Antigen Exposure Research Articles

Marginal Zone B Cells Facilitate CD4 T Cell Proliferation in Response to Factor VIII in Mice with Hemophilia Α

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies represents a significant barrier to FVIII replacement therapy in patients with hemophilia A. Despite this, the immune factors that regulate anti-FVIII antibody formation remain incompletely understood. We have previously shown that shortly after injection, FVIII co-localizes with marginal zone (MZ) B cells in the marginal sinus of the spleen in mice with hemophilia A. Moreover, mice depleted of MZ B cells did not produce detectable anti-FVIII IgG antibodies. Given the ability of MZ B cells to trap and rapidly respond to blood-borne antigens, produce both IgM and IgG antibodies and activate CD4 T cells, while also trafficking antigen to B cell follicles, these results suggest that MZ B cells likely play a critical role in the initiation and subsequent orchestration of an immune response to FVIII. In contrast to other model antigens, anti-FVIII antibody formation occurs only following multiple FVIII exposures in both human and mouse models. As CD4 T cells facilitate long-lasting antibody formation, and have previously been shown to play an important role in anti-FVIII antibody development (Bray GL, et al. Am J Hematol 1993; Reding MT, et al. Thromb Haemost 2000), it is possible that each FVIII exposure event eventually propagates a CD4 T cell response to a threshold necessary to generate an optimal antibody response. Consistent with this, we have previously shown that FVIII-specific CD4 T cell proliferation requires more than 1 prior exposure to FVIII. An improved understanding of the overall timing and factors influencing CD4 T cell activation in the immune response to FVIII is necessary for the development of strategies to prevent antibody formation. OBJECTIVE: Define the key factors that regulate CD4T cell activation following FVIII exposure and the number of exposure events required to observe FVIII-specific CD4 T cell proliferation. METHODS: Hemophilia A mice +/- CD4T cell depletion or MZ B cell depletion or S1PR1-TSS mice were challenged with 1-4 weekly exposures to FVIII. Anti-FVIII IgM and IgG was examined in plasma by enzyme-linked immunoassay (ELISA). To assess FVIII-specific CD4T cell proliferation, we engineered FVIII-OVA, which contains the ovalbumin peptide recognized by OTII T cell receptor transgenic T cells. CFSE-labeled OTII splenocytes were adoptively transferred into mice with hemophilia A, mice lacking B cells and MZ B cell-depleted mice, followed by administration of FVIII-OVA. RESULTS: Anti-FVIII IgG formation is CD4T cell-dependent, however IgM is readily produced in the absence of CD4T cells. In contrast, MZ B cell depletion prevented the formation of both IgM and IgG antibodies against FVIII. FVIII is unable to cause proliferation of CD4T cells or produce IgG antibodies following initial exposure, but instead significantly increases following the 3rd exposure to FVIII. Mapping the kinetics of antibody formation has uncovered that anti-FVIII IgM formation peaks and anti-FVIII IgG formation begins after the 3rd FVIII exposure, at the time of CD4 T cell proliferation. We next examined the influence of B cells on CD4 T cell proliferation, and observed that FVIII-specific CD4T cell proliferation is significantly impaired in B cell-deficient recipients. Moreover, in mice who have undergone MZ B cell depletion, there is likewise an absence of anti-FVIII IgM and IgG and a lack of measurable CD4 T cell proliferation. Finally, in S1PR1-TSS mice with MZ B cells that are unable to traffic antigen to the B cell follicle, anti-FVIII IgM and IgG formation occurs at the same rate as control mice. CONCLUSION: In summary, our findings suggest that MZ B cells are required for anti-FVIII IgM and IgG antibody formation as well as FVIII-specific CD4 T cell proliferation, which requires 3 antigen exposures. The finding that MZ B cells are not required to traffic antigen to the B cell follicle contributes to our hypothesis that MZ B cell production of IgM, with or without direct activation of CD4 T cells contributes to FVIII-specific CD4 T cell activation. These results suggest that targeted approaches designed to prevent anti-FVIII antibody formation may be most effectively achieved by defining key factors that regulate MZ B cell activation following initial FVIII exposure. In doing so, the present findings and future work will likely uncover important pathways to effectively target to reduce, or event prevent anti-FVIII antibody production.

Metabolic Abnormalities Associated with T-Cell Exhaustion in CLL Eµ-TCL1 Murine Model

Introduction. T-cell differentiation and immune responses are associated with major metabolic changes that control cell fate upon antigen exposure. Chronic lymphocytic leukemia (CLL) is characterized by a marked T-cell dysfunction that plays a major role in disease progression and failure of adoptive cell therapy. A handful of recent studies have demonstrated mitochondrial and metabolic defects in the T-cell compartment in CLL patients. However, to date, no studies have been done to validate such metabolic abnormalities in the Eµ-TCL1 model, the most established and reliable murine model for studying CLL. Therefore, we hypothesized that CD8+ T-cells derived from the Eµ-TCL1 adoptive transfer model exhibit metabolic and mitochondrial disturbance that might contribute to the overall dysfunctional state of these cells. Methods. To investigate the influence of leukemic burden on the differentiation phenotypes, metabolic state and gene expression profile of CD8+ T-cells, C57BL/6 mice adoptively transferred with Eµ-TCL1 leukemic splenocytes were euthanized at varying time points. Once disease was established, spleens were collected for analysis. Mice were divided into 3 groups; early, intermediate, and late disease, based on the peripheral blood leukemic burden at endpoint. Multicolor flow cytometry was used for identification of T-cell phenotypes and measurement of mitochondrial mass, membrane potential, reactive oxygen species (ROS) and cellular glucose uptake. For T-cell activation studies, we cultured splenocytes in the presence of CD3/CD28 dynabeads and 30IU/ml hIL-2 for 72 hours. Nanostring metabolic pathways gene expression analysis was done on splenic CD8+ T-cells isolated from early and late-stage Eµ-TCL1 adoptive transfer mice via fluorescence-activated cell sorting. Differential gene expression was measured using ROSALIND cloud-based software platform. Results. Unsupervised t-distributed stochastic neighborhood embedding (tSNE) and Xshift clustering analyses revealed a gradual enrichment of specific CD8+ T-cell populations along disease progression. These populations upregulated exhaustion-related markers (EOMES, TOX, PD-1 and KLRG-1) and significantly downregulated TCF-1 expression, a transcription factor responsible for cell self-renewal capacity. Further analysis demonstrated a significant downregulation of PGC-1α expression, a master regulator of mitochondrial function, in Eµ-TCL1 CD8+ T-cells with high EOMES and low TCF-1 expression levels (EOMEShi TCF-1lo) compared to EOMEShi TCF-1hi cells. At late disease stage, we noticed significant mitochondrial depolarization, an increase in mitochondrial ROS and marked reduction in glucose uptake capacity in the Eµ-TCL1 CD8+ T-cells. Post in vitro activation, there was a significant increase in Eµ-TCL1 CD8+ T-cell mitochondrial biogenesis, membrane potential and ROS production compared to wild type (WT) controls. However, Eµ-TCL1 cultures showed significant enrichment in EOMEShi TCF-1lo CD8+ T-cell population, that was not detected in the activated WT cells. Interestingly, this population exhibited marked downregulation of PGC-1α, SOD-2 and NRF-2 expression compared to the populations maintaining TCF-1 expression. Moreover, we found that Eµ-TCL1 CD8+ T-cells had increased frequency of effector memory phenotypes compared to more central memory cells in WT cultures. These effector memory populations accumulated significant levels of mitochondrial ROS and depolarized mitochondria compared to the central memory counterparts. Nanostring analysis revealed disease stage-dependent changes in gene expression, where glycolysis was the top differentially expressed pathway in the late versus early-stage CD8+ T-cells as indicated by the gene set global significance score. On the other hand, negative significance scores were assigned to AMPK and autophagy pathways indicating a probable downregulation of these pathways during late phases of the disease. Conclusion. Our results demonstrate mitochondrial and metabolic abnormalities in Eµ-TCL1-derived CD8+ T-cells and point to possible defects in cellular stress signaling response that might lead to accumulation of dysfunctional mitochondria and push the cells towards exhausted states.

Sars-Cov-2-Specific CD4+ and CD8+ T Cell Responses Can Originate from Cross-Reactive CMV-Specific T Cells

Introduction: SARS-CoV-2-specific CD4+ and CD8+ T cells play an important role in COVID-19 pathogenesis. In addition to de novo SARS-CoV-2-specific T cell responses in infected individuals, SARS-CoV-2-specific T cells have also been identified in samples frozen down before the pandemic. So far, pre-existing SARS-CoV-2 specific T cell responses has mostly been ascribed to cross-reactive T cells that were initially primed by other coronaviruses. The presence of these cross-reactive T cells is not surprising considering these human coronaviruses share a relatively high sequence homology. However, T cells are also able to cross-react towards dissimilar pathogens, called heterologous immunity. Given the high prevalence and magnitudes of pre-pandemic SARS-CoV-2-specific T cells, we hypothesized that pre-pandemic SARS-CoV-2 T cell immunity could be explained by cross-reactive cytomegalovirus (CMV)-specific T cells. Methods & Results: PBMCs from CMV seropositive and seronegative donors that were cryopreserved before the pandemic were incubated with SARS-CoV-2 peptide pools. To detect SARS-CoV-2-specific T cells using flow cytometry, CD4+ T cell responses were identified by upregulation of CD154 and CD137 whereas CD8+ T cell responses were identified by upregulation of CD137 and IFN-γ production. In the CMV seropositive group, clear CD4+ T cell responses against SARS-CoV-2 membrane and CD8+ T cell responses against SARS-CoV-2 spike were detected whilst this was not observed in CMV seronegative donors. To investigate whether these T cells were reactive against both SARS-CoV-2 and CMV, the SARS-CoV-2 reactive T cells were single cell sorted, clonally expanded, and tested in co-culture assays in which stimulator cells expressing the relevant human leukocyte antigen (HLA) alleles were pulsed with overlapping peptide pools, peptide libraries or single peptides. The results demonstrated that the pre-pandemic SARS-CoV-2 membrane-specific CD4+ T cells recognized both SARS-CoV-2 membrane and CMV-pp65 peptide pools in HLA-DRB3*02:02, and specifically recognized AGILRNLVPM peptide of CMV-pp65. The pre-pandemic SARS-CoV-2 spike-specific CD8+ T cells recognized spike peptide FVSNGTHWF (FVS) and, interestingly, dissimilar CMV-pp65 peptide IPSINVHHY (IPS) both presented by HLA-B*35:01. Importantly, these FVS/IPS-cross-reactive T cells displayed a higher avidity for CMV compared to SARS-CoV-2, which illustrates that these cells were originally primed against CMV since repeated antigen exposure results in a selection for high avidity T cell clonotypes. This selection cannot have occurred in response to SARS-CoV-2-specific peptides in SARS-CoV-2-unexposed individuals. PBMCs from pre-pandemic CMV-seropositive donors that express HLA-B*35:01 were screened for the presence of these dual IPS/FVS-specific CD8+ T cells by peptide-HLA tetramer technology. In multiple donors these cross-reactive T cells were detected, and isolation followed by direct T cell receptor (TCR) sequencing of the IPS/FVS-tetramer positive cells showed that these T cells express an amino acid-identical TCR, illustrating that this SARS-CoV-2/CMV cross-reactivity of the IPS/FVS T cells is caused by a public TCR. To investigate the potential of the IPS/FVS-specific T cells to kill virus-infected cells, and thereby reducing spreading of SARS-CoV-2, the T cells were incubated with SARS-CoV-2-infected airway epithelial Calu-3 cells. Intracellular SARS-CoV-2 RNA was reduced at low virus titers in IPS/FVS-specific T cell co-cultures, indicating that the cross-reactive T cells are killing SARS-CoV-2-infected cells and thereby reduce spreading of SARS-CoV-2 during initial infection. Circulating IPS/FVS-specific T cells were not phenotypically activated in two patients with advanced COVID-19, as measured by flow cytometry analysis of CD38 and HLA-DR expression, indicating that cross-reactive T cells may more likely play a role during early stage of infection. Conclusion: CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-pandemic immunity against SARS-CoV-2. Pre-print link: https://www.biorxiv.org/content/10.1101/2022.07.31.502203v1

The Enrichment of T-Cell Proliferation and Memory Gene Signatures of CD79A/CD40 Co-Stimulatory Domain Enhances CD19CAR-T Cell Efficacy

Introduction: CD28 or 4-1BB co-stimulated CD19 chimeric antigen receptor (CAR) T-cell has demonstrated remarkable outcomes in B-cell malignancies. Recently, we reported that a novel CD19CAR-T cell incorporated B-cell co-stimulatory molecules; CD79A/CD40 (CD19.79a.40z), exhibited superior anti-tumor activity in B-cell lymphoma model compared to CD28 or 4-1BB co-stimulated CAR-T cell. Despite these promising results, the molecular signatures of this novel CAR-T cell and its effects on the overall cellular state are not well understood. This study assessed mRNA expression using RNA-sequencing (RNA-seq) to interrogate the intrinsic transcriptional gene underlying CD19.79a.40z CAR-T cell response and identify the differentially expressed genes (DEGs) compared to CD19.28z or CD19.BBz CAR-T cell upon CD19-specific antigen exposure. Methods: We generated three CD19CAR structures contained different co-stimulatory domain (CD19.28z, CD19.BBz, CD19.79a.40z).The CAR genes were transfected into retroviral packaging cells and transduced into human CD3+ cells. We first screened the functions of each CAR-T cell structure in in vitro assay including cytotoxicity assay, T-cell proliferation assay, and T-cell phenotype assay to confirm whether each CAR-T cell structure contained proper functions. Total CAR-T 3 - 6 x 106 cells were harvested and extracted for RNA at post-stimulation with the target cell line on day seven. Nine samples were RNA-seq using Illumina Hiseq 4000. The short sequence reads were mapped to reference genome from GENCODE (GRCh38) and the expression count matrix was then computed using HTSeq. EdgeR program was used to perform DEGs analysis between three groups: CD19.79a.40z vs. CD19.28z, CD19.79a.40z vs. CD19.BBz and CD19.28z vs. CD19.BBz. The DEGs were selected at FDR <0.05 and fold change ≥ 1.5-fold difference. To identify the regulation of functionally enriched pathways, we performed both ranked gene list analyses on the whole gene expression level using the gene set enrichment analysis (GSEA) software from the Broad Institute and DEGs list separately analyzed for up- and down-regulated genes using g:Profiler. Results: A total of 374 DEGs (232 up-regulated and 142 down-regulated DEGs) and one up-regulated DEG were identified in CD19.79a.40z vs. CD19.28z CAR-T cell groups and CD19.79a.40z vs. CD19.BBz CAR-T cell groups respectively. We found 104 DEGs in which 32 DEGs were up-regulated and 72 DEGs were down-regulated in CD19.28z CAR-T cell compared to CD19.BBz CAR-T cell.Functional enrichment analysis using g;Profiler illustrated that CD19.79a.40z CAR-T cells consisted of down-regulated genes mediating apoptosis and exhaustion while up-regulated genes correlated with T-cell activation, proliferation, positive regulation of interferon production, and NF-κB pathway. Compared to either CD19.28z or CD19.BBz CAR-T cells, CD19.79a.40z CAR-T cells strongly enriched in genes associated with T-cell proliferation at the transcription level supported by the expression of BATF3, IL2, ZP3, TFRC,IRF1 and CD70. In addition, CD19.79a.40z CAR-T cell contained many down-regulated apoptosis-related genes, including ID3, PDCD1 and EOMES (Figure). GSEA revealed that CD19.79a.40z CAR-T cells enriched in both glycolysis and fatty acid metabolism, which are the underlying metabolic pathway closely linked to T-cell activation and proliferation. Regarding T-cell differentiation, CD19.79a.40z CAR-T cells were particularly enriched in naïve and memory-related genes compared to CD19.BBz CAR-T cell. Conclusions: This study provides a comprehensive insight into the understanding of gene expression and its related pathways that potentiates the superior anti-tumor functions by CD19CAR-T cell incorporated B-cell co-stimulatory domain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Highly Parallel Discovery of Synthetic Knockin Sequences for Enhanced Cancer Immunotherapies

Introduction T cells engineered to express transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as powerful treatment options for some malignancies. However, while CAR T cells have induced impressive initial response rates in patients with B-precursor acute lymphoblastic leukemia (ALL) and lymphoma, they fail to mediate long-term relapse-free survival in 40-60% of patients and have not been successful in most solid tumors. For both TCR- and CAR-based approaches, T cell persistence and long-term functionality can be diminished by chronic antigen stimulation or tonic signaling. One approach to enhance the efficacy of cell therapies that has been explored by several groups is the overexpression of specific transcription factors (e.g. AP-1/ATF transcription factors such as c-JUN or BATF); however, more systematic gain-of-function screens in human CAR T cells have not yet been done. Methods and Results Here, we introduce CRISPR-based modular pooled knockin (ModPoKI) screens to evaluate hundreds to thousands of different constructs in human T cells and their potential to overcome TCR or CAR T cell dysfunction. We generated two barcoded ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs) to identify those that confer a fitness advantage to TCR or CAR T cells. The libraries were combined with either an NY-ESO-1-specific TCR, an anti-CD19 CAR or a high affinity anti-GD2 CAR that is known to induce tonic signaling-based dysfunction. The libraries and the TCR/CAR were engineered as polycistronic sequences that were non-virally integrated into the TRAC (T cell receptor alpha chain constant) locus of primary human T cells. We performed >20 unique ModPoKI screens including bead-based single stimulation, repetitive stimulation with target cells and tonic stimulation (GD2 CAR model). While constructs containing known AP-1/ATF transcription factors BATF and BATF3 showed increased abundance across multiple screens, transcription factor AP4 (TFAP4) knockin (KI) constructs were more clearly enriched after repetitive stimulation and tonic signaling, suggesting potentially undescribed benefits in exhaustion-prone environments. We next validated single knockins of TFAP4 or a control construct (truncated nerve growth factor receptor, tNGFR) in combination with the GD2 or the CD19 CAR. TFAP4 KI led to increased levels of cytokine release, proliferation and target cell killing compared to conventional CAR T cells. Moreover, TFAP4 KI mediated increased tumor control and survival in an in vivo model. Interestingly, we observed elevated levels of CD25 (IL2RA) expression on TFAP4 KI T cells and enrichment of genes related to IL2/STAT5 pathways which could hint at a potential benefit in situations of IL-2 competition/presence of regulatory T cells. Lastly, we aimed to discover specific combinations of TFs that work in concert to enhance T cell fitness in the setting of tonic CAR signaling. We created a ~10,000-member library (102 x 102 - pairwise combinations of 100 transcription factors plus two controls) cloned into constructs with the GD2 CAR and performed a tonic signaling ModPoKI screen. Analysis of the constructs that increased the most in relative abundance highlighted that several of the top performing constructs included either TFAP4, BATF, BATF3 or a combination of TFAP4 and BATF(3), suggesting that TFAP4 and BATF(3) are key transcription factors that can coordinately drive increased T cell fitness upon repetitive stimulation. Validation analyses confirmed that the combinatorial KI of a BATF-TFAP4 construct confers highest cytotoxic capacity in vitro and in vivo compared to single knockin (BATF-RFP or RFP-TFAP4) or control knockin (RFP-tNGFR) GD2 CAR constructs. Conclusions In conclusion, ModPoKI screens allow for customizable parallel evaluation and functional characterization of hundreds to thousands of different T cell constructs. Using clinically relevant screening modalities, we nominated candidate genes that can synthetically re-write the T cell response to repetitive antigen exposure or tonic signaling and thus have the potential to program more effective TCR or CAR T cell therapies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Co-Stimulation of CD28/CD40 Signaling Molecule Potentiates CD19CAR-T Cell Functions and Stemness

Introduction: Adoptive T-cell therapy of gene-modified to express CD19 chimeric antigen receptor (CD19CAR) has achieved promising clinical outcomes in B-cell malignancies. However, the recurrences after therapies still occur due to the poor CAR-T persistence. Various approaches have been studied including signaling domain modification to improve T-cell efficacy. The nuclear factor-κB (NF-κB) which interestingly exists in both T- and B-cells is a crucial signaling pathway for T-cell persistence. Furthermore, CAR-T memory signatures drive robust T-cell proliferation. Consequently, we developed the third generation CD19CAR incorporated dual T- and B-cell costimulatory molecules; CD28 and CD40 (CD19.28.40z), to enhance CAR-T cell proliferation and tumoricidal activity. Methods: The anti-CD19CAR incorporated CD28/CD40 co-stimulatory domain; CD19scFv-CD28TM-CD28/CD40-CD3z-tEGFR, was generated. The CAR gene was subsequently cloned into retroviral packaging cells and transduced into primary CD3 cells. The CAR+-T cells were enriched using biotinylated anti-EGFR mAb and further expanded with anti-CD3/CD28 beads or gamma-irradiated EBV-LCL cells. These expanded cells were then used for downstream experiments compared with control CD19CARs including CD19.28z, CD19.BBz, and CD19.40z CAR-T cells. Results: All CD19CAR genes were successfully transduced into primary CD3cells. We first determined the downstream T-cell signaling and found the higher baseline pNF-kB and pRelB of CD19.28.40z CAR-T cells compared to others. We then investigated the ability of CD19 CAR T-cell proliferation by coculturing with gamma-irradiated CD19-expressing K562 cells. Surprisingly, the novel CD19.28.40z exhibited robust T-cell proliferation and persistence throughout 2 weeks of the culture regardless of IL-2 supplementation. The long-term co-culture assay unveiled that CD19.28.40z CAR-T cells could effectively eradicate tumor cell growth until the end of the experiment at an effector to target cell ratio of 1:16, compared to control CAR T-cells. In term of cytokine secretion assays, the similar level of IL-2, TNF-a, and IFN-g concentrations were observed among CAR constructs after being stimulated with target cells. To mimic persistent antigen stimulation in vivo, we performed three consecutive weekly antigen stimulation that exhibited the greater CD19.28.40z CAR-T cell expansion without influencing T-cell fate and exhaustion. We interrogated the intrinsic genes underlying CD19.28.40z CAR-T cell responses compared to conventional CAR-T cells following CD19 antigen exposures using RNA-sequencing. The results confirmed the significant up-regulated genes associated with T-cell proliferation and differentiation, NF-κB pathway, WNT pathway, and CD4+ phenotypes as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway (Figure 1). Regarding T-cell memory phenotypes, we observed the enrichment of genes toward T-cell stemness particularly SELL, IL-7r, TCF7, and KLF2 compared to conventional CAR-T cells. To evaluate the potential anti-tumor efficiency in vivo, we studied NALM-6- and Raji-bearing NSG mice treated with a low CD19CAR-T cell dosage to assess the functional limits of different CAR structures. The CD19.28.40z CAR-T cells demonstrated potent tumoricidal activity as well as prolonged overall survival in both NSG mice models compared to control CAR-T cells (Figure 2). Conclusions: The CD28/CD40 costimulatory molecule potentiates NF-κB signaling and memory signatures enhancing CD19CAR-T cell proliferation and persistence which translates into greater anti-tumor efficacy in both in vitro and xenograft models. The simplified structural modification of dual T-/B-cell signaling molecule could be advantageous to maximize the CAR-T cell functions and stemness. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD8+ T cell exhaustion in anti-tumour immunity: The new insights for cancer immunotherapy.

CD8+ T cells play a crucial role in anti-tumour immunity, but they often undergo exhaustion, which affects the anti-tumour activity of CD8+ T cells. The effect and mechanism of exhausted CD8+ T cells have become the focus of anti-tumour immunity research. Recently, a large number of studies have confirmed that long-term antigen exposure can induce exhaustion. Cytokines previously have identified their effects (such as IL-2 and IL-10) may play a dual role in the exhaustion process of CD8+ T cells, suggesting a new mechanism of inducing exhaustion. This review just focuses our current understanding of the biology of exhausted CD8+ T cells, including differentiation pathways, cellular characteristics and signalling pathways involved in inducing exhaustion, and summarizes how these can be applied to tumour immunotherapy.

The Challenging Diagnosis of Interstitial Lung Disease in Children-One Case Report and Literature Review.

Childhood interstitial lung disease (chILD) includes a heterogeneous spectrum of rare respiratory disorders in children associated with substantial morbi-mortality. Interstitial tissue, and other pulmonary structures, epithelium, blood vessels, or pleura are involved, resulting in a restrictive lung disfunction. Respiratory symptoms set in progressively and are often subtle, making thorough clinical history and physical examination fundamental. The etiology often is obscure. The clinical presentation mimics pneumonia or asthma, leading to a diagnostic delay. Challenging diagnosis may require genetic tests, bronchoalveolar lavage, or lung biopsy. Alongside general supportive therapeutic measures, anti-inflammatory, immunosuppressive or antifibrotic agents may be used, based on data derived from adult studies. However, if accurate diagnosis and treatment are delayed, irreversible chronic respiratory failure may ensue, impacting prognosis. The most frequent chILD is hypersensitivity pneumonitis (HP), although it is rare in children. HP is associated with exposure to an environmental antigen, resulting in inflammation of the airways. Detailed antigen exposure history and identification of the inciting trigger are the cornerstones of diagnostic. This article provides the current state of chILD, revealing specific features of HP, based on a clinical case report of a patient admitted in our clinic, requiring extensive investigations for diagnosis, with a favorable long-term outcome.

Factors Predictive for Immunomodulatory Therapy Response and Survival in Patients with Hypersensitivity Pneumonitis-Retrospective Cohort Analysis.

The aim of the present retrospective study was to establish predictive factors for treatment success and survival advantage in HP patients. We analyzed the short-term treatment outcome and overall survival in consecutive HP patients treated with prednisone alone or combined with azathioprine. The study group consisted of 93 HP patients, 54 (58%) with fibrotic HP and 39 (42%) with non-fibrotic HP. Mean (± SD) VCmax % pred. and TL,co % pred. before treatment initiation were 81.5 (±20.8)% and 48.3 (±15.7)%, respectively. Mean relative VCmax and TL,co change after 3-6 months of therapy were 9.5 (±18.8)% and 21.4 (±35.2)%, respectively. The short-term treatment outcomes were improvement in 49 (53%) patients, stabilization in 16 (17%) patients, and progression in 28 (30%) patients. Among those with fibrotic HP, improvement was noted in 19 (35%) cases. Significant positive treatment outcome predictors were fever after antigen exposure, lymphocyte count in broncho-alveolar lavage fluid (BALF) exceeding 54%, RV/TLC &gt; 120% pred., and ill-defined centrilobular nodules in high-resolution computed tomography (HRCT). An increased eosinophil count in BALF and fibrosis in HRCT were significant negative treatment outcome predictors. The presence of fibrosis in HRCT remained significant in a multivariate analysis. A positive response to treatment, as well as preserved baseline VCmax (% pred.) and TLC (% pred.), predicted longer survival, while fibrosis in HRCT was related to a worse prognosis. Immunomodulatory treatment may be effective in a significant proportion of patients with HP, including those with fibrotic changes in HRCT. Therefore, future trials are urgently needed to establish the role of immunosuppressive treatment in fibrotic HP.

Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1, encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]-expressing CD8+ CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8+ T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3+CD8+ and toward TCF1+CD8+ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.

Abstract 10847: Tolerability of Sars-cov-2 Mrna Vaccines and Infection for Patients With History of Kawasaki Disease

Introduction: Kawasaki disease (KD) and multisystem inflammatory syndrome in children from COVID-19 infections share overlapping symptoms, suggesting KD patients may be at risk for adverse outcomes from exposure to SARS-CoV-2 mRNA vaccine or infection. Through retrospective review of clinical outcome data, we determined tolerance of KD patients to SARS-CoV-2 antigen exposure. Methods: We evaluated two cohorts using an existing KD database of 826 patients and reviewed individual electronic medical records for the period spanning January 2020 through Jan 2022 via Epic Care Everywhere which includes Washington State immunization records. Vaccine cohort inclusion criteria were (1) age &amp;lt 21 years at immunization and (2) received one or more Pfizer-BioNTech or Moderna vaccine doses. The COVID-19 infection cohort included (1) age &amp;lt 21 years and (2) positive SARS-CoV-2 PCR or nuclear capsid IgG. Results: The vaccine cohort contained 153 patients with vaccine distribution by age subgroups shown in Figure 1. Pfizer-BioNTech vaccine was provided for 93.5% and the remaining received Moderna or a combination. 84.3% were fully vaccinated or received a third-dose booster. No clinically severe adverse events occurred including no vaccine-related hospitalizations or outpatient visits. The COVID-19 infection cohort included thirty-seven patients. No patients required hospitalization. The most common symptoms included low grade fever, fatigue, cough, and myalgia with resolution within a few days. Two patients, 9 and 19 years, had extended cough and fatigue for 3-4 weeks. One patient developed COVID-19 within 6 weeks of receiving IVIG for KD. Conclusions: COVID-19 infections and vaccinations are well tolerated in patients with preceding KD. No severe complications occurred in either cohort with only two patients exhibiting prolonged mild symptoms from COVID-19. This study provides assurance regarding vaccine safety and COVID-19 severity for parents of children with KD.

Homotypic Targeted Photosensitive Nanointerferer for Tumor Cell Cycle Arrest to Boost Tumor Photoimmunotherapy.

Recent advances in tumor immunotherapy mainly tend to remodel the immunosuppressive tumor microenvironment (TME) for immune enhancement. However, the complexity of TME makes it unlikely to achieve satisfactory therapeutic effects with any single intervention alone. Here, we focus on exposing intrinsic features of tumor cells to trigger direct pleiotropic antitumor immunity. We develop a photosensitive nanointerferer that is engineered with a nanoscale metal-organic framework decorated with tumor cell membranes for targeted delivery of a photosensitizer and small interfering RNA, which is used to knock down cyclin-dependent kinase 4 (Cdk4). Cdk4 blockade can arrest the cell cycle of tumor cells to facilitate antigen exposure and increase the expression level of programmed cell death protein ligand 1 (PD-L1). Under laser irradiation, photodynamic damage triggered by the nanointerferer induces the release of tumor antigens and recruitment of dendritic cells (DCs), thereby promoting the antitumor activity of CD8+ T cells in combination with anti-PD-L1 antibodies. Ultimately, these events markedly retard tumor progression in a mouse model of ectopic colon tumor with negligible adverse effects. This study provides an alternative treatment for effective antitumor immunity by exciting the intrinsic potential of tumor cells to initiate immune responses while reducing immune-related toxicities.

Photoacoustic mediated multifunctional tumor antigen trapping nanoparticles inhibit the recurrence and metastasis of ovarian cancer by enhancing tumor immunogenicity

The hypoimmunogenicity of tumors is one of the main bottlenecks of cancer immunotherapy. Enhancing tumor immunogenicity can improve the efficacy of tumor immunotherapy by increasing antigen exposure and presentation, and establishing an inflammatory microenvironment. Here, a multifunctional antigen trapping nanoparticle with indocyanine green (ICG), aluminum hydroxide (Al(OH)3) and oxaliplatin (OXA) (PPIAO) has been developed for tumor photoacoustic/ultrasound dual-modality imaging and therapy. The combination of photothermal/photodynamic therapy and chemotherapy induced tumor antigen exposure and release through immunogenic death of tumor cells. A timely capture and storage of antigens by aluminum hydroxide enabled dendritic cells to recognize and present those antigens spatiotemporally. In an ovarian tumor model, the photoacoustic-mediated PPIAO NPs combination therapy achieved a transition from “cold tumor” to “hot tumor” that promoted more CD8+ T lymphocytes activation in vivo and intratumoral infiltration, and successfully inhibited the growth of primary and metastatic tumors. An in situ tumor vaccine effect was produced from the treated tumor tissue, assisting mice against the recurrence of tumor cells. This study provided a simple and effective personalized tumor vaccine strategy for better treatment of metastatic and recurrent tumors. The developed multifunctional tumor antigen trapping nanoparticles may be a promising nanoplatform for integrating multimodal imaging monitoring, tumor treatment, and tumor vaccine immunotherapy.

RECALL URTICARIA IN PATIENT WITH CELIAC DISEASE

<h3>Introduction</h3> Recall urticaria refers to an urticarial response localized to an area of prior antigen exposure and reactivated by various stimuli, typically allergy immunotherapy. Our case describes a patient, ultimately diagnosed with celiac disease, who experiences episodes of recall urticaria at site of previous allergy immunotherapy after ingestion of wheat. <h3>Case Description</h3> The patient is a 28-year-old female with allergic rhinitis previously on allergy immunotherapy from 2015-2016. In June 2020, she began to experience urticaria on the posterior region of her left arm. The episodes occurred in the evening, resolving within 20 minutes after diphenhydramine treatment. She described the location of hives to be consistent with area of prior immunotherapy injections. In July 2020, she started cetirizine 10mg every morning and diphenhydramine 25mg every evening, which controlled her hives outside of recurrence with missed antihistamines doses. She was later diagnosed with celiac disease and initiated a gluten-free diet. She discontinued oral antihistamines in February 2021 and remained free from frequent episodes of hives. However, with accidental ingestion of wheat, she reports a recurrence of localized hives 5-6 hours later. <h3>Discussion</h3> The patient's history of spontaneous localized hives where she previously received allergy immunotherapy is consistent with recall urticaria. Given the correlation between ingestion of wheat and episodes of urticaria, this appears to be her trigger for recall urticaria. Further investigations are underway, including skin prick and patch testing, with potential oral challenge to wheat. The mechanism by which the immune or inflammatory response to gluten triggers her recall urticaria remains to be elucidated.

Divergent clonal differentiation trajectories of T cell exhaustion.

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

Exploring the immunomodulatory role of virtual memory CD8+ T cells: Role of IFN gamma in tumor growth control.

Virtual memory CD8+ T cells (TVM) have been described as cells with a memory-like phenotype but without previous antigen (Ag) exposure. TVM cells have the ability to respond better to innate stimuli rather than by TCR engagement, producing large amounts of interferon gamma (IFNγ) after stimulation with interleukin (IL)-12 plus IL-18. As a result of the phenotypic similarity, TVM cells have been erroneously included in the central memory T cell subset for many years. However, they can now be discriminated via the CD49d receptor, which is up-regulated only on conventional memory T cells (TMEM) and effector T cells (TEFF) after specific cognate Ag recognition by a TCR. In this work we show that systemic expression of IL-12 plus IL-18 induced an alteration in the normal TVM vs TMEM/TEFF distribution in secondary lymphoid organs and a preferential enrichment of TVM cells in the melanoma (B16) and the pancreatic ductal adenocarcinoma(KPC) tumor models. Using our KPC bearing OT-I mouse model, we observed a significant increase in CD8+ T cell infiltrating the tumor islets after IL-12+IL-18 stimulation with a lower average speed when compared to those from control mice. This finding indicates a stronger interaction of T cells with tumor cells after cytokine stimulation. These results correlate with a significant reduction in tumor size in both tumor models in IL-12+IL-18-treated OT-I mice compared to control OT-I mice. Interestingly, the absence of IFNγ completely abolished the high antitumor capacity induced by IL-12+IL-18 expression, indicating an important role for these cytokines in early tumor growth control. Thus, our studies provide significant new information that indicates an important role of TVM cells in the immune response against cancer.

Differential patterns of cross-reactive antibody response against SARS-CoV-2 spike protein detected for chronically ill and healthy COVID-19 naïve individuals

Immunity to previously encountered viruses can alter response to unrelated pathogens. We reasoned that similar mechanism may also involve SARS-CoV-2 and thereby affect the specificity and the quality of the immune response against the virus. Here, we employed high-throughput next generation phage display method to explore the link between antibody immune response to previously encountered antigens and spike (S) glycoprotein. By profiling the antibody response in COVID-19 naïve individuals with a diverse clinical history (including cardiovascular, neurological, or oncological diseases), we identified 15 highly antigenic epitopes on spike protein that showed cross-reactivity with antigens of seasonal, persistent, latent or chronic infections from common human viruses. We observed varying degrees of cross-reactivity of different viral antigens with S in an epitope-specific manner. The data show that pre-existing SARS-CoV-2 S1 and S2 cross-reactive serum antibody is readily detectable in pre-pandemic cohort. In the severe COVID-19 cases, we found differential antibody response to the 15 defined antigenic and cross-reactive epitopes on spike. We also noted that despite the high mutation rates of Omicron (B.1.1.529) variants of SARS-CoV-2, some of the epitopes overlapped with the described mutations. Finally, we propose that the resolved epitopes on spike if targeted by re-called antibody response from SARS-CoV-2 infections or vaccinations can function in chronically ill COVID-19 naïve/unvaccinated individuals as immunogenic targets to boost antibodies augmenting the chronic conditions. Understanding the relationships between prior antigen exposure at the antibody epitope level and the immune response to subsequent infections with viruses from a different strain is paramount to guiding strategies to exit the COVID-19 pandemic.

Hierarchical Clustering and Trajectory Analyses Reveal Viremia-Independent B-Cell Perturbations in HIV-2 Infection.

Time to AIDS in HIV-2 infection is approximately twice as long compared to in HIV-1 infection. Despite reduced viremia, HIV-2-infected individuals display signs of chronic immune activation. In HIV-1-infected individuals, B-cell hyperactivation is driven by continuous antigen exposure. However, the contribution of viremia to B-cell perturbations in HIV-2-infected individuals remains largely unexplored. Here, we used polychromatic flow cytometry, consensus hierarchical clustering and pseudotime trajectory inference to characterize B-cells in HIV-1- or HIV-2-infected and in HIV seronegative individuals. We observed increased frequencies of clusters containing hyperactivated T-bethighCD95highCD27int and proliferating T-bet+CD95highCD27+CD71+ memory B-cells in viremic HIV-1 (p < 0.001 and p < 0.001, respectively), viremic HIV-2 (p < 0.001 and p = 0.014, respectively) and in treatment-naïve aviremic HIV-2 (p = 0.004 and p = 0.020, respectively)-infected individuals, compared to seronegative individuals. In contrast, these expansions were not observed in successfully treated HIV-1-infected individuals. Finally, pseudotime trajectory inference showed that T-bet-expressing hyperactivated and proliferating memory B-cell populations were located at the terminal end of two trajectories, in both HIV-1 and HIV-2 infections. As the treatment-naïve aviremic HIV-2-infected individuals, but not the successfully ART-treated HIV-1-infected individuals, showed B-cell perturbations, our data suggest that aviremic HIV-2-infected individuals would also benefit from antiretroviral treatment.

Enhanced immune responses following heterologous vaccination with self-amplifying RNA and mRNA COVID-19 vaccines.

The optimal vaccination strategy to boost responses in the context of pre-existing immune memory to the SARS-CoV-2 spike (S) glycoprotein is an important question for global public health. To address this, we explored the SARS-CoV-2-specific humoral and cellular immune responses to a novel self-amplifying RNA (saRNA) vaccine followed by a UK authorised mRNA vaccine (BNT162b2) in individuals with and without previous COVID-19, and compared these responses with those who received an authorised vaccine alone. 35 subjects receiving saRNA (saRNA group) as part of the COVAC1 clinical trial and an additional 40 participants receiving an authorised SARS-CoV-2 vaccine only (non-saRNA group) were recruited. Antibody responses were measured by ELISA and a pseudoneutralisation assay for wildtype, Delta and Omicron variants. Cellular responses were measured by IFN-ƴ ELISpot and an activation induced marker (AIM) assay. Approximately 50% in each group had previous COVID-19 prior to vaccination, confirmed by PCR or antibody positivity on ELISA. All of those who received saRNA subsequently received a full course of an authorised vaccine. The majority (83%) of those receiving saRNA who were COVID-19 naïve at baseline seroconverted following the second dose, and those with previous COVID-19 had an increase in antibody titres two weeks following saRNA vaccination (median 27-fold), however titres were lower when compared to mRNA vaccination. Two weeks following the 2nd authorised mRNA vaccine dose, binding and neutralising antibody titres were significantly higher in the saRNA participants with previous COVID-19, compared to non-saRNA, or COVID-19 naive saRNA participants. Cellular responses were again highest in this group, with a higher proportion of spike specific CD8+ than CD4+ T cells when compared to those receiving the mRNA vaccine only. These findings suggest an immunological benefit of increased antigen exposure, both from natural infection and vaccination, particularly evident in those receiving heterologous vaccination with saRNA and mRNA.