65 research outputs found
Selection of DNA nanoparticles with preferential binding to aggregated protein target.
High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life's own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions
CD4+ T Cell Depletion during all Stages of HIV Disease Occurs Predominantly in the Gastrointestinal Tract
The mechanisms underlying CD4+ T cell depletion in human immunodeficiency virus (HIV) infection are not well understood. Comparative studies of lymphoid tissues, where the vast majority of T cells reside, and peripheral blood can potentially illuminate the pathogenesis of HIV-associated disease. Here, we studied the effect of HIV infection on the activation and depletion of defined subsets of CD4+ and CD8+ T cells in the blood, gastrointestinal (GI) tract, and lymph node (LN). We also measured HIV-specific T cell frequencies in LNs and blood, and LN collagen deposition to define architectural changes associated with chronic inflammation. The major findings to emerge are the following: the GI tract has the most substantial CD4+ T cell depletion at all stages of HIV disease; this depletion occurs preferentially within CCR5+ CD4+ T cells; HIV-associated immune activation results in abnormal accumulation of effector-type T cells within LNs; HIV-specific T cells in LNs do not account for all effector T cells; and T cell activation in LNs is associated with abnormal collagen deposition. Taken together, these findings define the nature and extent of CD4+ T cell depletion in lymphoid tissue and point to mechanisms of profound depletion of specific T cell subsets related to elimination of CCR5+ CD4+ T cell targets and disruption of T cell homeostasis that accompanies chronic immune activation
Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation
The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1β, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1β, TNF-α, and IFN-γ in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes
HLA B*5701-positive long-term nonprogressors/elite controllers are not distinguished from progressors by the clonal composition of HIV-specific CD8+ T cells
To better understand the qualitative features of effective human immunodeficiency virus (HIV)-specific immunity, we examined the TCR clonal composition of CD8+ T cells recognizing conserved HIV p24-derived epitopes in HLA-B*5701-positive long-term nonprogressors/elite controllers (LTNP/EC) and HLA-matched progressors. Both groups displayed oligoclonal HLA-B5701-restricted p24-specific CD8+ T-cell responses with similar levels of diversity and few public clonotypes. Thus, HIV-specific CD8+ T-cell responses in LTNP/EC are not differentiated from those of progressors on the basis of clonal diversity or TCR sharing
Acquisition of direct antiviral effector functions by CMV-specific CD4+T lymphocytes with cellular maturation
The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1β, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1β, TNF-α, and IFN-γ in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes
Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses
The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide–major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8+ T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8+ T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI–CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function
Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses
The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide–major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8+ T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8+ T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI–CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function
Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19.
Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100
Genetic Drivers of Heterogeneity in Type 2 Diabetes Pathophysiology
Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P \u3c 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care
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