Altered Thymic Function during Interferon Therapy in HCV-Infected Patients

Interferon alpha (IFNα) therapy, despite good efficacy in curing HCV infection, leads to major side effects, in particular inducement of a strong peripheral T-cell lymphocytopenia. We here analyze the early consequences of IFNα therapy on both thymic function and peripheral T-cell homeostasis in patients in the acute or chronic phase of HCV-infection as well as in HIV/HCV co-infected patients. The evolution of T-cell subsets and T-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of T-cell receptor excision circles (TREC) and estimation of intrathymic precursor T-cell proliferation during the first four months following the initiation of IFNα therapy. Beginning with the first month of therapy, a profound lymphocytopenia was observed for all T-cell subsets, including naïve T-cells and recent thymic emigrants (RTE), associated with inhibition of intrathymic precursor T-cell proliferation. Interleukin (IL)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. This was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble CD127. Decrease in IL-7 plasma concentration under IFNα therapy correlated with the decline in HCV viral load, thymic activity and RTE concentration in blood. These data demonstrate that IFNα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs T-cell homeostasis. Such a side effect might be detrimental for the continuation of IFNα therapy and may lead to an increased level of infectious risk, in particular in HIV/HCV co-infected patients. Altogether, this study suggests the therapeutic potential of IL-7 in the maintenance of peripheral T-cell homeostasis in IFNα-treated patients

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

International audienceObjectives: 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

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

International audienceInterferon-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

IFNα therapy leads to reduction in IL-7 plasma concentration.

<p>(A) IL-7 plasma levels were quantified in peripheral blood cells from acutely HCV-infected (light grey symbols), chronically HCV-infected (black symbols) and HIV/HCV co-infected (white symbols) patients at study entry, as compared to healthy donors (HCV-, dark grey symbols). **: p<0.001 for any HCV-infected patients group. (B) Evolution of plasma IL-7 levels over the first 4 months of IFNα therapy in acutely HCV-infected (left panels), chronically HCV-infected (central panels) and HIV/HCV co-infected (right panels) patients. Each line represents an individual patient. Statistical significances of the differences to baseline values (time 0), calculated on the absolute IL-7 plasma levels in each individual sample (Wilcoxon matched-pairs signed-ranks test) are shown on top. (C) Soluble CD127 was quantified in plasma from acutely HCV-infected (white symbols, top panel), chronically HCV-infected (black symbols, top panel) and HIV/HCV co-infected (bottom panel) patients at baseline (0) and M2. (D) CD127 expression was measured on circulating CD4⁺ (top panel) and CD8⁺ (bottom panel) and expressed as mean fluorescence intensity (left panels) and percentages of positive cells (right panels) over the 4 first months of IFNα therapy.</p

IFNα therapy leads to naïve T-cell lymphocytopenia.

<p>(A) CD4⁺ (top panel) and CD8⁺ (bottom panel) naïve T-cell counts were quantified in peripheral blood cells from acutely HCV-infected (light grey symbols), chronically HCV-infected (black symbols) and HIV/HCV co-infected (white symbols) patients at study entry, as compared to healthy donors (HCV-, dark grey symbols). (B) Evolution of CD4⁺ (top panels) and CD8⁺ (bottom panels) naïve T-cell counts during the first 4 months of IFNα therapy in acutely HCV-infected (left panels), chronically HCV-infected (central panels) and HIV/HCV co-infected (right panels) patients. Each line represents data from an individual patient. Statistical significances of the differences to baseline values (time 0), calculated on the absolute naïve T-cell counts in each individual sample, (Wilcoxon matched-pairs signed-ranks test) are shown on top. The horizontal bars represent median values.</p

Variations in IL-7 plasma levels correlate with evolution of RTE

<p><b>production.</b> Correlations. between variations in IL-7 plasma levels (ΔIL-7) and either variations in (A) total (CD4⁺ + CD8⁺) naïve T-cell counts (Δnaïve T-cell counts), (B) RTE defined as CD31^hi naïve CD4⁺ T-cells (ΔRTE CD4 counts), (C) the sj/βTREC ratio (Δsj/βTREC ratio), (D) the frequency of Ki-67⁺ cells in the RTE CD4⁺ T-cell subset (Δ%Ki-67⁺ in CD4⁺ RTEs) or (E) the number of circulating Ki-67⁺CD4⁺ RTEs (ΔKi-67⁺ RTE counts) between study entry and month 1 of therapy were calculated for acutely (black symbols) and chronically (white symbols) HCV-infected patients (left panels) and HIV/HCV co-infected patients (right panels). Correlation coefficients (Spearman's r) and the associated probabilities (p) are shown.</p

IFNα therapy leads to major impairment of thymic function.

<p>(A) The frequency of Ki-67 expressing cells in the CD4⁺ RTE subset (CD31^hi naïve T-cells) was measured in acutely HCV-infected (grey symbols, top panel), chronically HCV-infected (white symbols, top panel) and HIV/HCV co-infected (bottom panel) patients (central panels) and HIV/HCV co-infected (right panels) patients. Each line represents data from an individual patient. Statistical significances of the differences to baseline values (time 0), calculated on the absolute sj/βTREC ratio in each individual sample (Wilcoxon matched-pairs signed-ranks test) are shown on top. The horizontal bars represent median values.</p

Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing

International audienceHost versus graft reaction is a major impediment to CAR-T cell immune therapy in allogeneic settings. Authors show here that CAR-T cells, engineered to be deficient in MHC I expression but to express the NK inhibitor HLA-E, are resistant to destruction by both T and NK cells of the host. Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCR alpha beta- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications

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

© 2011 Wolters Kluwer Health | Lippincott Williams & WilkinsOBJECTIVE: 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/βTREC ratio (signal joint T-Cell Receptor Excision Circle frequency divided by DβJβTREC 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