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The novel coronavirus pandemic continues to cause significant morbidity and mortality around the world. Diverse clinical presentations prompted numerous attempts to predict disease severity to improve care and patient outcomes. Equally important is understanding the mechanisms underlying such divergent disease outcomes. Multivariate modeling was used here to define the most distinctive features that separate COVID-19 from healthy controls and severe from moderate disease. Using discriminant analysis and binary logistic regression models we could distinguish between severe disease, moderate disease, and control with rates of correct classifications ranging from 71 to 100%. The distinction of severe and moderate disease was most reliant on the depletion of natural killer cells and activated class-switched memory B cells, increased frequency of neutrophils, and decreased expression of the activation marker HLA-DR on monocytes in patients with severe disease. An increased frequency of activated class-switched memory B cells and activated neutrophils was seen in moderate compared to severe disease and control. Our results suggest that natural killer cells, activated class-switched memory B cells, and activated neutrophils are important for protection against severe disease. We show that binary logistic regression was superior to discriminant analysis by attaining higher rates of correct classification based on immune profiles. We discuss the utility of these multivariate techniques in biomedical sciences, contrast their mathematical basis and limitations, and propose strategies to overcome such limitations. [ABSTRACT FROM AUTHOR]

Chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from people living with HIV, here we expose the T cells to planar lipid bilayers containing ligands for T-cell receptor and a T-cell integrins and analyze the cellular morphology, dynamics of synaptic interface formation and patterns of the cellular degranulation. We find a large fraction of phenotypically naive T cells from chronically infected people are capable to form mature synapse with focused degranulation, a signature of a differentiated T cells. Further, differentiation of aberrant naive T cells may lead to the development of anomalous effector T cells undermining their capacity to control HIV and other pathogens that could be contained otherwise. HIV infection over time is thought to result in premature aging and aberrant immune responses including the induction of immunological senescence. Here the authors show altered formation of immune synapses by naive CD8+ T cells and dysregulated synapse functioning at late differentiated stages in people living with HIV. [ABSTRACT FROM AUTHOR]

Antiretroviral therapy (ART) is not curative due to the persistence of a reservoir of HIV-infected cells, particularly in tissues such as lymph nodes, with the potential to cause viral rebound after treatment cessation. In this study, fingolimod (FTY720), a lysophospholipid sphingosine-1-phosphate receptor modulator is administered to SIV-infected rhesus macaques at initiation of ART to block the egress from lymphoid tissues of natural killer and T-cells, thereby promoting proximity between cytolytic cells and infected CD4+ T-cells. When compared with the ART-only controls, FTY720 treatment during the initial weeks of ART induces a profound lymphopenia and increases frequencies of CD8+ T-cells expressing perforin in lymph nodes, but not their killing capacity; FTY720 also increases frequencies of cytolytic NK cells in lymph nodes. This increase of cytolytic cells, however, does not limit measures of viral persistence during ART, including intact proviral genomes. After ART interruption, a subset of animals that initially receives FTY720 displays a modest delay in viral rebound, with reduced plasma viremia and frequencies of infected T follicular helper cells. Further research is needed to optimize the potential utility of FTY720 when coupled with strategies that boost the antiviral function of T-cells in lymphoid tissues. Although antiretroviral therapy (ART) is able to successfully suppress plasma viremia in most people living with HIV, ART withdrawal typically results in viral replication and rebound. Authors investigate the effect, in terms of delay in viral replication, and immune cell dynamics in lymphoid tissue, of fingolimod (FTY720) administration at the time of ART initiation in SIV-infected rhesus macaques. [ABSTRACT FROM AUTHOR]

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale. Peptide-MHC (pMHC) tetramers are important tools for probing T cell repertoire and adaptive immune responses. Here the authors use a molecular chaperone, TAPBPR, to develop a high-throughput, multiplexible platform for pMHC tetramer generation to facilitate simultaneous assessments of T cell repertoire/antigen specificity and transcriptome. [ABSTRACT FROM AUTHOR]

HIV-1-associated neurocognitive disorder (HAND) remains a persistent problem despite antiretroviral therapy (ART), largely a result of continued inflammation in the periphery and the brain and neurotoxin release from activated myeloid cells in the CNS. CD14+CD16+ inflammatory monocytes, expanded in HIV infection, play a central role in the pathogenesis of HAND and have parallels with monocyte-dependent inflammatory mechanisms in atherosclerosis. Statins, through their HMG-CoA reductase inhibitor activity, have pleiotropic immunomodulatory properties that contribute to their benefit in atherosclerosis beyond lipid lowering. Here, we investigated whether statins would modulate the monocyte phenotype and function associated with HIV-1 neuropathogenesis. Treatment ex vivo with simvastatin and atorvastatin reduced the proportion of CD16+ monocytes in peripheral blood mononuclear cells, as well as in purified monocytes, especially CD14++CD16+ 'intermediate' monocytes most closely associated with neurocognitive disease. Statin treatment also markedly reduced expression of CD163, which is also linked to HAND pathogenesis. Finally, simvastatin inhibited production of monocyte chemoattractant protein-1 (MCP-1) and other inflammatory cytokines following LPS stimulation and reduced monocyte chemotaxis in response to MCP-1, a major driver of myeloid cell accumulation in the CNS in HAND. Together, these findings suggest that statin drugs may be useful to prevent or reduce HAND in HIV-1-infected subjects on ART with persistent monocyte activation and inflammation. [ABSTRACT FROM AUTHOR]

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses. [ABSTRACT FROM AUTHOR]

We isolated pure, viable populations of tumor-cytolytic T cells directly from patient blood samples using flow cytometric quantification of the surface mobilization of CD107a-an integral membrane protein in cytolytic granules-as a marker for degranulation after tumor stimulation. We show that tumor-cytolytic T cells are indeed elicited in patients after cancer vaccination, and that tumor reactivity is strongly correlated with efficient T-cell recognition of peptide-bearing targets. We combined CD107a mobilization with peptide-major histocompatibility complex (P-MHC) tetramer staining to directly correlate antigen specificity and cytolytic ability on a single-cell level. This showed that tumor-cytolytic T cells with high recognition efficiency represent only a minority of peptide-specific T cells elicited in patients after heteroclitic peptide vaccination. We were also able to expand these cells to high numbers ex vivo while maintaining their cytolytic potential. These techniques will be useful not only for immune monitoring of cancer vaccine trials, but also for adoptive cellular immunotherapy after ex vivo expansion. The ability to rapidly identify and isolate tumor-cytolytic T cells would be very useful in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Investigates the infection of human immunodeficiency virus (HIV)-specific CD4+ T cells by HIV. Stages of HIV disease; Effects of cell division and interferon-γ secretion on HIV infection of CD4+ T cells; Steps in T-cell stimulation and sorting.

The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study. [ABSTRACT FROM AUTHOR]

The mechanisms underlying possible increased HIV-1 acquisition in adenovirus 5 (Ad5)-seropositive subjects vaccinated with Ad5–HIV-1 vectors in the Merck STEP trial remain unclear. We find that Ad5 serostatus does not predict Ad5-specific CD4+ T cell frequency, and we did not observe durable significant differences in Ad5-specific CD4+ T cells between Ad5-seropositive and Ad5-seronegative subjects after vaccination. These findings indicate no causative role for Ad5-specific CD4+ T cells in increasing HIV-1 susceptibility in the STEP trial. [ABSTRACT FROM AUTHOR]

HIV-specific CD8+ T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8+ T cells were almost exclusively TIGIT+, had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIThi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4+ T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8+ T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future 'cure' strategies requiring potent HIV-specific CD8+ T cells. [ABSTRACT FROM AUTHOR]