Elderly human hematopoietic progenitor cells express cellular senescence markers and are more susceptible to pyroptosis.

The maintenance of effective immunity over time is dependent on the capacity of hematopoietic stem cells (HSCs) to sustain the pool of immunocompetent mature cells. Decline of immune competence with old age may stem from HSC defects, including reduced self-renewal potential and impaired lymphopoiesis, as suggested in murine models. To obtain further insights into aging-related alteration of hematopoiesis, we performed a comprehensive study of blood hematopoietic progenitor cells (HPCs) from older humans. In the elderly, HPCs present active oxidative phosphorylation and are pressed to enter cell cycling. However, p53-p21 and p15 cell senescence pathways, associated with telomerase activity deficiency, strong telomere attrition, and oxidative stress, are engaged, thus limiting cell cycling. Moreover, survival of old HPCs is impacted by pyroptosis, an inflammatory form of programmed cell death. Lastly, telomerase activity deficiency and telomere length attrition of old HPCs may be passed on to progeny cells such as naive T lymphocytes, further highlighting the poor hematopoietic potential of the elderly. This pre-senescent profile is characteristic of the multiple intrinsic and extrinsic factors affecting HPCs in elderly individuals and represents a major obstacle in terms of immune reconstitution and efficacy with advanced age.

Acute Simian Immunodeficiency Virus Infection Triggers Early and Transient Interleukin-7 Production in the Gut, Leading to Enhanced Local Chemokine Expression and Intestinal Immune Cell Homing.

The intestinal barrier, one of the first targets of HIV/simian immunodeficiency virus (SIV) is subjected to major physiological changes during acute infection. Having previously shown that pharmaceutical injection of interleukin-7 (IL-7) triggers chemokine expression in many organs leading to massive T-cell homing, in particular to the intestine, we here explored mucosal IL-7 expression as part of the cytokine storm occurring during the acute phase of SIV infection in rhesus macaques. Quantifying both mRNA and protein in tissues, we demonstrated a transient increase of IL-7 expression in the small intestine of SIV-infected rhesus macaques, starting with local detection of the virus by day 3 of infection. We also observed increased transcription levels of several chemokines in the small intestine. In infected macaques, ileal IL-7 expression correlated with the transcription of four of these chemokines. Among these chemokines, the macrophage and/or T-cell attractant chemokines CCL4, CCL25, and CCL28 also demonstrated increased transcription in uninfected IL-7-treated monkeys. Through immunohistofluorescence staining and image analysis, we observed increased CD8+ T-cell numbers and stable CD4+ T-cell counts in the infected lamina propria (LP) during hyperacute infection. Concomitantly, circulating CCR9+beta7+ CD4+ and CD8+ T-cells dropped during acute infection, suggesting augmented intestinal homing of gut-imprinted T-cells. Finally, CD4+ macrophages transiently decreased in the submucosa and concentrated in the LP during the first days of infection. Overall, our study identifies IL-7 as a danger signal in the small intestine of Chinese rhesus macaques in response to acute SIV infection. Through stimulation of local chemokine expressions, this overexpression of IL-7 triggers immune cell recruitment to the gut. These findings suggest a role for IL-7 in the initiation of early mucosal immune responses to SIV and HIV infections. However, IL-7 triggered CD4+ T-cells and macrophages localization at viral replication sites could also participate to viral spread and establishment of viral reservoirs.

Vitamin D supplementation is associated with reduced immune activation levels in HIV-1-infected patients on suppressive antiretroviral therapy.

BACKGROUND: A majority of HIV-1-infected patients present a severe deficit in vitamin D, which predicts short-term mortality. Vitamin D is a naturally synthesized hormone, with important immunomodulatory functions. In the general population, its deficit has been associated with increased markers of inflammation. Vitamin D deficit may therefore play a role in the establishment of elevated systemic immune activation, which persists despite suppressive antiretroviral therapy (ART) in HIV-infected patients, and is predictive of disease progression; and vitamin D supplementation may be beneficial in this context. METHODS: We performed both a cross-sectional study (vitamin D deficit versus normal level) and a longitudinal study (upon vitamin D supplementation for 6 to 12 months) of HIV-1-infected patients receiving suppressive ART. The primary outcome measure was the percentage of activated memory CD8(+) T cells in blood, which is a robust marker associated with disease progression. Secondary outcomes included general T-lymphocyte and B-lymphocyte phenotype. RESULTS: Although vitamin D deficiency had no influence on T-cell and B-cell subset distribution, we found an association between vitamin D and immune activation levels in HIV-1-infected patients. Vitamin D supplementation in vitamin D-deficient patients resulted in reduced immune activation levels. CONCLUSION: The present data support the rationale of vitamin D supplementation in the routine clinical management of HIV-1-infected patients, in order to decrease immune activation levels and possibly improve long-term survival.

Modified interferon-α subtypes production and chemokine networks in the thymus during acute simian immunodeficiency virus infection, impact on thymopoiesis.

OBJECTIVES: Thymus dysfunction characterizes human/simian immunodeficiency virus (SIV) infections and contributes to physiopathology. However, both the mechanisms involved in thymic dysfunction and its precise timing remain unknown. We here analyzed thymic function during acute SIV infection in rhesus macaques. DESIGN AND METHODS: Rhesus macaques were intravenously infected with SIVmac251 and bled every 2/3 days or necropsied at different early time points postinfection. Naive T-cell counts were followed by flow cytometry and their T-cell receptor excision circle content evaluated by qPCR. Thymic chemokines were quantified by reverse transcription-qPCR and localized by in-situ hybridization in thymuses collected at necropsy. Thymic interferon alpha (IFN-α) subtype production was quantified by reverse transcription-qPCR combined to heteroduplex tracking assay. The effect of thymic IFN-α subtypes was tested on sorted triple negative thymocytes cultured on OP9-hDL1 cells. RESULTS: A reduced intrathymic proliferation history characterizes T cells produced during the first weeks of infection. Moreover, we evidenced a profound alteration of both chemokines and IFN-α subtypes transcriptional patterns in SIV-infected thymuses. Finally, we showed that IFN-α subtypes produced in the infected thymuses inhibit thymocyte proliferation, still preserving their differentiation capacity. CONCLUSION: Thymopoiesis is deeply impacted from the first days of SIV infection. Reduced thymocyte proliferation - a time-consuming process - together with modified chemokine networks is consistent with thymocyte differentiation speed-up. This may transiently enhance thymic output, thus increasing naive T-cell counts and diversity and the immune competence of the host. Nonetheless, long-lasting modification of thymic physiology may lead to thymic exhaustion, as observed in late primary HIV infection.

CD4⁺ recent thymic emigrants are infected by HIV in vivo, implication for pathogenesis.

OBJECTIVE: The contribution of naive CD4⁺ T cells to the pool of HIV-infected cells remains poorly described. This study aimed at evaluating HIV infection in naive T-cell subsets in viremic and HAART-treated patients, together with various parameters implicated in naive T-cell homeostasis, in order to better understand infection in these subsets. DESIGN AND METHODS: HIV provirus was quantified in various FACS-sorted CD4/CD8 T-cell subsets [recent thymic emigrants (RTEs), non-RTE naives and memory T cells] purified from peripheral blood cells of untreated viremic and HAART-treated aviremic HIV-infected patients. HIV proviral DNA was quantified using a highly sensitive real-time PCR assay allowing detection of one HIV copy in 105 cells. Intrathymic precursor T-cell proliferation and circulating T-cell cycling were, respectively, evaluated through measurement of the sj/ßTREC ratio (signal joint T-Cell Receptor Excision Circle frequency divided by DßJßTREC frequency) and Ki-67 expression. Plasma interleukin (IL)-7 concentrations were measured by ELISA. RESULTS: RTEs and non-RTEs were equally HIV infected. Altogether, naive CD4⁺ T cells represented 0.24%-60% of the infected cells. In contrast, HIV DNA was undetectable in naive CD8⁺ T cells. RTE infection rate directly correlated with IL-7 plasma levels (r = 0.607, P = 0.0035) but was independent from plasma viral load, peripheral T-cell cycling and intrathymic precursor T-cell proliferation. CONCLUSION: We demonstrated that RTEs are effectively HIV infected. The similar infection rate observed in RTEs and other naive T cells, its relationship with plasma IL-7 levels, together with the lack of correlation between RTE infection and either thymic or peripheral proliferation, strongly suggests that RTE infection occurs either late during thymopoiesis or early on during their extrathymic maturation.

Interleukin-7 treatment counteracts IFN-α therapy-induced lymphopenia and stimulates SIV-specific cytotoxic T lymphocyte responses in SIV-infected rhesus macaques.

Interferon-α (IFN-α)-based therapy is presently the standard treatment for hepatitis C virus (HCV)-infected patients. Despite good effectiveness, this cytokine is associated with major side effects, including significant lymphopenia, that limits its use for HIV/HCV-coinfected patients. Interleukin-7 (IL-7) has recently shown therapeutic potential and safety in several clinical trials designed to demonstrate T-cell restoration in immunodeficient patients. The purpose of this study was to evaluate, in simian immunodeficiency virus-infected rhesus macaques, the relevance of IL-7 therapy as a means to overcoming IFN-α-induced lymphopenia. We showed that low-dose IFN-α treatment induced strong lymphopenia in chronically infected monkeys. In contrast, high-dose IFN-α treatment stimulated IL-7 production, leading to increased circulating T-cell counts. Moreover, IL-7 therapy more than abrogated the lymphopenic effect of low-dose IFN-α. Indeed, the association of both cytokines resulted in increased circulating T-cell counts, in particular in the naive compartments, as a consequence of central and peripheral homeostatic functions of the IL-7. Finally, reduced PD-1 expression by memory CD8(+) T cells and transient T-cell repertoire diversification were observed under IL-7 therapy. Our data strongly suggest that IL-7 immunotherapy will be of substantial benefit in the treatment of HIV/HCV coinfection and should enhance the likelihood of HCV eradication in poorly responding patients.

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells.

The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function.

Injection of glycosylated recombinant simian IL-7 provokes rapid and massive T-cell homing in rhesus macaques.

Interleukin-7 (IL-7), the principal cytokine implicated in thymopoiesis and peripheral T-cell homeostasis, is presently under evaluation in human diseases characterized by persistent lymphopenia. Unexpectedly, before the eventual IL-7-driven T-cell expansion, all treated patients showed a profound T-cell depletion 24 hours after injection. The current study uses the rhesus macaque model to investigate the mechanisms involved in this IL-7-induced T-cell depletion. We identify a new critical function of IL-7 that induces massive and rapid T-cell migration from the blood into various organs, including lymph nodes, parts of the intestine, and the skin. This homing process was initiated after the induction of chemokine receptor expression by circulating T cells and the production of corresponding chemokines in target organs. Finally, we demonstrate that the IL-7-induced cell cycling is initiated within these organs before T cells migrate back into the bloodstream, indicating that T-cell homing is required for in vivo IL-7 function.

Refinement of strategies for the development of a human cytomegalovirus dense body vaccine.

Development of a vaccine against human cytomegalovirus (HCMV) infection has been identified as a high priority goal in biomedical research, yet no vaccine has been licensed until now. Recombinant subviral dense bodies (recDB) are a promising basis for the establishment of such a vaccine. In this article, strategies for the generation of recDB, based on recombination-mediated genetic engineering of the 230 kb HCMV DNA genome in E. coli are outlined. Analysis of viral mutants that were constructed in this process provided the proof-of-principle that heterologous antigens can be packaged into recDB and that these particles prime CD8 T cell responses against the recombinant antigen upon their application to HLA-A2 transgenic mice.

Exogenous introduction of an immunodominant peptide from the non-structural IE1 protein of human cytomegalovirus into the MHC class I presentation pathway by recombinant dense bodies.

Exogenous introduction of particle-associated proteins of human cytomegalovirus (HCMV) into the major histocompatibility complex (MHC) class I presentation pathway by subviral dense bodies (DB) is an effective way to sensitize cells against CD8 T-cell (CTL) recognition and killing. Consequently, these particles have been proposed as a platform for vaccine development. We have developed a strategy to refine the antigenic composition of DB. For proof of principle, an HCMV recombinant (RV-VM3) was generated that encoded the immunodominant CTL determinant IE1TMY from the IE1 protein in fusion with the major constituent of DB, the tegument protein pp65. To generate RV-VM3, a bacterial artificial chromosome containing the HCMV genome was modified by applying positive/negative selection based on the expression of the bacterial galactokinase in conjunction with lambda Red-mediated homologous recombination. This method allowed the efficient and seamless insertion of the DNA sequence encoding IE1TMY in frame into the pp65 open reading frame (UL83) of the viral genome. RV-VM3 expressed its fusion protein to high levels. The fusion protein was packaged into DB and into virions. Its delivery into fibroblasts by these viral particles led to the loading of the MHC class I presentation pathway with IE1TMY and to efficient killing by specific CTLs. This demonstrated that a heterologous peptide, not naturally present in HCMV particles, can be processed from a recombinant, DB-derived protein to be subsequently presented by MHC class I. The results presented here provide a rationale for the optimization of a vaccine based on recombinant DB.