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Elite controllers (ECs) are people living with HIV (PLWH) able to control HIV replication without antiretroviral therapy and have been proposed as a model of a functional HIV cure. Much evidence suggests that this spontaneous control of HIV has a cost in terms of T cell homeostasis alterations. We performed a deep phenotypic study to obtain insight into T cell homeostasis disturbances in ECs maintaining long-term virologic and immunologic control of HIV (long-term elite controllers; LTECs). Forty-seven PLWH were included: 22 LTECs, 15 non-controllers under successful antiretroviral therapy (onART), and 10 non-controllers not receiving ART (offART). Twenty uninfected participants (UCs) were included as a reference. T cell homeostasis was analyzed by spectral flow cytometry and data were analyzed using dimensionality reduction and clustering using R software v3.3.2. Dimensionality reduction and clustering yielded 57 and 54 different CD4 and CD8 T cell clusters, respectively. The offART group showed the highest perturbation of T cell homeostasis, with 18 CD4 clusters and 15 CD8 clusters significantly different from those of UCs. Most of these alterations were reverted in the onART group. Interestingly, LTECs presented several disturbances of T cell homeostasis with 15 CD4 clusters and 13 CD8 clusters different from UC. Moreover, there was a specific profile of T cell homeostasis alterations associated with LTECs, characterized by increases in clusters of naïve T cells, increases in clusters of non-senescent effector CD8 cells, and increases in clusters of central memory CD4 cells. These results demonstrate that, compared to ART-mediated control of HIV, the spontaneous control of HIV is associated with several disturbances in CD4 and CD8 T cell homeostasis. These alterations could be related to the existence of a potent and efficient virus-specific T cell response, and to the ability to halt disease progression by maintaining an adequate pool of CD4 T cells.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) that control HIV replication without therapy. Among the mechanisms involved in this ability, natural killer (NK)-cells have recently gained much attention. We performed an in-deep phenotypic analysis of NK-cells to search for surrogate markers associated with the long term spontaneous control of HIV. Forty-seven PLWH (22 long-term EC [PLWH-long-term elite controllers (LTECs)], 15 noncontrollers receiving antiretroviral treatment [ART] [PLWH-onART], and 10 noncontrollers cART-naïve [PLWH-offART]), and 20 uninfected controls were included. NK-cells homeostasis was analyzed by spectral flow cytometry using a panel of 15 different markers. Data were analyzed using FCSExpress and R software for unsupervised multidimensional analysis. Six different subsets of NK-cells were defined on the basis of CD16 and CD56 expression, and the multidimensional analysis revealed the existence of 68 different NK-cells clusters based on the expression levels of the 15 different markers. PLWH-offART presented the highest disturbance of NK-cells homeostasis and this was not completely restored by long-term ART. Interestingly, long term spontaneous control of HIV (PLWH-LTEC group) was associated with a specific profile of NK-cells homeostasis disturbance, characterized by an increase of CD16 dim CD56 dim subset when compared to uninfected controls (UC) group and also to offART and onART groups (p < 0.0001 for the global comparison), an increase of clusters C16 and C26 when compared to UC and onART groups (adjusted p-value < 0.05 for both comparisons), and a decrease of clusters C10 and C20 when compared to all the other groups (adjusted p-value < 0.05 for all comparisons). These findings may provide clues to elucidate markers of innate immunity with a relevant role in the long-term control of HIV., (© 2024 Wiley Periodicals LLC.)

(1) Background: The role of hepatitis C virus (HCV) co-infection on the T-cell homeostasis disturbances in human immunodeficiency virus (HIV)-infected patients as well as its reversion after HCV eradication with direct acting antivirals (DAAs) therapy has not been yet clarified. We extensively analyzed the effect of HCV co-infection on immune parameters of HIV pathogenesis and its evolution after HCV eradication with DAAs. (2) Methods: Seventy individuals were included in the study-25 HIV-monoinfected patients, 25 HIV/HCV-coinfected patients and 20 HIV and HCV seronegative subjects. All patients were on antiretroviral therapy and undetectable HIV-viremia. Immune parameters, such as maturation, activation, apoptosis, senescence and exhaustion of T-cells were assessed by flow cytometry. Cross-sectional and longitudinal (comparing pre- and post-DAAs data in HIV/HCV coinfected patients) analyses were performed. Univariate and multivariate (general linear model and canonical discriminant analysis -CDA-) analyses were used to assess differences between groups. (3) Results-The CDA was able to clearly separate HIV/HCV coinfected from HIV-monoinfected patients, showing a more disturbed T-cells homeostasis in HIV/HCV patients, especially activation and exhaustion of T-cells. Interestingly, those perturbations were more marked in HIV/HCV patients with increased liver stiffness. Eradication of HCV with DAAs restored some but not all the T-cells homeostasis disturbances, with activation and exhaustion of effector CD8 T-cells remaining significantly increased three months after HCV eradication. (4) Conclusions-HCV co-infection significantly impacts on several immune markers of HIV pathogenesis, especially in patients with increased liver stiffness. Eradication of HCV with DAAs ameliorates but does not completely normalize these alterations. It is of utmost relevance to explore other mechanisms underlying the immune damage observed in HIV/HCV coinfected patients with control of both HIV and HCV replication.

A recent study has pointed out to CD32a as a potential biomarker of HIV-persistent CD4 cells. We have characterized the level and phenotype of CD32+ cells contained in different subsets of CD4 T-cells and its potential correlation with level of total HIV-DNA in thirty HIV patients (10 typical progressors naïve for cART, 10 cART-suppressed patients, and 10 elite controllers). Total HIV-DNA was quantified in different subsets of CD4 T-cells: Trm and pTfh cells. Level and immunephenotype of CD32+ cells were analyzed in these same subsets by flow cytometry. CD32 expression in Trm and pTfh subsets was similar in the different groups, and there was no significant correlation between the level of total HIV-DNA and the level of CD32 expression in these subsets. However, total HIV-DNA level was correlated with expression of CD127 (rho = -0.46, p = 0.043) and of CCR6 (rho = -0.418, p = 0.027) on CD32+ cells. Our results do not support CD32 as a biomarker of total HIV-DNA content. However, analyzing the expression of certain markers by CD32+ cells could improve the utility of this marker in the clinical setting, prompting the necessity of further studies to both validate our results and to explore the potential utility of certain markers expressed by CD32+ cells.

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103 + dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: Despite long-lasting HIV replication control, a significant proportion of elite controller (EC) patients may experience CD4 T-cell loss. Discovering perturbations in immunological parameters could help our understanding of the mechanisms that may be operating in those patients experiencing loss of immunological control., Methods: A case-control study was performed to evaluate if alterations in different T-cell homeostatic parameters can predict CD4 T-cell loss in ECs by comparing data from EC patients showing significant CD4 decline (cases) and EC patients showing stable CD4 counts (controls). The partial least-squares-class modeling (PLS-CM) statistical methodology was employed to discriminate between the two groups of patients, and as a predictive model., Results: Herein, we show that among T-cell homeostatic alterations, lower levels of naïve and recent thymic emigrant subsets of CD8 cells and higher levels of effector and senescent subsets of CD8 cells as well as higher levels of exhaustion of CD4 cells, measured prior to CD4 T-cell loss, predict the loss of immunological control., Conclusions: These data indicate that the parameters of T-cell homeostasis may identify those EC patients with a higher proclivity to CD4 T-cell loss. Our results may open new avenues for understanding the mechanisms underlying immunological progression despite HIV replication control, and eventually, for finding a functional cure through immune-based clinical trials.

HIV latency is the main barrier to HIV eradication. Peripheral T follicular helper (pTfh) cells have a prominent role in HIV persistence. Herein, we analyzed the HIV reservoir size within memory CD4+ T-cell subsets in patients with HIV replication control. Twenty HIV-infected patients with suppressed HIV replication were included, with 10 elite controllers (EC) and 10 treated (TX) individuals. The HIV reservoir size was analyzed in resting memory CD4+ T-cells (Trm), pTfh, and non-pTfh cells using an ultrasensitive digital-droplet-PCR assay. Inter-group and intra-group differences were tested using non-parametric tests. Compared with the TX patients, the EC patients had smaller HIV reservoir not only in Trm but also in pTfh and non-pTfh subsets of memory CD4+ T-cells. The largest differences were observed in pTfh cells (p = 0.025). The pTfh and non-pTfh cells harbored similar levels of HIV-DNA in the EC (p = 0.60) and TX patients (p = 0.17); however, the contribution to HIV-DNA levels in memory CD4+ T-cells varied among the pTfh and non-pTfh subsets in both groups of patients. The EC patients showed smaller HIV reservoir in memory CD4+ cells, especially in the pTfh subset, a population of cells with a pivotal role in the antiviral immune response, suggesting a potential link between low levels of infection in pTfh cells and the ability of the EC patients to spontaneously control HIV replication.

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation.

Background: The causes of HIV-vaccines failure are poorly understood. Therapeutic vaccination with modified vaccinia Ankara (MVA)-B in HIV-1-infected individuals did not control the virus upon analytical treatment interruption (ATI). We investigated whether the functional characteristics of HIV-specific CD8 T-cell responses stimulated by this vaccine, and the level of exhaustion of these cells might explain these results., Methods: Twenty-one HIV-1 chronically infected patients on combination antiretroviral therapy, included in the therapeutic vaccine trial RISVAC03, were studied: 13 immunized and eight controls. Functional characteristics, cytotoxic potential and exhaustion of HIV-specific CD8 T cells, were evaluated by polychromatic flow cytometry. Differences between groups were tested using nonparametric tests., Results: MVA-B vaccine induced an increase in HIV-specific CD8 T-cell response, but also increased their levels of exhaustion. At week 18 (following three immunizations) the level of response increased with respect to baseline (P = 0.02). A significant increase at weeks 18 and 24 (ATI) in granzyme B content was also observed. Interestingly, an increase in expression of exhaustion markers was found at weeks 18 (P = 0.006) and 24 (P = 0.01). However, there was no significant change in the functional profile of vaccine-induced CD8 cells. At week 36, in parallel to the rebound of plasma viremia after 12 weeks ATI, a significant increase in the level of CD8 response, in granzyme B content and in exhaustion markers expression, was observed in both groups., Conclusion: We show that therapeutic vaccination with MVA-B tilts the balance between activation and regulation of the response of HIV-specific CD8 T cells towards regulation, which impacts on the viral rebound after ATI.

The life-long maintenance of haematopoietic stem and progenitor cells (HSPCs) critically relies on environmental signals produced by cells that constitute the haematopoietic niche. Here we report a cell-intrinsic mechanism whereby haematopoietic cells limit proliferation within the bone marrow, and show that this pathway is repressed by E-selectin ligand 1 (ESL-1). Mice deficient in ESL-1 display aberrant HSPC quiescence, expansion of the immature pool and reduction in niche size. Remarkably, the traits were transplantable and dominant when mutant and wild-type precursors coexisted in the same environment, but were independent of E-selectin, the vascular receptor for ESL-1. Instead, quiescence is generated by unrestrained production of the cytokine TGFβ by mutant HSPC, and in vivo or in vitro blockade of the cytokine completely restores the homeostatic properties of the haematopoietic niche. These findings reveal that haematopoietic cells, including the more primitive compartment, can actively shape their own environment.

Polycomb group (PcG) proteins are key epigenetic regulators of hematopietic stem cell (HSC) fate. The PcG members Ezh2 and Ezh1 are important determinants of embryonic stem cell identity, and the transcript levels of these histone methyltransferases are inversely correlated during development. However, the role of Ezh1 in somatic stem cells is largely unknown. Here we show that Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response. Epigenomic and gene expression changes induced by Ezh1 deletion in senesced HSCs demonstrated that Ezh1-mediated PRC2 activity catalyzes monomethylation and dimethylation of H3K27. Deletion of Cdkn2a on the Ezh1 null background rescued HSC proliferation and survival. Our results suggest that Ezh1 is an important histone methyltransferase for HSC maintenance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recent evidence shows increased and decreased expression of Ezh2 in cancer, suggesting a dual role as an oncogene or tumour suppressor. To investigate the mechanism by which Ezh2-mediated H3K27 methylation leads to cancer, we generated conditional Ezh2 knock-in (Ezh2-KI) mice. Here we show that induced Ezh2 haematopoietic expression increases the number and proliferation of repopulating haematopoietic stem cells. Ezh2-KI mice develop myeloproliferative disorder, featuring excessive myeloid expansion in bone marrow and spleen, leukocytosis and splenomegaly. Competitive and serial transplantations demonstrate progressive myeloid commitment of Ezh2-KI haematopoietic stem cells. Transplanted self-renewing haematopoietic stem cells from Ezh2-KI mice induce myeloproliferative disorder, suggesting that the Ezh2 gain-of-function arises in the haematopoietic stem cell pool, and not at later stages of myelopoiesis. At the molecular level, Ezh2 regulates haematopoietic stem cell-specific genes such as Evi-1 and Ntrk3, aberrantly found in haematologic malignancies. These results demonstrate a stem cell-specific Ezh2 oncogenic role in myeloid disorders, and suggest possible therapeutic applications in Ezh2-related haematological malignancies.

Preservation of hematopoietic hierarchy requires a constant and reciprocal interplay between chromatin-specific epigenetic regulators and lineage-modifying transcription factors. The polycomb member Bmi1 is a key factor in hematopoietic stem cell (HSC) maintenance, but its specific physiological role in subsequent hematopoietic lineage-specific commitments is unclear. Here, we generated conditional Bmi1 knockout (Bmi1-KO) mice. Selective ablation of Bmi1 in the hematopoietic system induced extensive upregulation of Ikaros and concomitant Ikaros-dependent lymphoid-lineage transcriptional priming, which is marked by their loss of H2A ubiquitination and increased H3K4 trimethylation in Bmi1-KO long-term HSCs (LT-HSCs). Removal of Ikaros in Bmi1-null LT-HSCs significantly diminished the hematopoietic defects seen in conditional Bmi1-KO mice. These alterations resulted in recovering the Bmi1-KO exhausted quiescent stem-cell pool, whereas the block in Bmi1-KO lymphoid-progenitor differentiation was rescued, allowing the development of mature lymphoid cells. Together, our results indicate that Ikaros is a critical Bmi1 target in vivo that prevents premature lineage specification of HSCs.

Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues., Competing Interests: The authors have declared that no competing interests exist.

The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator.

PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar to N-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins.

We report the cloning of the full-length cDNA of a new murine protein kinase, mPKL12. The sequence reveals a 305-amino-acid protein that contains the characteristic subdomains of the kinase superfamily and particular homology indicating a ser/thr specificity. We have also identified its human homologue gene (94% identical) and the putative homologue proteins from Saccharomyces cerevisiae and Arabidoposis thaliana. These four sequences appear to form a new subfamily of protein kinases, close in size to the theoretical minimal catalytic domain, therefore suggesting that they could be the catalytic unit of a more complex holoenzyme. Using Escherichia coli-purified protein, we have demonstrated that the mPKL12 enzyme possesses an intrinsic kinase activity, capable of phosphorylating enolase and also of promoting autophosphorylation, with a ser/thr specificity. Tissue expression analysis of mPKL12 showed that it is ubiquitously expressed, although at very low levels. RT-PCR analysis of several cell lines also supports this view, therefore suggesting that PKL12 may play a role in a very general cellular function, probably related with the secretory pathway., (Copyright 1998 Academic Press.)