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In hospitalized COVID-19 patients, disease progression leading to acute kidney injury (AKI) may be driven by immune dysregulation. We explored the role of urinary cytokines and their relationship with kidney stress biomarkers in COVID-19 patients before and after the development of AKI. Of 51 patients, 54.9% developed AKI. The principal component analysis indicated that in subclinical AKI, epidermal growth factor (EGF) and interferon (IFN)-α were associated with a lower risk of AKI, while interleukin-12 (IL-12) and macrophage inflammatory protein (MIP)-1β were associated with a higher risk of AKI. After the manifestation of AKI, EGF and IFN-α remained associated with a lower risk of AKI, while IL-1 receptor (IL-1R), granulocyte-colony stimulating factor (G-CSF), interferon-gamma-inducible protein 10 (IP-10) and IL-5 were associated with a higher risk of AKI. EGF had an inverse correlation with kidney stress biomarkers. Subclinical AKI was characterized by a significant up-regulation of kidney stress biomarkers and proinflammatory cytokines. The lack of EGF regenerative effects and IFN-α antiviral activity seemed crucial for renal disease progression. AKI involved a proinflammatory urinary cytokine storm.

Background: Respiratory failure in severe coronavirus disease 2019 (COVID-19) is associated with a severe inflammatory response. Acetylcholine (ACh) reduces systemic inflammation in experimental bacterial and viral infections. Pyridostigmine increases the half-life of endogenous ACh, potentially reducing systemic inflammation. We aimed to determine if pyridostigmine decreases a composite outcome of invasive mechanical ventilation (IMV) and death in adult patients with severe COVID-19., Methods: We performed a double-blinded, placebo-controlled, phase 2/3 randomized controlled trial of oral pyridostigmine (60 mg/day) or placebo as add-on therapy in adult patients admitted due to confirmed severe COVID-19 not requiring IMV at enrollment. The primary outcome was a composite of IMV or death by day 28. Secondary outcomes included reduction of inflammatory markers and circulating cytokines, and 90-day mortality. Adverse events (AEs) related to study treatment were documented and described., Results: We recruited 188 participants (94 per group); 112 (59.6%) were men; the median (IQR) age was 52 (44-64) years. The study was terminated early due to a significant reduction in the primary outcome in the treatment arm and increased difficulty with recruitment. The primary outcome occurred in 22 (23.4%) participants in the placebo group vs. 11 (11.7%) in the pyridostigmine group (hazard ratio, 0.47, 95% confidence interval 0.24-0.9; P = 0.03). This effect was driven by a reduction in mortality (19 vs. 8 deaths, respectively)., Conclusion: Our data indicate that adding pyridostigmine to standard care reduces mortality among patients hospitalized for severe COVID-19., (© 2022. The Author(s).)

Follicular-helper T cells (T FH ) are an essential arm of the adaptive immune system. Although T FH were first discovered through their ability to contribute to antibody affinity maturation through co-stimulatory interactions with B cells, new light has been shed on their ability to remain a complex and functionally plastic cell type. Due to a lack sample availability, however, many studies have been limited to characterizing T FH in mice or non-canonical tissue types, such as peripheral blood. Such constraints have resulted in a limited, and sometimes contradictory, understanding of this fundamental cell type. One subset of T FH receiving attention in chronic infection are CXCR3-expressing T FH cells (CXCR3 + T FH ) due to their abnormal accumulation in secondary lymphoid tissues. Their function and clonal relationship with other T FH subsets in lymphoid tissues during infection, however, remains largely unclear. We thus systematically investigated this and other subsets of T FH within untreated HIV-infected human lymph nodes using Mass CyTOF and a combination of RNA and TCR repertoire sequencing. We show an inflation of the CXCR3 + T FH compartment during HIV infection that correlates with a lower HIV burden. Deeper analysis into this population revealed a functional shift of CXCR3 + T FH away from germinal center T FH (GC-T FH ), including the altered expression of several important transcription factors and cytokines. CXCR3 + T FH also upregulated cell migration transcriptional programs and were clonally related to peripheral T FH populations. In combination, these data suggest that CXCR3 + T FH have a greater tendency to enter circulation than their CXCR3 - counterparts, potentially functioning through distinct modalities that may lead to enhanced defense., Competing Interests: NJ is a Scientific Advisor and holds equity interest in ImmuDX, LLC, and Immune Arch, Inc. In the interest of full disclosure, DBW reports that he serves on Advisories for AstraZeneca, Geneos, Advaccinepharma, he participates in BOD service for Inovio. Remuneration received by DBW for these services includes SRA funding, direct payments, stock or stock options disclosed here. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 He, Malone, Wendel, Ma, Del Alcazar, Weiner, De Jager, Del Río-Estrada, Ablanedo-Terrazas, Reyes-Terán, Su and Jiang.)

A high proportion of critically ill patients with COVID-19 develop acute kidney injury (AKI) and die. The early recognition of subclinical AKI could contribute to AKI prevention. Therefore, this study was aimed at exploring the role of the urinary biomarkers NGAL and [TIMP-2] × [IGFBP7] for the early detection of AKI in this population. This prospective, longitudinal cohort study included critically ill COVID-19 patients without AKI at study entry. Urine samples were collected on admission to critical care areas for determination of NGAL and [TIMP-2] × [IGFBP7] concentrations. The demographic information, comorbidities, clinical, and laboratory data were recorded. The study outcomes were the development of AKI and mortality during hospitalization. Of the 51 individuals that were studied, 25 developed AKI during hospitalization (49%). Of those, 12 had persistent AKI (23.5%). The risk factors for AKI were male gender (HR = 7.57, 95% CI: 1.28-44.8; p = 0.026) and [TIMP-2] × [IGFBP7] ≥ 0.2 (ng/mL) 2 /1000 (HR = 7.23, 95% CI: 0.99-52.4; p = 0.050). Mortality during hospitalization was significantly higher in the group with AKI than in the group without AKI ( p = 0.004). Persistent AKI was a risk factor for mortality (HR = 7.42, 95% CI: 1.04-53.04; p = 0.046). AKI was frequent in critically ill COVID-19 patients. The combination of [TIMP-2] × [IGFBP7] together with clinical information, were useful for the identification of subclinical AKI in critically ill COVID-19 patients. The role of additional biomarkers and their possible combinations for detection of AKI in ritically ill COVID-19 patients remains to be explored in large clinical trials.

The main barrier to a cure for HIV is the persistence of long-lived and proliferating latently infected CD4 + T-cells despite antiretroviral therapy (ART). Latency is well characterized in multiple CD4 + T-cell subsets, however, the contribution of regulatory T-cells (Tregs) expressing FoxP3 as well as immune checkpoints (ICs) PD-1 and CTLA-4 as targets for productive and latent HIV infection in people living with HIV on suppressive ART is less well defined. We used multiplex detection of HIV DNA and RNA with immunohistochemistry (mIHC) on formalin-fixed paraffin embedded (FFPE) cells to simultaneously detect HIV RNA and DNA and cellular markers. HIV DNA and RNA were detected by in situ hybridization (ISH) (RNA/DNAscope) and IHC was used to detect cellular markers (CD4, PD-1, FoxP3, and CTLA-4) by incorporating the tyramide system amplification (TSA) system. We evaluated latently infected cell lines, a primary cell model of HIV latency and excisional lymph node (LN) biopsies collected from people living with HIV (PLWH) on and off ART. We clearly detected infected cells that coexpressed HIV RNA and DNA (active replication) and DNA only (latently infected cells) in combination with IHC markers in the in vitro infection model as well as LN tissue from PLWH both on and off ART. Combining ISH targeting HIV RNA and DNA with IHC provides a platform to detect and quantify HIV persistence within cells identified by multiple markers in tissue samples from PLWH on ART or to study HIV latency., (Copyright © 2021 Elsevier B.V. All rights reserved.)

CXCR5 is a key marker of follicular helper T (T FH ) cells. Using primary lymph nodes (LNs) from HIV-infected patients, we identified a population of CXCR5 - CD4 + T cells with T FH -cell-like features. This CXCR5 - subset becomes expanded in severe HIV infection and is characterized by the upregulation of activation markers and high PD-1 and ICOS surface expression. Integrated analyses on the phenotypic heterogeneity, functional capacity, T cell receptor (TCR) repertoire, transcriptional profile, and epigenetic state of CXCR5 - PD-1 + ICOS + T cells revealed a shared clonal relationship with T FH cells. CXCR5 - PD-1 + ICOS + T cells retained a poised state for CXCR5 expression and exhibited a migratory transcriptional program. TCR sequence overlap revealed a contribution of LN-derived CXCR5 - PD-1 + ICOS + T cells to circulating CXCR5 - CD4 + T cells with B cell help function. These data link LN pathology to circulating T cells and expand the current understanding on the diversity of T cells that regulate B cell responses during chronic inflammation., (Published by Elsevier Inc.)

Recent evidence has revealed that PD-L1 is expressed in two functional forms, namely, a membrane-bound form (mPD-L1) and a soluble form (sPD-L1). The identification of the soluble form of PD-L1 represents the discovery of a new potential mechanism for the activation of the PD-1 pathway that may mediate a physiological apoptotic mechanism through a cell-cell signalling-independent pathway and may also favour T cell dysfunction during HIV infection. Since the presence of sPD-L1 has not been well established in the scenario of chronic viral infection, we investigated the presence of sPD-L1 in the plasma of viraemic HIV + individuals and the potential mechanism that promotes its production. We report the following: 1) the level of the soluble form of PD-L1 is increased in the plasma of viraemic HIV + individuals, 2) the level of the soluble form of PD-L1 in viraemic HIV + individuals correlates with markers of microbial product translocation and inflammation, 3) the expression of the membrane-bound form of PD-L1 on conventional dendritic cells from viraemic HIV + individuals correlates with the levels of soluble PD-L1 and MMP-2, and 4) monocyte-derived dendritic cells not only increase their expression of mPD-L1 and MMP-2 but also produce sPD-L1 after LPS and TNF-α stimulation, as demonstrated by functional in vitro experiments, which provides insight into the potential source of sPD-L1 production., (Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Follicular helper CD4 + T cells (T FH ) play an integral role in promoting B cell differentiation and affinity maturation. Whereas T FH cell frequencies are increased in lymph nodes (LNs) from individuals infected with HIV, humoral immunity remains impaired during chronic HIV infection. Whether HIV inhibits T FH responses in LNs remains unclear. Advances in this area have been limited by the difficulty of accessing human lymphoid tissues. Here, we combined high-dimensional mass cytometry with T cell receptor repertoire sequencing to interrogate the composition of T FH cells in primary human LNs. We found evidence for intact antigen-driven clonal expansion of T FH cells and selective utilization of specific complementarity-determining region 3 (CDR3) motifs during chronic HIV infection, but the resulting T FH cells acquired an activation-related T FH cell signature characterized by interleukin-21 (IL-21) dominance. These IL-21 + T FH cells contained an oligoclonal HIV-reactive population that preferentially accumulated in patients with severe HIV infection and was associated with aberrant B cell distribution in the same LN. These data indicate that T FH cells remain capable of responding to HIV antigens during chronic HIV infection but become functionally skewed and oligoclonally restricted under persistent antigen stimulation., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cytolytic CD8 T cells play a crucial role in the control and elimination of virus-infected cells and are a major focus of HIV cure efforts. However, it has been shown that HIV-specific CD8 T cells are infrequently found within germinal centers (GCs), a predominant site of active and latent HIV infection. We demonstrate that HIV infection induces marked changes in the phenotype, frequency, and localization of CD8 T cells within the lymph node (LN). Significantly increased frequencies of CD8 T cells in the B cell follicles and GCs were found in LNs from treated and untreated HIV-infected individuals. This profile was associated with persistent local immune activation but did not appear to be directly related to local viral replication. Follicular CD8 (fCD8) T cells, despite compromised cytokine polyfunctionality, showed good cytolytic potential characterized by high ex vivo expression of granzyme B and perforin. We used an anti-HIV/anti-CD3 bispecific antibody in a redirected killing assay and found that fCD8 T cells had better killing activity than did non-fCD8 T cells. Our results indicate that CD8 T cells with potent cytolytic activity are recruited to GCs during HIV infection and, if appropriately redirected to kill HIV-infected cells, could be an effective component of an HIV cure strategy., (Copyright © 2017, American Association for the Advancement of Science.)

The immune response induced by dendritic cells (DC) during the HIV infection has been of remarkable interest because of the therapeutic potential of DC for vaccine development. However, their beneficial or detrimental contribution in HIV infection remains unclear. The activation state of DC in lymph nodes (LN) is essential to induce T cell responses against HIV. In the present study, we characterized the immunophenotype and function of conventional (cDC) and plasmacytoid (pDC) dendritic cells from peripheral blood (PB) and LN of HIV(+) individuals. We observed that the frequency of PB pDC was decreased and exhibited an immature phenotype; whereas in the LN, activated pDC accumulated (CD40(+) and CD83(+)). In addition, the frequency of PB cDC from HIV(+) individuals was decreased and exhibited an immature phenotype, whereas LN harbored activated and mature cDC (CD40(+), CD83(+), CD80(+) and CD86(+)). However, an increased number of PD-L1(+) cDC was also observed in the LN. Moreover, pDC and cDC were able to produce inflammatory cytokines (IFN-α, TNF-α and IL-12) after TLR stimulation. These findings suggests that LN cDC expressing PD-L1 from HIV(+) individuals may negatively impact the generation of HIV-specific T cells and that DC might be contributing to tissue chronic immune activation., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

HIV(+) subjects are reported to have increased soluble CD14 (sCD14) in plasma, an indicator of microbial translocation. We evaluated if microbial translocation has a differential impact on the activation and function of conventional dendritic cells (cDC) from viraemic HIV(+) subjects and HIV(+) controllers (CTs). The HIV(+) subjects were classified into two groups according to their plasma viral load (pVL): CT and viraemic. Subjects without HIV were included as controls (HIV(-) ). The frequencies and phenotypes of cDC from these subjects were evaluated by multi-parameter flow cytometry. In addition, peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) or single-stranded RNA40 (ssRNA40), the phenotype of the cDC and the intracellular production of tumour necrosis factor (TNF)-α by the cDC were evaluated by flow cytometry. We observed a partial activation phenotype for the cDC in the viraemic subjects and CTs ex vivo and after LPS activation, which showed differences in the expression of CD40 and CD86. Furthermore, in response to LPS the cDC from the viraemic subjects produced more TNF-α compared to the cDC from CTs. Interestingly, the percentage of TNF-α(+) cDC was found to be correlated positively with the pVL. The partial activation of cDC and the over-production of TNF-α in response to LPS in viraemic HIV(+) subjects might be related to the increased chronic activation observed in these subjects. In contrast, cDC from CTs seem to have a regulated response to LPS, indicating that they respond differently to chronic immune activation. These results may have implications in the development of HIV therapies and vaccines using DC., (© 2014 British Society for Immunology.)