients treated with immune checkpoint inhibitors (ICI). Neither clinicopathologic features nor biological mechanisms associated with HP have been identified. EXPERIMENTAL DESIGN: Among 187 patients with non-small cell lung cancer (NSCLC) treated with ICI at our institute, cases with HP were identified according to clinical and radiologic criteria. Baseline histologic samples from patients treated with ICI were evaluated by IHC for myeloid and lymphoid markers. T-cell-deficient mice, injected with human lung cancer cells and patient-derived xenografts (PDX) belonging to specific mutational subsets, were assessed for tumor growth after treatment with antibodies against mouse and human programmed death receptor-1 (PD-1). The immune microenvironment was evaluated by flow cytometry and IHC. RESULTS: Among 187 patients, 152 were evaluable for clinical response. We identified four categories: 32 cases were defined as responders (21%), 42 patients with stable disease (27.7%), 39 cases were defined as progressors (25.7%), and 39 patients with HP (25.7%). Pretreatment tissue samples from all patients with HP showed tumor infiltration by M2-like CD163+CD33+PD-L1+ clustered epithelioid macrophages. Enrichment by tumor-associated macrophages (TAM) was observed, even in tumor nodules from immunodeficient mice injected with human lung cancer cells and with PDXs. In these models, tumor growth was enhanced by treatment with anti-PD-1 but not anti-PD-1 F(ab)2 fragments. CONCLUSIONS: These results suggest a crucial role of TAM reprogramming, upon Fc receptor engagement by ICI, eventually inducing HP and provide clues on a distinctive immunophenotype potentially able to predict HP.See related commentary by Knorr and Ravetch, p. 904.

Fibroblast Growth Factor Binding Protein 3 (FGFBP3) impacts carbohydrate and lipid metabolism.

Secreted FGF binding proteins (FGFBP) mobilize locally-acting paracrine FGFs from their extracellular storage. Here, we report that FGFBP3 (BP3) modulates fat and glucose metabolism in mouse models of metabolic syndrome. BP3 knockout mice exhibited altered lipid metabolism pathways with reduced hepatic and serum triglycerides. In obese mice the expression of exogenous BP3 reduced hyperglycemia, hepatosteatosis and weight gain, blunted de novo lipogenesis in liver and adipose tissues, increased circulating adiponectin and decreased NEFA. The BP3 protein interacts with endocrine FGFs through its C-terminus and thus enhances their signaling. We propose that BP3 may constitute a new therapeutic to reverse the pathology associated with metabolic syndrome that includes nonalcoholic fatty liver disease and type 2 diabetes mellitus.

The non-small cell lung cancer immune landscape: emerging complexity, prognostic relevance and prospective significance in the context of immunotherapy.

Immunotherapy of non-small cell lung cancer (NSCLC), by immune checkpoint inhibitors, has profoundly improved the clinical management of advanced disease. However, only a fraction of patients respond and no effective predictive factors have been defined. Here, we discuss the prospects for identification of such predictors of response to immunotherapy, by fostering an in-depth analysis of the immune landscape of NSCLC. The emerging picture, from several recent studies, is that the immune contexture of NSCLC lesions is a complex and heterogeneous feature, as documented by analysis for frequency, phenotype and spatial distribution of innate and adaptive immune cells, and by characterization of functional status of inhibitory receptor+ T cells. The complexity of the immune landscape of NSCLC stems from the interaction of several factors, including tumor histology, molecular subtype, main oncogenic drivers, nonsynonymous mutational load, tumor aneuploidy, clonal heterogeneity and tumor evolution, as well as the process of epithelial-mesenchymal transition. All these factors contribute to shape NSCLC immune profiles that have clear prognostic significance. An integrated analysis of the immune and molecular profile of the neoplastic lesions may allow to define the potential predictive role of the immune landscape for response to immunotherapy.

The Role of Fibroblast Growth Factor-Binding Protein 1 in Skin Carcinogenesis and Inflammation.

Fibroblast growth factor-binding protein 1 (FGFBP1) is a secreted chaperone that mobilizes paracrine-acting FGFs, stored in the extracellular matrix, and presents them to their cognate receptors. FGFBP1 enhances FGF signaling including angiogenesis during cancer progression and is upregulated in various cancers. Here we evaluated the contribution of endogenous FGFBP1 to a wide range of organ functions as well as to skin pathologies using Fgfbp1-knockout mice. Relative to wild-type littermates, knockout mice showed no gross pathologies. Still, in knockout mice a significant thickening of the epidermis associated with a decreased transepidermal water loss and increased proinflammatory gene expression in the skin was detected. Also, skin carcinogen challenge by 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate resulted in delayed and reduced papillomatosis in knockout mice. This was paralleled by delayed healing of skin wounds and reduced angiogenic sprouting in subcutaneous matrigel plugs. Heterozygous green fluorescent protein (GFP)-knock-in mice revealed rapid induction of gene expression during papilloma induction and during wound healing. Examination of wild-type skin grafted onto Fgfbp1 GFP-knock-in reporter hosts and bone marrow transplants from the GFP-reporter model into wild-type hosts revealed that circulating Fgfbp1-expressing cells migrate into healing wounds. We conclude that tissue-resident and circulating Fgfbp1-expressing cells modulate skin carcinogenesis and inflammation.

Blood Pressure Control by a Secreted FGFBP1 (Fibroblast Growth Factor-Binding Protein).

Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2-/- mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2-/- model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure.

Early Effector T Lymphocytes Coexpress Multiple Inhibitory Receptors in Primary Non-Small Cell Lung Cancer.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed "early effector cells" (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non-small cell lung cancer. Cancer Res; 77(4); 851-61. ©2016 AACR.

Late failure of a split-thickness skin graft in the setting of homozygous factor V Leiden mutation: a case report and correlative animal model from the Wound Etiology and Healing (WE-HEAL) study.

We present the case of a 53-year-old Caucasian male smoker with remote history of left lower extremity deep venous thrombosis (DVT) and a strong family history of thrombosis, who presented to the Center for Wound Healing at MedStar Georgetown University Hospital with spontaneous left leg ulceration. Prothrombotic evaluation showed homozygosity for the factor V Leiden (FVL) mutation. Therapeutic anticoagulation was commenced with warfarin (Coumadin®) and the patient underwent successful debridement and Apligraf® followed by split-thickness skin graft (STSG) of two wounds. He had an uneventful postoperative course and on the 27th postoperative day the grafts were 95% intact. However, by postoperative day 41 there was 10% graft loss, and over the subsequent 2 weeks both grafts necrosed. On further questioning, it transpired that the patient had discontinued his warfarin on postoperative day 37 because he thought that it was no longer necessary. The patient is enrolled in the Wound Etiology and Healing (WE-HEAL) study, and at the time of the original graft, residual skin fragments from the STSG were transplanted onto a nude mouse for development of an animal model of wound healing. The mouse graft was successful and was harvested at postoperative day 87 for pathological examination. We review the mechanisms by which prothrombotic states, particularly FVL mutation, can contribute to skin graft failure and delayed wound healing. This case highlights the importance of considering prothrombotic conditions in patients with spontaneous leg ulcerations and the impact of therapeutic anticoagulation on healing. It further allows us to demonstrate the efficacy of the animal model in which residual fragments of STSG tissue are utilised for transplant onto nude mice for manipulation in the laboratory.

Utility of a human-mouse xenograft model and in vivo near-infrared fluorescent imaging for studying wound healing.

To study the complex cellular interactions involved in wound healing, it is essential to have an animal model that adequately mimics the human wound microenvironment. Currently available murine models are limited because wound contraction introduces bias into wound surface area measurements. The purpose of this study was to demonstrate utility of a human-mouse xenograft model for studying human wound healing. Normal human skin was harvested from elective abdominoplasty surgery, xenografted onto athymic nude (nu/nu) mice, and allowed to engraft for 3 months. The graft was then wounded using a 2-mm punch biopsy. Wounds were harvested on sequential days to allow tissue-based markers of wound healing to be followed sequentially. On the day of wound harvest, mice were injected with XenoLight RediJect cyclooxygenase-2 (COX-2) probe and imaged according to package instructions. Immunohistochemistry confirms that this human-mouse xenograft model is effective for studying human wound healing in vivo. Additionally, in vivo fluorescent imaging for inducible COX-2 demonstrated upregulation from baseline to day 4 (P = 0·03) with return to baseline levels by day 10, paralleling the reepithelialisation of the wound. This human-mouse xenograft model, combined with in vivo fluorescent imaging provides a useful mechanism for studying molecular pathways of human wound healing.