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.
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.
Elimination of lymphoid tissue reservoirs is a key component of HIV eradication strategies. CD8+ T cells play a critical role in control of HIV, but their functional attributes in lymph nodes (LNs) ...remain unclear. Here, we show that memory, follicular CXCR5+, and HIV-specific CD8+ T cells from LNs do not manifest the properties of cytolytic CD8+ T cells. While the frequency of follicular CXCR5+ CD8+ T cells was strongly inversely associated with peripheral viremia, this association was not dependent on cytolytic CXCR5+ CD8+ T cells. Moreover, the poor cytolytic activity of LN CD8+ T cells was linked to a compartmentalized dissociation between effector programming and the transcription factor T-bet. In line with this, activation of LN CD8+ T cells only partially induced the acquisition of cytolytic functions relative to peripheral blood CD8+ T cells. These results suggest that a state of immune privilege against CD8+ T cell-mediated cytolysis exists in lymphoid tissue, potentially facilitating the persistence of HIV.
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•CD8+ T cells in quiescent lymphoid tissues do not express markers of cytotoxicity•Lymphoid tissue HIV-specific CD8+ T cells do not possess full cytolytic markers•Noncytolytic CXCR5+ CD8+ T cells in lymphoid tissue associate with viral control
Reuter et al. show that lymphoid tissue CD8+ T cells from HIV-infected and uninfected individuals do not possess phenotypic, functional, or transcriptional regulatory properties of cytolytic T cells equivalent to those found in circulation. Their findings suggest that the failure to eliminate HIV could be related to compartmentalized CD8+ T cell function favoring noncytolytic responses in lymphoid tissue.
CD38 expression on CD56dimCD16+ NK cells is strongly associated with HIV‐1 disease progression, and affected by the individualˈs immunologic state.
The main predictor of HIV‐1 disease progression is ...CD8+ T cell activation, characterized by elevated expression of CD38 and HLA‐DR. NK cells are also activated in viremic HIV‐1‐infected individuals. However, the relationship between NK cell activation and HIV‐1 disease progression remains undefined. We characterized NK cell activation and its association with disease progression in treatment of naive HIV‐1‐infected individuals, who naturally maintained low/undetectable viremia (elite and viremic controllers), compared with progressors and AIDS subjects, and treated individuals. Our results show that CD38 expression on NK cells, predominantly in the cytotoxic CD56dimCD16+ subset, is associated with HIV‐1 disease progression (CD4+ T cell count and pVL), T cell activation (percentage of CD38+HLA‐DR+ T cells), sCD14, inflammation, and innate immune activation. Moreover, NK cell activation is increased in HIV‐1‐infected subjects progressing to AIDS but not in elite and viremic controllers. ART partially reduces the proportion of activated NK cells. Furthermore, our results show that individuals, who naturally control viremia, maintain low levels of innate immune activation similar to those of uninfected controls.
Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on ...abundance of specific lymphoid subsets explain these correlations.
Multiple regression was used to determine the relationship between abundance of specific blood lymphoid cell types, age, sex, requirement for hospitalization, duration of hospitalization, and elevation of blood markers of systemic inflammation, in adults hospitalized for severe COVID-19 (n = 40), treated for COVID-19 as outpatients (n = 51), and in uninfected controls (n = 86), as well as in children with COVID-19 (n = 19), recovering from COVID-19 (n = 14), MIS-C (n = 11), recovering from MIS-C (n = 7), and pediatric controls (n = 17).
This observational study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan - T cell subsets decrease less than 2-fold - and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis. Among controls, the percentage of ILCs that produced amphiregulin was higher in females than in males, and people hospitalized with COVID-19 had a lower percentage of ILCs that produced amphiregulin than did controls.
These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance contributes to increased COVID-19 severity with age and in males.
This work was supported in part by the Massachusetts Consortium for Pathogen Readiness and NIH grants R37AI147868, R01AI148784, F30HD100110, 5K08HL143183.
Recent advances in high-resolution multiparametric flow cytometry enable ever deeper analysis of human lymphocyte subsets that require rigorous methodology development and optimization. Here, we ...detail methods to characterize glycosylated Sialyl-LewisX (SLeX)- or cutaneous lymphocyte-associated antigen (CLA)-expressing CD4+ T cells using two separate multiparametric flow cytometry panels enabling the identification of memory subsets, Th subsets, and expression of diverse activation markers and chemokine receptors. The proposed protocol allows optimal resolution of the measured parameters while minimizing background in a 25-parameter experiment.
For complete details on the use and execution of this protocol, please refer to Colomb et al. (2020).
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•25-parameter panels for glycosylated CD4+ T cell deep immune profiling•Characterization of memory subsets and Th profiles on CD4+ T cells•Quantification of expression levels of many activation markers and chemokine receptors•Optimal resolution and minimal background
Recent advances in high-resolution multiparametric flow cytometry enable ever deeper analysis of human lymphocyte subsets that require rigorous methodology development and optimization. Here, we detail methods to characterize glycosylated Sialyl-LewisX (SLeX)- or cutaneous lymphocyte-associated antigen (CLA)-expressing CD4+ T cells using two separate multiparametric flow cytometry panels enabling the identification of memory subsets, Th subsets, and expression of diverse activation markers and chemokine receptors. The proposed protocol allows optimal resolution of the measured parameters while minimizing background in a 25-parameter experiment.
A comprehensive understanding of the phenotype of persistent HIV-infected cells, transcriptionally active and/or transcriptionally inactive, is imperative for developing a cure. The relevance of ...cell-surface glycosylation to HIV persistence has never been explored. We characterize the relationship between cell-surface glycomic signatures and persistent HIV transcription in vivo. We find that the cell surface of CD4+ T cells actively transcribing HIV, despite suppressive therapy, harbors high levels of fucosylated carbohydrate ligands, including the cell extravasation mediator Sialyl-LewisX (SLeX), compared with HIV-infected transcriptionally inactive cells. These high levels of SLeX are induced by HIV transcription in vitro and are maintained after therapy in vivo. Cells with high-SLeX are enriched with markers associated with HIV susceptibility, signaling pathways that drive HIV transcription, and pathways involved in leukocyte extravasation. We describe a glycomic feature of HIV-infected transcriptionally active cells that not only differentiates them from their transcriptionally inactive counterparts but also may affect their trafficking abilities.
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•Persistent HIV transcription modulates the glycosylation of infected cells•Cells actively transcribing HIV harbor high levels of fucosylated carbohydrates•HIV transcription induces the fucosylated extravasation mediator, Sialyl-LewisX•Cells with high Sialyl-LewisX are enriched with pathways involved in trafficking
Cell-surface glycans play a critical role in cell functions and fate. Nevertheless, the relevance of host glycosylation to HIV persistence is unknown. Colomb et al. characterized the cell-surface glycomes of HIV-infected cells during therapy and identified glycomic signatures of these cells that may affect cell trafficking and therefore HIV persistence.
Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 ...patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.
The durability of immune memory after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccination remains unclear. In this study, we longitudinally profiled vaccine ...responses in SARS-CoV-2–naïve and –recovered individuals for 6 months after vaccination. Antibodies declined from peak levels but remained detectable in most subjects at 6 months. By contrast, mRNA vaccines generated functional memory B cells that increased from 3 to 6 months postvaccination, with the majority of these cells cross-binding the Alpha, Beta, and Delta variants. mRNA vaccination further induced antigen-specific CD4
and CD8
T cells, and early CD4
T cell responses correlated with long-term humoral immunity. Recall responses to vaccination in individuals with preexisting immunity primarily increased antibody levels without substantially altering antibody decay rates. Together, these findings demonstrate robust cellular immune memory to SARS-CoV-2 and its variants for at least 6 months after mRNA vaccination.
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