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Endothelial cells (ECs) are central to vascular health but also interact with and regulate the immune system. Changes in endothelial state enable immune cells to migrate into the tissue to facilitate repair and fight infection. ECs modulate the function of immune cells through the expression of adhesion molecules, chemokines, major histocompatibility complex (MHC), and an array of co-stimulatory and inhibitor molecules. These interactions allow ECs to act as antigen presenting cells (APCs) and influence the outcome of immune recognition. This study elucidates how EC microenvironment, vascular cell biology, and immune response are not only connected but interdependent. More specifically, we explored how cell-substratum interactions influence EC antigen presentation and co-stimulation, and how these differences affect allorecognition in animal models of cell transplantation. Investigation of EC state was carried out using RNA sequencing while assessment of the allogeneic response includes measurements of immune cell cytotoxic ability, T cell proliferation, cytokine release, serum antibodies, and histological staining. Differences in substratum led to divergent EC phenotypes which in turn influenced immune response to transplanted cells, both due to the physical barrier of matrix-adhesion and differences in expression of surface markers. ECs grown in 2D on tissue culture plastic or in 3D on collagen scaffolds had significantly different basal levels of MHC expression, co-stimulatory and adhesion molecules. When treated with cytokines to mimic an inflammatory state, ECs did not converge to a single phenotype but rather responded differently based on their substratum. Generally, 3D ECs were more responsive to inflammatory stimuli than 2D ECs. These unique expression patterns measured in vitro also influence immune recognition in vivo . ECs grown in 2D were more likely to provoke a cytotoxic response while 3D ECs induced T cell proliferation. ECs are uniquely configured to sense not only local flow and mechanical forces but a range of markers related to systemic state, including immune function. ECs interact with immune cells with differing results depending on the environment in which the EC-lymphocyte interaction occurs. Therefore, understanding this relationship is essential to predicting and modifying the outcome of EC-immune interacts. We specifically examined the relationship between EC substratum and allorecognition.

Relapsed and/or refractory acute myeloid leukemia (AML) post-allogeneic hematopoietic cell transplantation (HCT) is usually fatal. We previously reported that post-HCT immunotherapy with Epstein-Barr virus (EBV)-specific donor CD8 + T cells engineered to express a Wilms Tumor Antigen 1-specific T-cell receptor (T TCR-C4 ) appeared to prevent relapse in high-risk patients. In this phase I/II clinical trial (NCT01640301), we evaluated safety (primary endpoint), persistence and efficacy (secondary endpoints) of EBV- or Cytomegalovirus (CMV)-specific T TCR-C4 in fifteen patients with active AML post-HCT. Infusions were well tolerated, with no dose-limiting toxicities or serious adverse events related to the product. However, T TCR-C4 cells did not clearly improve outcomes despite EBV-specific T TCR-C4 cells showing enhanced potential for prolonged persistence compared to CMV-specific T TCR-C4 . Investigating the fate of persisting T TCR-C4 , we identified a shift towards natural killer-like (NKL) terminal differentiation, distinct from solid tumor-associated canonical exhaustion programs. In one patient, treatment with azacitidine appeared to mitigate this NKL skewing, promoting T TCR-C4 persistence. These findings suggest that AML drives a distinct form of T-cell dysfunction, highlight the need for targeted approaches that preserve T-cell fitness, ultimately improving the efficacy of cellular therapies for AML. Relapsed and/or refractory acute myeloid leukemia (AML) postallogeneic hematopoietic cell transplantation has limited treatment options. Here the authors report the clinical results and immune correlates of a phase I/II trial of adoptively transferred virus-specific donor CD8 + T cells engineered to express a WT1-specific T cell receptor in patients with acute myeloid leukemia and active disease post-allogeneic hematopoietic cell transplantation.

T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to \"resist\" M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ-independent T cell responses to M. tuberculosis-specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and \"resistance\" to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO 2 ) that can achieve a sterility assurance level 10 −6 in decellularized lung matrix. The effects of ScCO 2 treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO 2 did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO 2 , indicating that ScCO 2 produces a matrix that is stable during storage. The current study's results indicate that ScCO 2 can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes.

BackgroundT cells genetically modified to express a high affinity T cell receptor (TCR) have considerable potential for the treatment of cancer, however identifying immunogenic, tumor-specific targets remains a challenge. Recent transcriptomic analysis has shown that splicing mutations are frequently found across many different cancer types and can be drivers of tumorigenesis. Heterozygous mutations in SRSF2 (Serine and arginine Rich Splicing Factor 2), a protein involved in pre-mRNA splicing are prevalent across multiple types of high-risk myeloid neoplasms.1 The P95H mutation results in altered binding affinity of SRSF2 for cognate RNA site in target transcripts, producing abnormal proteins. Indeed, cell lines modified to express a SFSR2-P95H/+ mutation showed mis-regulation of hundreds of splicing events potentially exposing a large pool of unique, tumor-associated peptides that could be used to develop effective treatments (figure 1).MethodsTo identify presented peptides, HLA-specific single chain dimers, which have the transmembrane domain replaced with a His-Tag allowing for secretion of the peptide/HLA in the supernatant, are transduced into SRSF2 wild type and mutant cells lines for downstream mass-spectrometry analysis (figure 2).2 We used this versatile platform to discover HLA-specific peptides resulting from alternative splicing events induced by a mutation in SRSF2. We then used recently published computational analysis to select promising mutant-derived peptides by aligning mass spectrometry-derived peptides to RNAseq data from our cell lines as well as broader tumor transcriptome databases.3 Finally, we tested our candidate peptides for their ability to induce tumor-specific killing by growing out T cell lines from healthy donors through repeated cycles of peptide stimulation (figure 3).ResultsUsing eluted single chain dimers, we identified thousands of peptides exclusive to mutant P95H/+ K562s cells for three different HLAs (HLA- A2, A11, and A24) that together cover 71% of phenotype of the US. We found that 11% of peptides are exclusive to the mutant cells (figure 4). Next, computational analysis of RNA sequencing data from wild type and mutant cells narrowed the list of peptides to those which resulted from alternate splicing events, against which T cell lines could be developed. Validating the identified peptides, we found multiple peptides which were able to trigger T cell activation and proliferation.ConclusionsThis work demonstrates a robust method to discover novel HLA/peptide pairs and extend the reach of engineered TCR-T cell therapies. These techniques can easily be adapted for other mutations associated with RNA processing including SF3B1, U2AF1 and DDX41.AcknowledgementsThis work was funded by the Fred Hutch Immunotherapy Integrated Research Center (IIRC) and the MPN Research Foundation. The authors wish to thank Kathryn Finton for developing the ARTEMIS system, Deborah Baker for writing and editing support, and the staff of the Fred Hutch flow core for making sure experiments run smoothly.ReferencesKim E, et al. SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition. Cancer Cell. 2015;27:617–630.Finton K, et al. ARTEMIS: A Novel Mass-Spec Platform for HLA-Restricted Self and Disease-Associated Peptide Discovery. Front Immunol. 2021;12:658372.Pan Y, et al. IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing. Proc National Acad Sci. 2023;120:e2221116120.Abstract 1399 Figure 1Alternative splicing resulting in targetable peptides. Alternatively spliced RNA that is translated into a protein and processed by the proteasome may produce mutation-specific peptides. When loaded into an HLA and presented on the surface, these peptide-HLA (pHLA) complexes can be recognized by TCR-T cell therapiesAbstract 1399 Figure 2Single chain dimer peptide discovery pipeline. Single chain dimers (SCDs) for each HLA of interest are transduced into SRSF2 WT and P95H K562s. Transduced cells are sorted and cultured, usually with the addition of IFNy and TNFα. During the culture period, SCDs are secreted into the supernatant which is collected at the end of the culture. Peptides are then eluted from the SCDs and identified using mass spectometryAbstract 1399 Figure 3T cell line discovery pipeline. Dendritic cells (DCs) are isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and matured using a cytokine treatment. DCs are loaded with peptides of interest and cultured with donor-matched CD8+ T cells for two weeks. This cycle is repeated 1–2 more times using peptide-loaded PBMCs. After overnight stimulation with peptides, CD137+ (41BB) cells are sorted and expanded. These T cell lines are then tested for their ability to respond to SRSF2 wild type and mutant (P95H) K562 lines. This protocol aims to identify lines that kill mutant cells while sparing wild type cellsAbstract 1399 Figure 4Venn diagram comparisons of eluted peptides. The three HLAs selected have distinct binding preferences (left). Combining peptides from the three HLAs, approximately 11% of peptides are exclusive to the SRSF2 mutant (P95H) cells (right). These peptides are potential candidates for the development of TCR-T cells. Results from 2x A2, 3x All, and 3x A24 biological replicates

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (T S ) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index T S cells and tracked their frequency in bulk TCRβ repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory T S cell clonotypes, most of which were CD8 + T cells, while also eliciting diverse T S cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8 + and CD4 + TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRβ repertoires and paired-chain TCRɑβ sequences from droplet sequencing of T S cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens. Koelle and colleagues use an activation marker-dependent approach to determine the recruitment of TCR by three doses of mRNA vaccination in individuals previously infected with SARS-CoV-2.

BackgroundPatients with melanoma refractory to PD-1 inhibitors have limited therapeutic options. Personalized neoantigen vaccines can induce tumor-specific T-cell responses, but their efficacy in the PD-1-refractory setting remains uncertain. This phase I study evaluated the immunologic impact of the neoantigen peptide vaccine PNV-021 in PD-1-refractory melanoma.MethodsEight patients received up to 20 neoantigen peptides, predicted to bind HLA class I/II, every four weeks alongside Poly-ICLC and nivolumab. Peripheral blood mononuclear cells (PBMCs) were collected every 4 weeks and tumor biopsies were collected before and 12 weeks post-vaccination. Tumor biopsies underwent single-cell RNA and TCR sequencing. In four patients with either progressive disease (Pt5), stable disease (Pt13), or partial responses (Pt6, Pt16), vaccine-specific T cell responses were assessed by ELISpot using PBMCs. The 25–55 most expanded or de novo tumor-associated TCRs at week 12 were reconstituted in Jurkat reporter cells and tested for reactivity against patient-derived tumor cell lines and autologous B cells pulsed with vaccine peptides or TAAs. Reactivity was defined by reporter activation.ResultsVaccination induced expansion and persistence of neoantigen-reactive T cells in peripheral blood (figure 1a). Week 12 biopsies from all four patients showed increased immune-cell infiltration compared to enrollment biopsies (figure 1b). In Pt5 (PD), 47% (26/55) of reconstituted tumor expanded lymphocyte (TEL) TCRs were reactive to the tumor cell line; eight targeted vaccine neoantigens (HSP90AB1 A498V, CDC37 A232V), and three were cross-reactive to the MelanA TAA. In Pt13 (SD), 64% (16/25) of TELs were tumor-reactive; none were specific vaccine peptide, while three targeted TAAs (tyrosinase, MelanA, PRAME). For Pt16 (PR), 32% (8/25) of TELs were tumor-reactive; the most abundant TCR recognized a vaccine-targeted neoantigen (ERN1.00 P97S), and one recognized tyrosinase. For Pt6 (PR), 70% (33/47) of TELs were tumor-reactive; one recognized a non-vaccine neoantigen (LDLR G287S), and ten targeted TAAs including gp100, tyrosinase, or tyrosinase-related protein 2 (TYRP2). Across patients, tumor-reactive clones—regardless of antigen specificity—expressed activation/exhaustion-associated genes (CXCL13, ENTPD1) (figure 2).ConclusionsPNV-021 neoantigen vaccination induced strong immune activation in PD-1 refractory melanoma. All patients showed increased immune infiltration within the tumor, with TILs recognizing both vaccine-targeted neoantigens and non-targeted neoantigens/TAAs. These results suggest that PNV-021 alongside polyICLC and nivolumab induces/expands tumor-specific responses beyond targeted epitopes. Analyses of response and resistance mechanisms are ongoing.Abstract 602 Figure 1Immune responses in patients assessed by ex vivo IFN-γ ELISpot of PBMCs at multiple time points and multiplex immunofluorescence staining of tumor biopsies before and after vaccine treatment. a) Neoantigen-reactive T cells expanded in the blood of all patients post-vaccination (mean ± SEM, 2-3 wells/timepoint). b) Week 12 biopsies from all patients showed increased CD163+ macrophage and CD3+ T cell infiltration compared to enrollment biopsies[Image Omitted. See PDF.]Abstract 602 Figure 2Uniform Manifold Approximation and Projection (UMAP) of the phenotypic distribution of tumor-reactive T cells. Tumor-reactive TCRs expressed genes associated with T cell activation and exhaustion, including the previously characterized tumor-reactive T cell signature markers CXCL13 and ENTPD1[Image Omitted. See PDF.]

Clinical applications of skin substitutes primarily include burn injuries and chronic wounds. Tissue‐engineered vessels appear to be the next generation of small‐diameter bypass grafts, addressing the growing need for alternative materials for small‐caliber arterial replacement. There have been many different large‐animal studies, many of which have produced encouraging results that have made it possible to investigate putative bone tissue engineering strategies. Synthetic polymers such as PLA and PGA were initially investigated as cartilage scaffolds early in the study of cartilage tissue engineering. In addition to replacing damaged cardiac tissue, cardiac tissue engineering can be used to reconstruct pediatric congenital defects. The objective of urinary tissue engineering is to provide an alternative source of tissue that is more similar to native bladder or urethra. Continued progress toward reliable and reproducible graft success can position corneal tissue engineering as an important alternative to conventional donor based techniques.

Environmental DNA (eDNA) sampling to monitor wildlife presence has mostly focused on water but increasingly includes soil, air, and creative biotic substrates like flowers and spiderwebs. Biotic substrates are unique in that they also provide insight into ecological interactions. Here we explore the ability of eDNA from songbird nests to reveal avian trophic ecology, such as nestling diet and nest predator identity, in addition to local insect biodiversity. Twenty‐two nests comprising five New World sparrow species and two nonsparrow passerines were collected in a montane sagebrush steppe ecosystem shortly after confirmed nest predation events. A novel protocol was used to extract eDNA from whole nests, and each nest was sequenced twice—with and without a blocking oligonucleotide. The blocker was designed with alternating locked nucleic acids to specifically inhibit sparrow amplification and improve detection of rare species. A total of 126 species were detected, and the blocker proved highly effective, reducing sparrow reads ~100% with no discernable coblocking of nonsparrow passerines. Species richness in sparrow nests increased by 31% with the blocker when using a minimum read threshold of 10 copies. Most detected species were insects, including likely prey items and ectoparasites of nestling birds. Predators were detected in 36% of nests. We discuss the merits of this rich and unique data source and considerations for future implementation. We present a novel method for extracting eDNA from whole bird nests and a LNA blocking oligonucleotide designed to reduce metabarcoding reads from nest building sparrows. Metabarcoding of 22 nests from a montane sagebrush steppe ecosystem detected 126 species, primarily insects, including putative diet items, ectoparasites, and confirmed nest predators.

On October 1, 2018, Maryland's extreme risk protection order (ERPO) law took effect. This was the first ERPO law in the United States to authorize clinicians to initiate a civil court process to temporarily prohibit people behaving dangerously and at risk of engaging in violence from purchasing and possessing firearms. This is the first publication reporting results from a survey of physicians about ERPOs. To assess Maryland physicians' knowledge, past use, and likely future use of ERPOs, and to identify barriers to physicians' use of ERPOs and strategies to address those barriers. This survey study conducted at The Johns Hopkins Hospital in Baltimore, Maryland, surveyed physicians, including emergency medicine physicians, pediatricians, and psychiatrists, using a 15-question online instrument between June 15, 2019, and July 1, 2019. Data analysis was performed in July 2019. Maryland's ERPO law. Knowledge, use, and likely use of ERPOs, barriers to use, and strategies to address those barriers. Ninety-two of 353 physicians invited (26.1%) completed the survey; 1 respondent reported having filed an ERPO petition. Sixty-six respondents (71.7%) described themselves as not at all familiar with ERPOs. After reading a brief description of the ERPO law, 85 respondents (92.4%) indicated that they encounter patients whom they would consider for an ERPO at least a few times per year. Fifty-five respondents (59.8%) reported that they would be very or somewhat likely to file an ERPO petition when they identify a qualifying patient. Respondents identified time as the main barrier to using ERPOs (not enough time to complete paperwork, 57 respondents [62.6%]; not enough time to attend hearing at courthouse, 64 respondents [70.3%]), followed by concern that filing an ERPO would negatively affect their relationship with the patient (36 respondents [39.6%]). Having a coordinator to manage the process (80 respondents [87.0%]), training (79 respondents [85.9%]), participating in court hearings remotely (68 respondents [73.9%]), and having access to legal counsel (59 respondents [64.1%]) were all selected by large majorities of respondents as strategies to address barriers to ERPO use. Awareness of ERPOs among physicians in the sample was low. Physicians are treating patients who would qualify for an ERPO, and respondents in the sample indicated a willingness to use ERPOs. Training, providing access to legal counsel, designating a clinician to process petitions, and allowing clinicians to participate remotely in court hearings were strategies respondents identified to address barriers to ERPO use. These survey findings identify concrete solutions for addressing barriers to physician use of ERPOs.