Despite marked improvements in the modern treatment era, human immunodeficiency virus (HIV)-infected individuals, particularly those who initiated antiretroviral therapy (ART) at advanced disease stages, continue to have increased age-related morbidity and mortality, compared with the general population. Immune activation and inflammation persist despite suppressive ART and predict many of these morbidities. The goal of this review is to examine the evidence suggesting a link between the persistent inflammatory state and morbidity and mortality in this setting, to describe the impact of early ART initiation on these factors, and to highlight important unanswered questions for the field. We also advance a hypothesis to explain why some morbidities-and their root inflammatory drivers-may be prevented more than others by early ART initiation.

While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naïve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naïve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naïve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naïve counterparts.

Background

Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures.

Results

Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets.

Conclusions

We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Stimulant use may accelerate HIV disease progression through biological and behavioral pathways. However, scant research with treated HIV-positive persons has examined stimulant-associated alterations in pathophysiologic processes relevant to HIV pathogenesis. In a sample of 55 HIV-positive, methamphetamine-using sexual minority men with a viral load less than 200 copies/mL, we conducted RNA sequencing to examine patterns of leukocyte gene expression in participants who had a urine sample that was reactive for stimulants (n = 27) as compared to those who tested non-reactive (n = 28). Results indicated differential expression of 32 genes and perturbation of 168 pathways in recent stimulant users. We observed statistically significant differential expression of single genes previously associated with HIV latency, cell cycle regulation, and immune activation in recent stimulant users (false discovery rate p < 0.10). Pathway analyses indicated enrichment for genes associated with inflammation, innate immune activation, neuroendocrine hormone regulation, and neurotransmitter synthesis. Recent stimulant users displayed concurrent elevations in plasma levels of tumor necrosis factor - alpha (TNF-α) but not interleukin 6 (IL-6). Further research is needed to examine the bio-behavioral mechanisms whereby stimulant use may contribute to HIV persistence and disease progression.

A man with newly diagnosed AIDS presented with months of back pain and fever. Computed tomography (CT) results demonstrated aortitis with periaortic tissue thickening. DNA amplification of biopsy tissue revealed Bartonella quintana, and Bartonella serologies were subsequently noted to be positive. The patient improved with prolonged doxycycline and rifabutin treatment. This case illustrates how molecular techniques are increasingly important in diagnosing Bartonella infections.

T cells residing in mucosal tissues play important roles in homeostasis and defense against microbial pathogens. The gut and female reproductive tract (FRT) are both tolerogenic environments, but they differ in the kinds of foreign Ags they need to tolerate. How these different environments influence the properties of their T cells is poorly understood, but important for understanding women's health. We recruited antiretroviral therapy-suppressed women living with HIV who donated, within one visit, blood and tissue samples from the ileum, colon, rectosigmoid, endometrium, endocervix, and ectocervix. With these samples, we conducted 36-parameter cytometry by time of flight phenotyping of T cells. Although gut and FRT T cells shared features discriminating them from their blood counterparts, they also harbored features distinguishing them from one another. These included increased proportions of CD69⁺ T resident memory cells of the T effector memory phenotype, as well as preferential coexpression of CD69 and CD103, on the gut-derived cells. In contrast, CD69⁺CD103⁺ T resident memory CD8⁺ T cells from FRT, but not those from gut, preferentially expressed PD1. We further determined that a recently described population of CXCR4⁺ T inflammatory mucosal cells differentially expressed multiple other chemokine receptors relative to their blood counterparts. Our findings suggest that T cells resident in different tolerogenic mucosal sites take on distinct properties.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56^brightTCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features.

Background

Unlike cytomegalovirus (CMV) infection and aging, human immunodeficiency virus (HIV) decreases the proportion of CD28(-)CD8(+) T cells expressing CD57. Whether this abnormality predicts mortality in treated HIV infection and can be reversed by early antiretroviral therapy (ART) remains unknown.

Methods

We sampled recently HIV-infected individuals (<6 months) and HIV-uninfected controls and compared longitudinal changes in the proportion of CD28(-)CD8(+) T cells expressing CD57 between those who initiated ART early (<6 months) vs later (≥2 years). We also assessed the relationship between this phenotype and mortality in a nested case-control study of ART-suppressed chronically infected individuals.

Results

Compared to HIV-uninfected controls (n = 15), individuals who were recently infected with HIV had lower proportions of CD28(-)CD8(+) T cells expressing CD57 (P < .001), and these proportions increased during ART. The early ART group (n = 33) achieved normal levels, whereas the later ART group (n = 30) continued to have lower levels than HIV-uninfected controls (P = .02). Among 141 ART-suppressed participants in the SOCA study, those in the lowest quartile of CD28(-)CD8(+) T cells expressing CD57 had 5-fold higher odds of mortality than those in the highest quartile (95% CI, 1.6-15.9, P = .007).

Conclusions

Abnormally low proportions of CD28(-)CD8(+) T cells expressing CD57 predict increased mortality during treated HIV infection and may be reversed with early ART initiation.

Convalescing coronavirus disease 2019 (COVID-19) patients mount robust T cell responses against SARS-CoV-2, suggesting an important role of T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells and predominantly Tcm cells with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can proliferate homeostatically, and can persist for over 2 months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection and support an important role of SARS-CoV-2-specific T cells in host control of COVID-19.