High frequencies of polyfunctional CD8+ NK cells in chronic HIV-1 infection are associated with slower disease progression.

UNLABELLED: Natural killer (NK) cells are effector and regulatory innate immune cells and play a critical role in the first line of defense against various viral infections. Although previous reports have indicated the vital contributions of NK cells to HIV-1 immune control, nongenetic NK cell parameters directly associated with slower disease progression have not been defined yet. In a longitudinal, retrospective study of 117 untreated HIV-infected subjects, we show that higher frequencies as well as the absolute numbers of CD8(+) CD3(-) lymphocytes are linked to delayed HIV-1 disease progression. We show that the majority of these cells are well-described blood NK cells. In a subsequent cross-sectional study, we demonstrate a significant loss of CD8(+) NK cells in untreated HIV-infected individuals, which correlated with HIV loads and inversely correlated with CD4(+) T cell counts. CD8(+) NK cells had modestly higher frequencies of CD57-expressing cells than CD8(-) cells, but CD8(+) and CD8(-) NK cells showed no differences in the expression of a number of activating and inhibiting NK cell receptors. However, CD8(+) NK cells exhibited a more functional profile, as detected by cytokine production and degranulation. IMPORTANCE: We demonstrate that the frequency of highly functional CD8(+) NK cells is inversely associated with HIV-related disease markers and linked with delayed disease progression. These results thus indicate that CD8(+) NK cells represent a novel NK cell-derived, innate immune correlate with an improved clinical outcome in HIV infection.

Dynamics of virological and immunological markers of HIV persistence after allogeneic haematopoietic stem-cell transplantation in the IciStem cohort: a prospective observational cohort study.

BACKGROUND: Allogeneic haematopoietic stem-cell transplantation (allo-HSCT) markedly reduces HIV reservoirs, but the mechanisms by which this occurs are only partly understood. In this study, we aimed to describe the dynamics of virological and immunological markers of HIV persistence after allo-HSCT. METHODS: In this prospective observational cohort study, we analysed the viral reservoir and serological dynamics in IciStem cohort participants with HIV who had undergone allo-HSCT and were receiving antiretroviral therapy, ten of whom had received cells from donors with the CCR5Δ32 mutation. Participants from Belgium, Canada, Germany, Italy, the Netherlands, Spain, Switzerland, and the UK were included in the cohort both prospectively and retrospectively between June 1, 2014 and April 30, 2019. In the first 6 months after allo-HSCT, participants had monthly assessments, with annual assessments thereafter, with the protocol tailored to accommodate for the individual health status of each participant. HIV reservoirs were measured in blood and tissues and HIV-specific antibodies were measured in plasma. We used the Wilcoxon signed-rank test to compare data collected before and after allo-HSCT in participants for whom longitudinal data were available. When the paired test was not possible, we used the Mann-Whitney U test. We developed a mathematical model to study the factors influencing HIV reservoir reduction in people with HIV after allo-HSCT. FINDINGS: We included 30 people with HIV with haematological malignancies who received a transplant between Sept 1, 2009 and April 30, 2019 and were enrolled within the IciStem cohort and included in this analysis. HIV reservoirs in peripheral blood were reduced immediately after full donor chimerism was achieved, generally accompanied by undetectable HIV-DNA in bone marrow, ileum, lymph nodes, and cerebrospinal fluid, regardless of donor CCR5 genotype. HIV-specific antibody levels and functionality values declined more slowly than direct HIV reservoir values, decaying significantly only months after full donor chimerism. Mathematical modelling suggests that allogeneic immunity mediated by donor cells is the main viral reservoir depletion mechanism after massive reservoir reduction during conditioning chemotherapy before allo-HSCT (half-life of latently infected replication-competent cells decreased from 44 months to 1·5 months). INTERPRETATION: Our work provides, for the first time, data on the effects of allo-HSCT in the context of HIV infection. Additionally, we raise the question of which marker can serve as the last reporter of the residual viraemia, postulating that the absence of T-cell immune responses might be a more reliable marker than antibody decline after allo-HSCT. FUNDING: amfAR (American Foundation for AIDS Research; ARCHE Program), National Institutes of Health, National Institute of Allergy and Infectious Diseases, and Dutch Aidsfonds.

In-depth virological and immunological characterization of HIV-1 cure after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation.

Despite scientific evidence originating from two patients published to date that CCR5Δ32/Δ32 hematopoietic stem cell transplantation (HSCT) can cure human immunodeficiency virus type 1 (HIV-1), the knowledge of immunological and virological correlates of cure is limited. Here we characterize a case of long-term HIV-1 remission of a 53-year-old male who was carefully monitored for more than 9 years after allogeneic CCR5Δ32/Δ32 HSCT performed for acute myeloid leukemia. Despite sporadic traces of HIV-1 DNA detected by droplet digital PCR and in situ hybridization assays in peripheral T cell subsets and tissue-derived samples, repeated ex vivo quantitative and in vivo outgrowth assays in humanized mice did not reveal replication-competent virus. Low levels of immune activation and waning HIV-1-specific humoral and cellular immune responses indicated a lack of ongoing antigen production. Four years after analytical treatment interruption, the absence of a viral rebound and the lack of immunological correlates of HIV-1 antigen persistence are strong evidence for HIV-1 cure after CCR5Δ32/Δ32 HSCT.

HIV infection is associated with a preferential decline in less-differentiated CD56dim CD16+ NK cells.

HIV-1 infection is characterized by loss of CD56(dim) CD16(+) NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57(-) and CD57(dim) cells but not of CD57(bright) cells on CD56(dim) CD16(+) NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27(+), HLA-DR(+), Ki-67(+), and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.

Decreased Frequency of Intestinal CD39+ γδ+ T Cells With Tissue-Resident Memory Phenotype in Inflammatory Bowel Disease.

The ectoenzymes CD39 and CD73 play a major role in controlling tissue inflammation by regulating the balance between adenosine triphosphate (ATP) and adenosine. Still, little is known about the role of these two enzymes and ATP and its metabolites in the pathophysiology of inflammatory bowel disease (IBD). We isolated mononuclear cells from peripheral blood and lamina propria of the large intestine of patients diagnosed with IBD and of healthy volunteers. We then comprehensively analyzed the CD39 and CD73 expression patterns together with markers of activation (HLA-DR, CD38), differentiation (CCR7, CD45RA) and tissue-residency (CD69, CD103, CD49a) on CD4+, CD8+, γδ+ T cells and mucosa-associated invariant T cells using flow cytometry. CD39 expression levels of γδ+ and CD8+ T cells in lamina propria lymphocytes (LPL) were much higher compared to peripheral blood mononuclear cells. Moreover, the frequency of CD39+ CD4+ and CD8+, but not γδ+ LPL positively correlated with T-cell activation. The frequency of CD39+ cells among tissue-resident memory LPL (Trm) was higher compared to non-Trm for all subsets, confirming that CD39 is a marker for the tissue-resident memory phenotype. γδ+ Trm also showed a distinct cytokine profile upon stimulation - the frequency of IFN-γ+ and IL-17A+ cells was significantly lower in γδ+ Trm compared to non-Trm. Interestingly, we observed a decreased frequency of CD39+ γδ+ T cells in IBD patients compared to healthy controls (p = 0.0049). Prospective studies need to elucidate the exact role of this novel CD39+ γδ+ T-cell population with tissue-resident memory phenotype and its possible contribution to the pathogenesis of IBD and other inflammatory disorders.

Vulnerability to reservoir reseeding due to high immune activation after allogeneic hematopoietic stem cell transplantation in individuals with HIV-1.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only medical intervention that has led to an HIV cure. Whereas the HIV reservoir sharply decreases after allo-HSCT, the dynamics of the T cell reconstitution has not been comprehensively described. We analyzed the activation and differentiation of CD4+ and CD8+ T cells, and the breadth and quality of HIV- and CMV-specific CD8+ T cell responses in 16 patients with HIV who underwent allo-HSCT (including five individuals who received cells from CCR5Δ32/Δ32 donors) to treat their underlying hematological malignancy and who remained on antiretroviral therapy (ART). We found that reconstitution of the T cell compartment after allo-HSCT was slow and heterogeneous with an initial expansion of activated CD4+ T cells that preceded the expansion of CD8+ T cells. Although HIV-specific CD8+ T cells disappeared immediately after allo-HSCT, weak HIV-specific CD8+ T cell responses were detectable several weeks after transplant and could still be detected at the time of full T cell chimerism, indicating that de novo priming, and hence antigen exposure, occurred during the time of T cell expansion. These HIV-specific T cells had limited functionality compared with CMV-specific CD8+ T cells and persisted years after allo-HSCT. In conclusion, immune reconstitution was slow, heterogeneous, and incomplete and coincided with de novo detection of weak HIV-specific T cell responses. The initial short phase of high T cell activation, in which HIV antigens were present, may constitute a window of vulnerability for the reseeding of viral reservoirs, emphasizing the importance of maintaining ART directly after allo-HSCT.

HCV-specific CD4+ T cells of patients with acute and chronic HCV infection display high expression of TIGIT and other co-inhibitory molecules.

The combined regulation of a network of inhibitory and activating T cell receptors may be a critical step in the development of chronic HCV infection. Ex vivo HCV MHC class I + II tetramer staining and bead-enrichment was performed with baseline and longitudinal PBMC samples of a cohort of patients with acute, chronic and spontaneously resolved HCV infection to assess the expression pattern of the co-inhibitory molecule TIGIT together with PD-1, BTLA, Tim-3, as well as OX40 and CD226 (DNAM-1) of HCV-specific CD4+ T cells, and in a subset of patients of HCV-specific CD8+ T cells. As the main result, we found a higher expression level of TIGIT+ PD-1+ on HCV-specific CD4+ T cells during acute and chronic HCV infection compared to patients with spontaneously resolved HCV infection (p < 0,0001). Conversely, expression of the complementary co-stimulatory receptor of TIGIT, CD226 (DNAM-1) was significantly decreased on HCV-specific CD4+ T cells during chronic infection. The predominant phenotype of HCV-specific CD4+ T cells during acute and chronic infection was TIGIT+, PD-1+, BTLA+, Tim-3-. This comprehensive phenotypic study confirms TIGIT together with PD-1 as a discriminatory marker of dysfunctional HCV-specific CD4+ T cells.

Comparison of the integrin α4ß7 expression pattern of memory T cell subsets in HIV infection and ulcerative colitis.

Anti-α4ß7 therapy with vedolizumab (VDZ) has been suggested as possible immune intervention in HIV. Relatively little is known about the α4ß7-integrin (α4ß7) expression of different T-cell subsets in different anatomical compartments of healthy individuals, patients with HIV or inflammatory bowel disease (IBD). Surface expression of α4ß7 as well as the frequency of activation, homing and exhaustion markers of T cells were assessed by multicolour flow cytometry in healthy volunteers (n = 15) compared to HIV infected patients (n = 52) or patients diagnosed with ulcerative colitis (UC) (n = 14), 6 of whom treated with vedolizumab. In addition, lymph nodal cells (n = 6), gut-derived cells of healthy volunteers (n = 5) and patients with UC (n = 6) were analysed. Additionally, we studied longitudinal PBMC samples of an HIV patient who was treated with vedolizumab for concomitant UC. Overall, only minor variations of the frequency of α4ß7 on total CD4+ T cells were detectable regardless of the disease status or (VDZ) treatment status in peripheral blood and the studied tissues. Peripheral α4ß7+ CD4+ T cells of healthy individuals and patients with UC showed a higher activation status and were more frequently CCR5+ than their α4ß7- counterparts. Also, the frequency of α4ß7+ cells was significantly lower in peripheral blood CD4+ effector memory T cells of HIV-infected compared to healthy individuals and this reduced frequency did not recover in HIV patients on ART. Conversely, the frequency of peripheral blood naïve α4ß7+ CD4+ T cells was significantly reduced under VDZ treatment. The results of the current study will contribute to the understanding of the dynamics of α4ß7 expression pattern on T cells in HIV and UC and will be useful for future studies investigating VDZ as possible HIV cure strategy.

CD32 Expression of Different Memory T Cell Subpopulations in the Blood and Lymph Nodal Tissue of HIV Patients and Healthy Controls Correlates With Immune Activation.

BACKGROUND: Recently, CD32 has been described to be a specific surface marker of latently HIV-infected CD4 T cells, but little is known about the frequency and distribution of CD32 expression on naive and memory CD8 and CD4 T cell populations in HIV patients and healthy individuals. METHODS: We studied peripheral blood samples of 36 HIV-1-infected patients [23 viremic patients / 13 antiretroviral therapy(ART)-treated] and healthy individuals (n = 14) as well as cells from lymph nodes (8 HIV infected, 5 controls) using a multiparametric flow cytometry panel determining surface expression of CD3, CD8, CD4, CD45RA, CCR7, CD27, CD25, CD127, CCR5, CCR6, CXCR4, CD38, HLA-DR, TIGIT, and PD-1. RESULTS: Overall, expression of CD32 on total peripheral CD4 T cells between viremic HIV patients, ART-treated and healthy individuals only slightly differed (mean values 1.501%, 0.2785%, and 0.2343%, respectively). However, the level of expression was significantly higher in peripheral and lymph nodal memory CD4 T cell subpopulations of viremic patients compared with ART-treated patients and healthy controls. CD32 CD4 T cells showed higher immune activation and higher expression of CXCR4 than their CD32 counterparts. Furthermore, expression of CD32 on total CD4 T cells and memory T cell populations correlated with general immune activation regardless of the infection status. CONCLUSIONS: Follow-up studies will have to further evaluate CD32 as marker of latently HIV-infected CD4 T cells since other host-related variables such as immune activation seem to influence CD32 expression regardless of the infection status.

Brief Report: Increased Frequency of CD39+ CD56bright Natural Killer Cells in HIV-1 Infection Correlates With Immune Activation and Disease Progression.

The expression pattern of the ectonucleotidases CD39 and CD73 on natural killer (NK) cells was examined in peripheral blood mononuclear cell of 61 HIV-1-infected patients. Increased frequencies of CD39CD56 NK cells were detectable in untreated HIV patients, which was associated with high viral load, low CD4 T-cell count, and CD8 T-cell activation. Additionally, levels of CD39 on NK cells were inducible by in vitro stimulation of NK cells, correlating with aryl hydrocarbon receptor and interleukin 10 expression. Here, we provide the first evidence of increased CD39CD56 NK cell frequencies during HIV infection, which might have consequences for NK cell function and HIV pathogenesis.

Down-regulation of CD73 on B cells of patients with viremic HIV correlates with B cell activation and disease progression.

Recently, alterations of the T cell expression of the ectonucleotidases, CD39 and CD73, during HIV infection have been described. Here, peripheral (n = 70) and lymph nodal B cells (n = 10) of patients with HIV at different stages of disease as well as uninfected individuals were analyzed via multicolor flow cytometry with regard to expression of CD39 and CD73 and differentiation, proliferation, and exhaustion status. Patients with chronic, untreated HIV showed a significantly decreased frequency of CD73-expressing B cells (P < 0.001) compared with healthy controls. Decreased frequencies of CD39+CD73+ B cells in patients with HIV correlated with low CD4+ counts (P < 0.0256) as well as increased proliferation and exhaustion status as determined by Ki-67 and programmed death-1 expression. Down-regulation of CD73 was observed in naive and memory B cells as determined by CD27 and CD21. Neither HIV elite controller patients nor antiretroviral therapy-treated patients had significantly lower CD39 and CD73 expression on B cells compared with healthy controls. Of importance, low CD73+ expression on B cells was associated with modulated in vitro B cell function. Further in vivo studies are warranted to evaluate the in vivo role of phenotypic loss of CD73 in B cell dysregulation in HIV.

Parallel assessment of Th17 cell frequencies by surface marker co-expression versus ex vivo IL-17 production in HIV-1 infection.

INTRODUCTION: Th17 cells can either be identified by co-staining of surface markers or by intracellular cytokine staining (ICS) for IL-17 production. Discrepancies regarding the published frequencies of Th17 cells in peripheral blood mononuclear cells (PBMC) of HIV patients may partly be due to the different methodologies used. METHODS: Cryopreserved PBMC from healthy controls and HIV-infected subjects, including treated (cART) and viremic patients, were split and analyzed side-by-side by flow cytometry for expression of surface markers CCR6, CXCR3, CCR4, and CD161, or for intracellular expression of IL-17A and IFNγ after stimulation. RESULTS: The characterization of Th17 cells as CXCR3 - CCR6 + CCR4 + CD161+ yielded considerably higher frequencies than the corresponding frequencies obtained by characterization via cytokines (IL-17 + IFNγ-), regardless of the HIV status. However, the overall frequencies delivered by the two methods significantly correlated. The relative frequency of Th17 cells within the CD4+ T cell compartment was preserved in HIV infection but there was a significant decrease in the absolute Th17 number, which was restored after initiation of cART, paralleling CD4+ T cell recovery. Absolute Th17 numbers inversely correlated with HIV viral load. CONCLUSION: The definition of Th17 cells by surface markers might overestimate their frequency in comparison to functional assessment of IL-17 production by ICS, regardless of the HIV infection status. However, both methods yield proportionate results with reduced absolute numbers of Th17 cells in untreated HIV disease, reflecting the depletion of total CD4+ T cells in viremic HIV patients, and restoration with cART. © 2016 International Clinical Cytometry Society.

Loss of CCR7 expression on CD56(bright) NK cells is associated with a CD56(dim)CD16⁺ NK cell-like phenotype and correlates with HIV viral load.

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56(dim)CD16⁺ and CD56⁻CD16⁺ NK cells. However, the impact of HIV-infection on CD56(bright) NK cells is less well understood. Here we report a rise of CD56(bright) NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7⁻CD56(bright) NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56(dim)CD16⁺ NK cells. Furthermore, CD56(bright) NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56(bright) NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.

Phenotypically and functionally distinct subsets contribute to the expansion of CD56-/CD16+ natural killer cells in HIV infection.

OBJECTIVE: Chronic HIV infection has been associated with activation and increased turnover of natural killer (NK) cells as well as with disturbed homeostasis of the NK cell compartment, including loss of CD56(+) NK cells and accumulation of dysfunctional CD56(-)/CD16(+) NK cells. We performed a comprehensive phenotypical and functional characterization of this population. DESIGN: A cross-sectional study was performed to analyze CD56(-)/CD16(+) NK cells from 34 untreated HIV-infected and 15 seronegative individuals. METHODS: NK cells were analyzed by flow cytometry. Degranulation was assessed by measuring their expression of CD107a after stimulation with K562 cells, interleukin-12 and interleukin-15. RESULTS: CD56(-)/CD16(+) NK cells are heterogeneous and composed of two populations, namely CD122(-)/CCR7(+) cells and CD122(-)/CCR7(+) cells. We show that expanded CD122(+) but not CCR7(+) cells in HIV-seropositive individuals are characterized by expression of senescence marker CD57 similarly to CD56(dim)/CD16(+) NK cells along with expression of KIRs, CD8, perforin and granzyme B. Despite expression of perforin and granzyme B, CD57 expressing cells exhibited less numbers of degranulating cells as measured by CD107a, indicating their functional impairment. However, there was no correlation between expansion of total CD56(-)/CD16(+) NK cells or the distinct subpopulations and viral load or CD4 cell count. CONCLUSION: These data indicate that expansion of CD56(-)/CD16(+) cells in HIV infection is driven by a distinct subset within this population with high expression of terminal differentiation marker with a phenotype resembling CD56(-)/CD16(+) NK cells.