Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

Extricating human tumour immune alterations from tissue inflammation.

Immunotherapies have achieved remarkable successes in the treatment of cancer, but major challenges remain1,2. An inherent weakness of current treatment approaches is that therapeutically targeted pathways are not restricted to tumours, but are also found in other tissue microenvironments, complicating treatment3,4. Despite great efforts to define inflammatory processes in the tumour microenvironment, the understanding of tumour-unique immune alterations is limited by a knowledge gap regarding the immune cell populations in inflamed human tissues. Here, in an effort to identify such tumour-enriched immune alterations, we used complementary single-cell analysis approaches to interrogate the immune infiltrate in human head and neck squamous cell carcinomas and site-matched non-malignant, inflamed tissues. Our analysis revealed a large overlap in the composition and phenotype of immune cells in tumour and inflamed tissues. Computational analysis identified tumour-enriched immune cell interactions, one of which yields a large population of regulatory T (Treg) cells that is highly enriched in the tumour and uniquely identified among all haematopoietically-derived cells in blood and tissue by co-expression of ICOS and IL-1 receptor type 1 (IL1R1). We provide evidence that these intratumoural IL1R1+ Treg cells had responded to antigen recently and demonstrate that they are clonally expanded with superior suppressive function compared with IL1R1- Treg cells. In addition to identifying extensive immunological congruence between inflamed tissues and tumours as well as tumour-specific changes with direct disease relevance, our work also provides a blueprint for extricating disease-specific changes from general inflammation-associated patterns.

An In Vivo Model of Human Macrophages in Metastatic Melanoma.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

Innate immune signatures to a partially-efficacious HIV vaccine predict correlates of HIV-1 infection risk.

The pox-protein regimen tested in the RV144 trial is the only vaccine strategy demonstrated to prevent HIV-1 infection. Subsequent analyses identified antibody and cellular immune responses as correlates of risk (CoRs) for HIV infection. Early predictors of these CoRs could provide insight into vaccine-induced protection and guide efforts to enhance vaccine efficacy. Using specimens from a phase 1b trial of the RV144 regimen in HIV-1-uninfected South Africans (HVTN 097), we profiled innate responses to the first ALVAC-HIV immunization. PBMC transcriptional responses peaked 1 day post-vaccination. Type I and II interferon signaling pathways were activated, as were innate pathways critical for adaptive immune priming. We then identified two innate immune transcriptional signatures strongly associated with adaptive immune CoR after completion of the 4-dose regimen. Day 1 signatures were positively associated with antibody-dependent cellular cytotoxicity and phagocytosis activity at Month 6.5. Conversely, a signature present on Days 3 and 7 was inversely associated with Env-specific CD4+ T cell responses at Months 6.5 and 12; rapid resolution of this signature was associated with higher Env-specific CD4+ T-cell responses. These are the first-reported early immune biomarkers of vaccine-induced responses associated with HIV-1 acquisition risk in humans and suggest hypotheses to improve HIV-1 vaccine regimens.

Development of a clinically relevant ovarian cancer model incorporating surgical cytoreduction to evaluate treatment of micro-metastatic disease.

OBJECTIVES: Mouse models of ovarian cancer commonly transfer large numbers of tumor cells into the peritoneal cavity to establish experimental metastatic disease, which may not adequately model early metastatic spread from a primary tumor site. We hypothesized we could develop an ovarian cancer model that predictably represents micro-metastatic disease. METHODS: Murine ID8VEGF ovarian cancer cells were transduced to express enhanced luciferase (eLuc) to enable intravital detection of microscopic disease burden and injected beneath the ovarian bursa of C57Bl/6 mice. At 6 or 10 weeks after orthotopic injection, when mice had detectable metastases, hysterectomy and bilateral salpingo-oophorectomy was performed to remove all macroscopic disease, and survival monitored. Immunohistochemistry and gene expression profiling were performed on primary and metastatic tumors. RESULTS: eLuc-transduced ID8VEGF cells were brighter than cells transduced with standard luciferase, enabling in vivo visualization of microscopic intra-abdominal metastases developing after orthotopic injection. Primary surgical cytoreduction removed the primary tumor mass but left minimal residual disease in all mice. Metastatic sites that developed following orthotopic injection were similar to metastatic human ovarian cancer sites. Gene expression and immune infiltration were similar between primary and metastatic mouse tumors. Surgical cytoreduction prolonged survival compared to no surgery, with earlier cytoreduction more beneficial than delayed, despite micro-metastatic disease in both settings. CONCLUSIONS: Mice with primary ovarian tumors established through orthotopic injection develop progressively fatal metastatic ovarian cancer, and benefit from surgical cytoreduction to remove bulky disease. This model enables the analysis of therapeutic regimens designed to target and potentially eradicate established minimal residual disease.

Integrated Analysis of Proteomic and Transcriptomic Data Highlights Late Fetal Muscle Maturation Process.

In pigs, the perinatal period is the most critical time for survival. Piglet maturation, which occurs at the end of gestation, is an important determinant of early survival. Skeletal muscle plays a key role in adaptation to extra-uterine life, e.g. motor function and thermoregulation. Progeny from two breeds with extreme neonatal mortality rates were analyzed at 90 and 110 days of gestation (dg). The Large White breed is a highly selected breed for lean growth and exhibits a high rate of neonatal mortality, whereas the Meishan breed is fatter and more robust and has a low neonatal mortality. Our aim was to identify molecular signatures underlying late fetal longissimus muscle development. First, integrated analysis was used to explore relationships between co-expression network models built from a proteomic data set (bi-dimensional electrophoresis) and biological phenotypes. Second, correlations with a transcriptomic data set (microarrays) were investigated to combine different layers of expression with a focus on transcriptional regulation. Muscle glycogen content and myosin heavy chain polymorphism were good descriptors of muscle maturity and were used for further data integration analysis. Using 89 identified unique proteins, network inference, correlation with biological phenotypes and functional enrichment revealed that mitochondrial oxidative metabolism was a key determinant of neonatal muscle maturity. Some proteins, including ATP5A1 and CKMT2, were important nodes in the network related to muscle metabolism. Transcriptomic data suggest that overexpression of mitochondrial PCK2 was involved in the greater glycogen content of Meishan fetuses at 110 dg. GPD1, an enzyme involved in the mitochondrial oxidation of cytosolic NADH, was overexpressed in Meishan. Thirty-one proteins exhibited a positive correlation between mRNA and protein levels in both extreme fetal genotypes, suggesting transcriptional regulation. Gene ontology enrichment and Ingenuity analyses identified PPARGC1A and ESR1 as possible transcriptional factors positively involved in late fetal muscle maturation.

Comparing the intestinal transcriptome of Meishan and Large White piglets during late fetal development reveals genes involved in glucose and lipid metabolism and immunity as valuable clues of intestinal maturity.

BACKGROUND: Maturity of intestinal functions is critical for neonatal health and survival, but comprehensive description of mechanisms underlying intestinal maturation that occur during late gestation still remain poorly characterized. The aim of this study was to investigate biological processes specifically involved in intestinal maturation by comparing fetal jejunal transcriptomes of two representative porcine breeds (Large White, LW; Meishan, MS) with contrasting neonatal vitality and maturity, at two key time points during late gestation (gestational days 90 and 110). MS and LW sows inseminated with mixed semen (from breed LW and MS) gave birth to both purebred and crossbred fetuses. We hypothesized that part of the differences in neonatal maturity between the two breeds results from distinct developmental profiles of the fetal intestine during late gestation. Reciprocal crossed fetuses were used to analyze the effect of parental genome. Transcriptomic data and 23 phenotypic variables known to be associated with maturity trait were integrated using multivariate analysis with expectation of identifying relevant genes-phenotypic variable relationships involved in intestinal maturation. RESULTS: A moderate maternal genotype effect, but no paternal genotype effect, was observed on offspring intestinal maturation. Four hundred and four differentially expressed probes, corresponding to 274 differentially expressed genes (DEGs), more specifically involved in the maturation process were further studied. In day 110-MS fetuses, Ingenuity® functional enrichment analysis revealed that 46% of DEGs were involved in glucose and lipid metabolism, cell proliferation, vasculogenesis and hormone synthesis compared to day 90-MS fetuses. Expression of genes involved in immune pathways including phagocytosis, inflammation and defense processes was changed in day 110-LW compared to day 90-LW fetuses (corresponding to 13% of DEGs). The transcriptional regulator PPARGC1A was predicted to be an important regulator of differentially expressed genes in MS. Fetal blood fructose level, intestinal lactase activity and villous height were the best predicted phenotypic variables with probes mostly involved in lipid metabolism, carbohydrate metabolism and cellular movement biological pathways. CONCLUSIONS: Collectively, our findings indicate that the neonatal maturity of pig intestine may rely on functional development of glucose and lipid metabolisms, immune phagocyte differentiation and inflammatory pathways. This process may partially be governed by PPARGC1A.

Handling missing rows in multi-omics data integration: multiple imputation in multiple factor analysis framework.

BACKGROUND: In omics data integration studies, it is common, for a variety of reasons, for some individuals to not be present in all data tables. Missing row values are challenging to deal with because most statistical methods cannot be directly applied to incomplete datasets. To overcome this issue, we propose a multiple imputation (MI) approach in a multivariate framework. In this study, we focus on multiple factor analysis (MFA) as a tool to compare and integrate multiple layers of information. MI involves filling the missing rows with plausible values, resulting in M completed datasets. MFA is then applied to each completed dataset to produce M different configurations (the matrices of coordinates of individuals). Finally, the M configurations are combined to yield a single consensus solution. RESULTS: We assessed the performance of our method, named MI-MFA, on two real omics datasets. Incomplete artificial datasets with different patterns of missingness were created from these data. The MI-MFA results were compared with two other approaches i.e., regularized iterative MFA (RI-MFA) and mean variable imputation (MVI-MFA). For each configuration resulting from these three strategies, the suitability of the solution was determined against the true MFA configuration obtained from the original data and a comprehensive graphical comparison showing how the MI-, RI- or MVI-MFA configurations diverge from the true configuration was produced. Two approaches i.e., confidence ellipses and convex hulls, to visualize and assess the uncertainty due to missing values were also described. We showed how the areas of ellipses and convex hulls increased with the number of missing individuals. A free and easy-to-use code was proposed to implement the MI-MFA method in the R statistical environment. CONCLUSIONS: We believe that MI-MFA provides a useful and attractive method for estimating the coordinates of individuals on the first MFA components despite missing rows. MI-MFA configurations were close to the true configuration even when many individuals were missing in several data tables. This method takes into account the uncertainty of MI-MFA configurations induced by the missing rows, thereby allowing the reliability of the results to be evaluated.

Muscle transcriptomic investigation of late fetal development identifies candidate genes for piglet maturity.

BACKGROUND: In pigs, the perinatal period is the most critical time for survival. Piglet maturation, which occurs at the end of gestation, leads to a state of full development after birth. Therefore, maturity is an important determinant of early survival. Skeletal muscle plays a key role in adaptation to extra-uterine life, e.g. glycogen storage and thermoregulation. In this study, we performed microarray analysis to identify the genes and biological processes involved in piglet muscle maturity. Progeny from two breeds with extreme muscle maturity phenotypes were analyzed at two time points during gestation (gestational days 90 and 110). The Large White (LW) breed is a selected breed with an increased rate of mortality at birth, whereas the Meishan (MS) breed produces piglets with extremely low mortality at birth. The impact of the parental genome was analyzed with reciprocal crossed fetuses. RESULTS: Microarray analysis identified 12,326 differentially expressed probes for gestational age and genotype. Such a high number reflects an important transcriptomic change that occurs between 90 and 110 days of gestation. 2,000 probes, corresponding to 1,120 unique annotated genes, involved more particularly in the maturation process were further studied. Functional enrichment and graph inference studies underlined genes involved in muscular development around 90 days of gestation, and genes involved in metabolic functions, such as gluconeogenesis, around 110 days of gestation. Moreover, a difference in the expression of key genes, e.g. PCK2, LDHA or PGK1, was detected between MS and LW just before birth. Reciprocal crossing analysis resulted in the identification of 472 genes with an expression preferentially regulated by one parental genome. Most of these genes (366) were regulated by the paternal genome. Among these paternally regulated genes, some known imprinted genes, such as MAGEL2 or IGF2, were identified and could have a key role in the maturation process. CONCLUSION: These results reveal the biological mechanisms that regulate muscle maturity in piglets. Maturity is also under the conflicting regulation of the parental genomes. Crucial genes, which could explain the biological differences in maturity observed between LW and MS breeds, were identified. These genes could be excellent candidates for a key role in the maturity.

Enhancing immunogenic responses through CDK4/6 and HIF2α inhibition in Merkel cell carcinoma.

Approximately 50% of Merkel cell carcinoma (MCC) patients facing this highly aggressive skin cancer initially respond positively to PD-1-based immunotherapy. Nevertheless, the recurrence of MCC post-immunotherapy emphasizes the pressing need for more effective treatments. Recent research has highlighted Cyclin-dependent kinases 4 and 6 (CDK4/6) as pivotal cell cycle regulators gaining prominence in cancer studies. This study reveals that the CDK4/6 inhibitor, palbociclib can enhance PD-L1 gene transcription and surface expression in MCC cells by activating HIF2α. Inhibiting HIF2α with TC-S7009 effectively counteracts palbociclib-induced PD-L1 transcription and significantly intensifies cell death in MCC. Simultaneously, co-targeting CDK4/6 and HIF2α boosts ROS levels while suppressing SLC7A11, a key regulator of cellular redox balance, promoting ferroptosis- a form of immunogenic cell death linked to iron. Considering the rising importance of immunogenic cell death in immunotherapy, this strategy holds promise for improving future MCC treatments, markedly increasing immunogenic cell death various across various MCC cell lines, thus advancing cancer immunotherapy.

Long-term Remissions Following CD20-Directed Chimeric Antigen Receptor-Adoptive T-cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy produces high response rates in refractory B-cell non-Hodgkin lymphoma, but long-term data are minimal to date. In this study, we present long-term follow-up of a pilot trial testing a CD20-targeting third-generation CAR in patients with relapsed B-cell lymphomas following cyclophosphamide-only lymphodepletion. Two of the three patients in the trial, with mantle cell lymphoma and follicular lymphoma, had remissions lasting more than 7 years, though they ultimately relapsed. The absence of B-cell aplasia in both patients suggested a lack of functional CAR T-cell persistence, leading to the hypothesis that endogenous immune responses were responsible for these long-term remissions. Correlative immunologic analyses supported this hypothesis, with evidence of new humoral and cellular antitumor immune responses proximal to clinical response time points. Collectively, our results suggest that CAR T-cell therapy may facilitate epitope spreading and endogenous immune response formation in lymphomas. Significance: Two of three patients treated with CD20-targeted CAR T-cell therapy had long-term remissions, with evidence of endogenous antitumor immune response formation. Further investigation is warranted to develop conditions that promote epitope spreading in lymphomas.

Adolescent BCG revaccination induces a phenotypic shift in CD4+ T cell responses to Mycobacterium tuberculosis.

A recent clinical trial demonstrated that Bacille Calmette-Guérin (BCG) revaccination of adolescents reduced the risk of sustained infection with Mycobacterium tuberculosis (M.tb). In a companion phase 1b trial, HVTN 602/Aeras A-042, we characterize in-depth the cellular responses to BCG revaccination or to a H4:IC31 vaccine boost to identify T cell subsets that could be responsible for the protection observed. High-dimensional clustering analysis of cells profiled using a 26-color flow cytometric panel show marked increases in five effector memory CD4+ T cell subpopulations (TEM) after BCG revaccination, two of which are highly polyfunctional. CITE-Seq single-cell analysis shows that the activated subsets include an abundant cluster of Th1 cells with migratory potential. Additionally, a small cluster of Th17 TEM cells induced by BCG revaccination expresses high levels of CD103; these may represent recirculating tissue-resident memory cells that could provide pulmonary immune protection. Together, these results identify unique populations of CD4+ T cells with potential to be immune correlates of protection conferred by BCG revaccination.

Tumor Regression Following Engineered Polyomavirus-Specific T Cell Therapy in Immune Checkpoint Inhibitor-Refractory Merkel Cell Carcinoma.

Although immune check-point inhibitors (CPIs) revolutionized treatment of Merkel cell carcinoma (MCC), patients with CPI-refractory MCC lack effective therapy. More than 80% of MCC express T-antigens encoded by Merkel cell polyomavirus, which is an ideal target for T-cell receptor (TCR)-based immunotherapy. However, MCC often repress HLA expression, requiring additional strategies to reverse the downregulation for allowing T cells to recognize their targets. We identified TCRMCC1 that recognizes a T-antigen epitope restricted to human leukocyte antigen (HLA)-A*02:01. Seven CPI-refractory metastatic MCC patients received CD4 and CD8 T cells transduced with TCRMCC1 (TTCR-MCC1) preceded either by lymphodepleting chemotherapy or an HLA-upregulating regimen (single-fraction radiation therapy (SFRT) or systemic interferon gamma (IFNγ)) with concurrent avelumab. Two patients who received preceding SFRT and IFNγ respectively experienced tumor regression. One experienced regression of 13/14 subcutaneous lesions with 1 'escape' lesion and the other had delayed tumor regression in all lesions after initial progression. Although TTCR-MCC1 cells with an activated phenotype infiltrated tumors including the 'escape' lesion, all progressing lesions transcriptionally lacked HLA expression. While SFRT/IFNγ did not immediately upregulate tumor HLA expression, a secondary endogenous antigen-specific T cell infiltrate was detected in one of the regressing tumors and associated with HLA upregulation, indicating in situ immune responses have the potential to reverse HLA downregulation. Indeed, supplying a strong co-stimulatory signal via a CD200R-CD28 switch receptor allows TTCR-MCC1 cells to control HLA-downregulated MCC cells in a xenograft mouse model, upregulating HLA expression. Our results demonstrate the potential of TCR gene therapy for metastatic MCC and propose a next strategy for overcoming epigenetic downregulation of HLA in MCC.

Dysfunctional effector memory CD8 T cells in the bronchoalveolar compartment of people living with HIV.

Mechanisms by which HIV causes susceptibility to respiratory pathogens remain incompletely understood. We obtained whole blood and bronchoalveolar lavage (BAL) from people with latent TB infection in the presence or absence of antiretroviral-naïve HIV co-infection. Transcriptomic and flow cytometric analyses demonstrated HIV-associated cell proliferation plus type I interferon activity in blood and effector memory CD8 T-cells in BAL. Both compartments displayed reduced induction of CD8 T-cell-derived IL-17A in people with HIV, associated with elevated T-cell regulatory molecule expression. The data suggest that dysfunctional CD8 T-cell responses in uncontrolled HIV contribute to susceptibility to secondary bacterial infections, including tuberculosis.

Mucosal viral infection induces a regulatory T cell activation phenotype distinct from tissue residency in mouse and human tissues.

Regulatory T cells (Tregs) mediate immune homeostasis, yet also facilitate nuanced immune responses during infection, balancing pathogen control while limiting host inflammation. Recent studies have identified Treg populations in non-lymphoid tissues that are phenotypically distinct from Tregs in lymphoid tissues (LT), including performance of location-dependent roles. Mucosal tissues serve as critical barriers to microbes while performing unique physiologic functions, so we sought to identify distinct phenotypical and functional aspects of mucosal Tregs in the female reproductive tract. In healthy human and mouse vaginal mucosa, we found that Tregs are highly activated compared to blood or LT Tregs. To determine if this phenotype reflects acute activation or a general signature of vaginal tract (VT)-residency, we infected mice with HSV-2 to discover that VT Tregs express granzyme-B (GzmB) and acquire a VT Treg signature distinct from baseline. To determine the mechanisms that drive GzmB expression, we performed ex vivo assays to reveal that a combination of type-I interferons and interleukin-2 is sufficient for GzmB expression. Together, we highlight that VT Tregs are activated at steady state and become further activated in response to infection; thus, they may exert robust control of local immune responses, which could have implications for mucosal vaccine design.

Achieving intracellular cytokine staining assay concordance on two continents to assess HIV vaccine-induced T-cell responses.

The HIV Vaccine Trials Network (HVTN) conducts clinical trials on 4 continents in pursuit of a safe and effective HIV vaccine. Cellular immune responses to vaccination that define vaccine immunogenicity and/or immune correlates of protection can be measured using multiparameter intracellular cytokine staining (ICS) assays. The HVTN cellular immunology laboratory, located in Seattle, WA, conducts ICS assays for vaccine trials according to Good Clinical Laboratory Practices (GCLP). In 2013, the HVTN established a second GCLP compliant cellular immunology laboratory in Cape Town, South Africa to assess vaccine immunogenicity for HVTN trials conducted on the African continent. To ensure ICS readouts in the 2 laboratories were directly comparable, we conducted concordance testing using PBMC from healthy controls and vaccine trial participants. Despite standardized procedures and instrumentation, shared quality control measures and quality assurance oversight, several factors impacted our ability to obtain close agreement in T-cell responses measured in the 2 laboratories. One of these was the type of fetal bovine serum (FBS) used in the assay, which impacted lymphocyte cell viability and background responses. In addition, the differences in supernatant removal technique also significantly affected our ability to detect positive responses to vaccine antigens. Standardization of these factors allowed us to achieve and maintain ICS assay concordance across the 2 laboratories over multiple years, accelerating our efforts to evaluate HIV vaccines. The insights gained in this process are valuable for assay transfer efforts by groups of investigators that need to directly compare data generated in different laboratories around the globe.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

The human memory T cell compartment changes across tissues of the female reproductive tract.

Memory CD4 T cells in tissues fulfill numerous functions that are critical for local immune homeostasis and protection against pathogens. Previous studies have highlighted the phenotypic and functional heterogeneity of circulating and tissue-resident memory CD4 T cells across different human tissues such as skin, lung, liver, and colon. Comparatively little is known in regard to memory CD4 T cells across tissues of the female reproductive tract (FRT). We examined CD4 T cells in donor-matched vaginal, ecto- and endocervical tissues, which differ in mucosal structure and exposure to external environmental stimuli. We hypothesized that this could be reflected by tissue-specific differences in the memory CD4 T cell compartment. We found differences in CD4 subset distribution across these tissues. Specifically, CD69+CD103+ CD4 T cells were significantly more abundant in vaginal than cervical tissues. In contrast, the transcriptional profiles of CD4 subsets were fairly conserved across FRT tissues. CD69+CD103+ CD4 T cells showed a TH17 bias independent of tissue niche. Our data suggest that FRT tissues affect T cell subset distribution but have limited effects on the transcriptome of each subset. We discuss the implications for barrier immunity in the FRT.

Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function.

Chimeric antigen receptor (CAR)-modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we directly compared TCR and CAR signaling in primary human T cells. Global phosphoproteomic analysis revealed that key T cell signaling proteins-such as CD3δ, CD3ε, and CD3γ, which comprise a portion of the T cell co-receptor, as well as the TCR adaptor protein LAT-were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying a commonplace 4-1BB/CD3ζ CAR sequence to better engage CD3ε and LAT using embedded CD3ε or GRB2 domains resulted in enhanced T cell activation in vitro in settings of a low density of antigen, and improved efficacy in in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling.