RESUMO
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 (TS) 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 TS cells and tracked their frequency in bulk TCRß 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 TS cell clonotypes, most of which were CD8+ T cells, while also eliciting diverse TS 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 TCRß repertoires and paired-chain TCRÉß sequences from droplet sequencing of TS cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevenção & controle , Linfócitos T CD8-Positivos , Vacinação , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Anticorpos AntiviraisRESUMO
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.
Assuntos
Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/imunologia , Uganda , Adulto , Masculino , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/genética , Feminino , Tuberculose/imunologia , Tuberculose/microbiologia , Linfócitos T/imunologia , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Células Clonais/imunologia , Resistência à Doença/imunologia , Resistência à Doença/genética , Adulto Jovem , Antígenos de Histocompatibilidade Classe IRESUMO
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.
Assuntos
Células Endoteliais , Ativação Linfocitária , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Comunicação Celular , Citocinas/metabolismo , Células Endoteliais/metabolismo , Transplante HomólogoRESUMO
Endothelial cells (ECs) sense and adapt to their environment, allowing them to display a range of functional phenotypes and promote vascular homeostasis across organ systems. However, many of these cues are lost when cells are cultured in vitro. This work explores how substratum interactions influence cellular phenotype. Culture conditions, specifically 2D culture on tissue culture polystyrene (TCP) versus 3D culture on collagen scaffolds, had a much greater effect on EC phenotype than did in vivo cell source. The 3D ECs responded to hypoxic gradients by inducing the expression of HIF1-a while 2D ECs underwent endothelial-to-mesenchymal transition. In comparing the effect of culture condition on EC phenotype and function to its effect on epithelial cells (EPs) and fibroblasts (FBs), it is evident that ECs are not simply vascular EPs but are unique in their response. For cell types like ECs, which are particularly responsive to their microenvironment, traditional culture on TCP is insufficient for revealing in vivo behavior. Further applying these findings, we found that culture-condition differentially affected the expression of immunomodulatory factors and in an in vivo model of allotransplantation, 3D culture reduced the cytotoxic response of host immune cells to all three cell types compared to 2D culture.
Assuntos
Células Endoteliais , Poliestirenos , Células Cultivadas , Colágeno/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Fenótipo , Poliestirenos/metabolismoRESUMO
Almost three years into the SARS-CoV-2 pandemic, hybrid immunity is highly prevalent worldwide and more protective than vaccination or prior infection alone. Given emerging resistance of variant strains to neutralizing antibodies (nAb), it is likely that T cells contribute to this protection. To understand how sequential SARS-CoV-2 infection and mRNA-vectored SARS-CoV-2 spike (S) vaccines affect T cell clonotype-level expansion kinetics, we identified and cross-referenced TCR sequences from thousands of S-reactive single cells against deeply sequenced peripheral blood TCR repertoires longitudinally collected from persons during COVID-19 convalescence through booster vaccination. Successive vaccinations recalled memory T cells and elicited antigen-specific T cell clonotypes not detected after infection. Vaccine-related recruitment of novel clonotypes and the expansion of S-specific clones were most strongly observed for CD8+ T cells. Severe COVID-19 illness was associated with a more diverse CD4+ T cell response to SARS-CoV-2 both prior to and after mRNA vaccination, suggesting imprinting of CD4+ T cells by severe infection. TCR sequence similarity search algorithms revealed myriad public TCR clusters correlating with human leukocyte antigen (HLA) alleles. Selected TCRs from distinct clusters functionally recognized S in the predicted HLA context, with fine viral peptide requirements differing between TCRs. Most subjects tested had S-specific T cells in the nasal mucosa after a 3rd mRNA vaccine dose. The blood and nasal T cell responses to vaccination revealed by clonal tracking were more heterogeneous than nAb boosts. Analysis of bulk and single cell TCR sequences reveals T cell kinetics and diversity at the clonotype level, without requiring prior knowledge of T cell epitopes or HLA restriction, providing a roadmap for rapid assessment of T cell responses to emerging pathogens.
RESUMO
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 (ScCO2) that can achieve a sterility assurance level 10(-6) in decellularized lung matrix. The effects of ScCO2 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 ScCO2 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 ScCO2, indicating that ScCO2 produces a matrix that is stable during storage. The current study's results indicate that ScCO2 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.
Assuntos
Dióxido de Carbono/farmacologia , Pulmão/fisiologia , Esterilização/métodos , Células A549 , Animais , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Fenômenos Mecânicos/efeitos dos fármacos , Ácido Peracético/farmacologia , Ratos Sprague-Dawley , Alicerces Teciduais/químicaRESUMO
Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.