Single-nucleotide polymorphism-defined class I and class III major histocompatibility complex genetic subregions contribute to natural long-term nonprogression in HIV infection.

We performed a genome-wide association study comparing a cohort of 144 human immunodeficiency virus (HIV type 1-infected, untreated white long-term nonprogressors (LTNPs) with a cohort of 605 HIV-1-infected white seroconverters. Forty-seven single-nucleotide polymorphisms (SNPs), located from class I to class III major histocompatibility complex (MHC) subregions, show statistical association (false discovery rate, <0.05) with the LTNP condition, among which 5 reached genome-wide significance after Bonferonni correction. The MHC LTNP-associated SNPs are ordered in ≥4 linkage disequilibrium blocks; interestingly, an MHC class III linkage disequilibrium block (defined by the rs9368699 SNP) seems specific to the LTNP phenotype.

Analysis of CD16+CD56dim NK cells from CLL patients: evidence supporting a therapeutic strategy with optimized anti-CD20 monoclonal antibodies.

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FcγRIIIA) is well preserved in CD16(+)CD56(dim) cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FcγRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FcγR engagement in CLL patients.

Fully functional NK cells after unrelated cord blood transplantation.

Promising results of umbilical cord blood transplantation (UCBT) from unrelated donors have been reported in patients with hematologic disorders. These transplants, having potential to trigger beneficial donor-versus-recipient natural killer (NK) cell-mediated alloreaction, we have conducted the first extensive analysis of the phenotypic and functional properties of NK cells after UCBT. NK cells from 25 patients with high-risk hematologic malignancies were compared with cells derived from both healthy adult and CB cells. We found that following UCBT, NK cells display not only some phenotypic features associated with maturity but also unique characteristics that make them fully functional against leukemic blasts. We propose that this full functionality of alloreactive donor-derived NK may drive graft-versus-leukemia reactions after UCBT.

HLA-E upregulation on IFN-gamma-activated AML blasts impairs CD94/NKG2A-dependent NK cytolysis after haplo-mismatched hematopoietic SCT.

Natural killer (NK) cells generated after haploidentical hematopoietic SCT in patients with AML are characterized by specific phenotypic features and impaired functioning that may affect transplantation outcome. We show that IFN-gamma produced by immature CD56(bright) NK cells upregulates cell surface expression of HLA-E on AML blasts and that this upregulation protects leukemic cells from NK-mediated cell lysis through the mediation of CD94/NKG2A, an inhibitory receptor overexpressed on NK cells after haploidentical SCT. Two years after transplantation, however, maturing NK cells were functionally active, as evidenced by high cytotoxicity and poor IFN-gamma production. This implies that maturation of NK cells is the key to improved immune responses and transplantation outcome.

Transient depletion of dividing T lymphocytes in mice induces the emergence of regulatory T cells and dominant tolerance to islet allografts.

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long-term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor-alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2- to 3-fold increase in the proportion of CD4(+)CD25(+)Foxp3(+) Treg, within 3 weeks that persisted only in allograft-bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno-intervention based on disturbance of T-cell homeostasis and subsequent increase in Treg proportion.

Involvement of mature donor T cells in the NK cell reconstitution after haploidentical hematopoietic stem-cell transplantation.

We previously demonstrated that natural killer (NK) cells generated after haploidentical stem-cell transplantation (SCT) are blocked at an immature state characterized by phenotypic features and impaired functioning and that this may affect transplantation outcome. We hypothesize that the absence of mature donor T cells in the graft may affect NK cell differentiation. NK cells from 21 transplant recipients who underwent either partial (pTCD; n=11) or extensive (eTCD; n=10) T-cell depletion were compared with NK cells from their healthy donors. We report that despite the strong graft-versus-host disease (GvHD) reaction, pTCD patients, with T cells present during SCT, had a better clinical outcome than patients with eTCD transplants. In addition, the frequency of CD3- CD56(bright) and NKG2A+ NK cells was much lower in pTCD than in eTCD patients after transplantation, and the level of cytotoxicity against primary haplo-mismatched blasts was significantly more pronounced after pTCD than eTCD transplants. These finding strongly suggest that mature donor T cells in the graft may play a key role in NK cell differentiation in vivo, after haploidentical SCT.

Polymorphisms in CCR5 chemokine receptor gene in Japan.

Mutations in the human CC chemokine receptor 5 (CCR5) gene may alter the expression or function of the protein product, thereby altering chemokine binding/signalling or human immunodeficiency virus type 1 (HIV-1) infection of the cells that normally express CCR5 protein. We performed a systematic survey of natural sequence variations in an 8.1-kb region of the entire CCR5 gene as well as CCR2V64I in 50 Japanese subjects and evaluated the effects of those variations on CCR5 promoter activity. We also analysed CCR5 promoters and CCR2V64I in 80 more Japanese and 186 Thais. There was no 32-bp deletion observed in Caucasians, but two types of non-synonymous substitutions were found in CCR5 genes of Japanese. Our results showed several novel characteristics of the CCR2-CCR5 haplotype structure that were not reported from studies on Caucasians and African-Americans. Specifically, we were able to show that the G allele at position -2852 from the CCR5 open reading frame in Japanese and Thais is the representative of the CCR5 promoter haplotype that was reported to be associated with rapid progression to acquired immune deficiency syndrome (AIDS) in HIV-1-infected individuals. Furthermore, nearly all non-synonymous polymorphisms in Japanese CCR5 occurred in haplotypes with elevated promoter activity. We thus hypothesized that there was a certain selective pressure favouring low levels of CCR5 expression during human evolution.

Effects of the molecular weight of peg molecules (8, 20 and 35 KDA) on cell function and allograft survival prolongation in pancreatic islets transplantation.

The normopotassic solution SCOT (Macopharma, France) used for the isolation of the islets of Langerhans may improve both graft function and survival. We believe that this is due to the immunoprotective properties of polyethyleneglycol (PEG) (20 kDa; 1.5 mM/L), which is contained in this solution. However, the optimal PEG chain length remains to be determined. Three extracellular type solutions (SCOT without PEG) containing various PEG-8 kDa, 20 kDa, or 35 kDa- at 1.5 mM/L were compared in vitro for viscosity and osmolarity as well as in vivo using a murine model of pancreatic islet allotransplantation. We compared the effects of the various solutions on functional cell recovery (primary nonfunction rate, PNF) and immunoprotection (allograft survival time). We showed that the viscosity of PEG 35 kDa solutions was too high for physiological use. PEG 20 kDa solution provided the best graft function (0% PNF, P < .05). PEG 8 kda and 20 kDa solutions significantly increased allograft survival time compared to the PEG 35 kDa solution (P < .05). Graft survival was similar with PEG 20 kDa and PEG 8 kDa solutions: 27.50 +/- 3.70 days versus 23.13 +/- 4.39 days (NS). However, the number of PNF with PEG 8 kDa solution (50%) was significantly higher (P < .01) than that with the PEG 20 kDa solution (0%). These preliminary results indicated that the optimal chain length at 1.5 mM/L of PEG is 20 kDa.

Polymorphism in the proximal promoter region of the perforin gene and its impact on the course of HIV infection.

Cytotoxic T lymphocytes (CTLs) play an essential role in the control of viral replication during human immunodeficiency virus (HIV) infection. However, the efficacy of the CTL response varies between individuals. We tested the hypothesis that genetic polymorphisms in the lytic effector molecule perforin could influence the progression of HIV infection. The perforin gene was screened for single nucleotide polymorphisms (SNPs) by denaturing high-performance liquid chromatography (dHPLC). Correlations were sought between perforin genotype, perforin expression and lytic function of CD8+ T lymphocytes from HIV-positive patients. Association of perforin genotype with disease progression was investigated in 426 seroconverters enrolled in the French SEROCO cohort. AIDS-free survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test. Three SNPs were found in the proximal promoter region of the perforin gene: 63G (allelic frequency 0.029), 112G (allelic frequency 0.071) and 1012T (allelic frequency 0.070). The presence of the 1012T genotype correlated with fewer perforin+ cells among circulating CD8+ CTL. However, CTL lines from HIV(-positive) individuals heterozygous for the perforin 1012T SNP displayed normal lysis of target cells, and within the SEROCO cohort, patients heterozygous for the 1012T SNP showed normal disease progression. However, 1012T/T homozygotes showed a tendency towards slower disease progression (P = 0.08). In conclusion, polymorphism in the perforin gene is limited, and although the 1012T genotype appears to influence perforin expression, it was not conclusively associated with disease progression in HIV infection.

32-nucleotide deletion, associated with defence against hiv/aids, is a predominant mutation of CCR5 gene in the population of Georgia.

There is a special interest to investigate genetic peculiarities in the populations with a low HIV seroprevalence. Despite of presence of high-risk conditions for rapid spread of HIV/AIDS epidemics in Georgia, the prevalence of this infection in the country remains very low. We studied polymorphisms of CCR5 gene in Georgians. Blood samples from 190 women randomly selected from the cohort of pregnant women involved in the program of prevention of mother-to-child HIV transmission in Georgia have been investigated. Two-step PCR was used to amplify the whole CCR5 genetic sequence. Detection of mutations and polymorphisms was done by dHPLC. All samples showing specific patterns by dHPLC, were sequenced to identify the exact nature of the mutation. It was shown that CCR5-delta32 mutation is a predominant alteration of CCR5 gene among Georgians. All subjects bearing this mutation were heterozygotes. Frequency of delta32 CCR5 allele in the population of Georgia was equal to 5%. Only one case of R223Q mutation and two cases of mutations in the non-coding region of CCR5 gene were also found. Our findings differ from the existing data showing the absence of the CCR5-delta32 mutation among Georgians and provide further support to the hypothesis on a Northeastern European origin of this mutation and North to South gradient of its distribution.

The effect of methylprednisolone treatment on the cardiopulmonary bypass-induced systemic inflammatory response.

OBJECTIVE: Cardiac surgery with cardiopulmonary bypass (CPB) is associated with an inflammatory response caused by contact of blood with artificial surfaces of the extracorporeal circuit, ischemia-reperfusion injury, and release of endotoxin. The inflammatory reaction involves activation of complement leucocytes, and endothelial cells with secretion of cytokines, proteases, arachidonic acid metabolites, and generation of oxygen derived free radicals (OFR) by polymorphonuclear neutrophils (PMN). Although this inflammatory response to CPB often remains at subclinical levels, it can also lead to major organ dysfunction. A number of studies have demonstrated that treatment of patients with a high-dose (30 mg/kg) of corticosteroids (methylprednisolone) attenuates the CPB-induced SIR and improves the outcome of patients undergoing cardiac surgery. However, large doses of steroids can cause abnormal metabolic responses such as metabolic acidosis and hyperglycemia. In the present study, we examined the efficacy of low doses of methylprednisolone (5 and 10 mg/kg) to attenuate the CPB-induced inflammatory response, during and after heart operations. METHODS: Thirty-six adult patients undergoing cardiac surgery, were randomized into three groups: (1) control group: group A; (2) methylprednisolone, 5 mg/kg body weight: group B; and (3) methylprednisolone, 10 mg/kg body weight: group C. Plasma levels of the cytokines interleukin-6 (IL-6) and TNF-alpha were analyzed by enzyme-linked immunosorbent assay, before, during, and after CPB. OFR production was determined by cytofluorometry (FACS) at the same end points. RESULTS: No significant differences in age, body weight, CPB time, and cross-clamp time were observed among the three groups. CPB induced a marked increased in cytokine release and OFR generation. Low-dose of methylprednisolone (5 mg/kg) effectively reduced the increase in TNF-alpha and IL-6 secretion (P<0.05 compared to control group) after release of the cross-clamp. However, OFR generation was significantly reduced with a greater dose of methylprednisolone (10 mg/kg). CONCLUSIONS: The results indicate that a single low-dose of methylprednisolone (10 mg/kg) reduces the inflammatory reaction during and after CPB, by inhibition of proinflammatory cytokine release and OFR generation after release of the aortic cross-clamp.

Common variable immunodeficiency patient classification based on impaired B cell memory differentiation correlates with clinical aspects.

Common variable immunodeficiency (CVID) is a very heterogeneous syndrome defined by impaired immunoglobulin production. The functional classification of CVID patients on the basis of in vitro immunoglobulin production is time consuming and has never shown any predictive value. We propose a classification based on the quantitative repartition of naive/memory B cells according to the dual expression of IgD and CD27. Fifty-seven patients were categorized into three groups: Group MB2 (11 patients, 19%) with normal memory B cells; Group MB1 (19 patients, 33%) with defective switched memory (IgD-CD27+) but normal nonswitched memory B cells (IgD+CD27+); Group MB0 (27 patients, 47%) with almost no memory B cells. In addition, a downexpression of activation markers (CD25, CD21, CD80, CD86) on B cells characterized the group MB1 patients and was associated with an upexpression of activation markers (HLA-DR, CD95, CD57) on T cells. This classification correlates with some clinical aspects showing a higher prevalence of splenomegaly (16/27, 59%), lymphoid proliferation (13/27, 48%) and granulomatous disease (12/27, 44%) in group MB0. Splenomegaly was also frequent in group MB1 (8/19, 42%). In contrast, autoimmunity was observed with similar prevalence in all three groups. Moreover, by analyzing B cell phenotype, immunoglobulin transcript expression, and somatic mutations, we propose different putative mechanisms responsible for impaired B cell activation and memory differentiation in this syndrome.

[Chemokines and immunomodulation: applications for HIV infections]. / Chimiokines et immunomodulation: applications à l'infection VIH.

The genetic control of HIV infection by the host involves a certain number of genes, among which those which code for chemokines/chemokines receptors, cytokines, MHC. Genes such as CCR5, CCR2, SDF1, and more recently CX3CR1 received great attention from several laboratories including ours, since they play a role as HIV coreceptor and, as such, on the infectivity of the host. In addition, it was shown that the polymorphism of these genes influences the evolution of infection, whether they have a protective or deleterious effect. Results obtained by our laboratory on the genetic polymorphism and its implication in HIV infection will be reported herein. Furthermore, to better understand their role, we looked for the capacities that the chemokines may have to play an immunomodulatory function, independently of their chemoattractive effect. In two examples, we showed that chemokines influence notably the cellular immune functions, such as CD8 cytotoxicity (Rantes/CCR3) and gamma interferon production (fractalkine/CX3CR1). Globally, the results indicate that chemokines/chemokines receptors polymorphism represent important epidemiological factors, but also contributes to evaluate the prognosis of HIV infection, through a better understanding of the disease physiopathology.

Aiolos and Ikaros: regulators of lymphocyte development, homeostasis and lymphoproliferation.

Aiolos and Ikaros encode hemopoietic-specific zinc finger transcription factors that are important regulators of lymphocyte differentiation. Aiolos and Ikaros play a critical role in regulating B and T cell development. Gene targeting studies in mice have shown that inactivation of Ikaros family proteins leads to a complete absence of T, B, NK and dendritic cells, whereas a reduction of Ikaros activity induce hyperproliferation and lymphomas. Aiolos knock-out mice have quantitatively normal lymphoid cells but have chronically activated B cells producing autoantibodies and develop lymphomas with increased frequency. These proteins are involved in the control of gene expression and, associated to nuclear complexes, participate in nucleosome remodeling. This protein family governs cell fate decisions and regulates homeostasis through complex isoforms expression and dimerization. Changes in this regulatory network may reflect differentiation and proliferation adjustments made in lymphoid progenitors and precursors. The direct involvement of aberrant Ikaros protein expression in human hematological oncogenesis, although suggested by several studies, remains to be settled at the genomic level. These points will be discussed in the present review.

Reconstitution of CD4+ T lymphocytes in HIV-infected individuals following antiretroviral therapy.

Immune reconstitution during antiretroviral therapy has recently been shown to depend upon multiple factors at work in T cell homeostasis, amongst which the reduction of thymus dysfunction and of immune hyperactivation are instrumental. The optimism that has been raised by the restoration of hosts' defenses against opportunistic pathogens is, however, balanced by the poor immunity restored against HIV; thus, innovative immune interventions are required.

Early immune responses accompanying human asymptomatic Ebola infections.

In a recent study we identified certain asymptomatic individuals infected by Ebola virus (EBOV) who mounted specific IgG and early and strong inflammatory responses. Here, we further characterized the primary immune response to EBOV during the course of asymptomatic infection in humans. Inflammatory responses occurred in temporal association with anti-inflammatory phase composed by soluble antagonist IL-1RA, circulating TNF receptors, IL-10 and cortisol. At the end of the inflammatory process, mRNA expression of T-cell cytokines (IL-2 and IL-4) and activation markers (CD28, CD40L and CTLA4) was up-regulated, strongly suggesting T-cell activation. This T-cell activation was followed by EBOV-specific IgG responses (mainly IgG3 ang IgG1), and by marked and sustained up-regulation of IFN gamma, FasL and perforin mRNA expression, suggesting activation of cytotoxic cells. The terminal down-regulation of these latter markers coincided with the release of the apoptotic marker 41/7 NMP in blood and with the disappearance of viral RNA from PBMC, suggesting that infected cells are eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-V beta repertoire usage showed that TCR-V beta 12 mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus.

Investigation of human spleen dendritic cell phenotype and distribution reveals evidence of in vivo activation in a subset of organ donors.

Although the mouse spleen dendritic cell (DC) is perhaps the most intensively studied DC type, little has been published concerning its human equivalent. In this report, rare event flow cytometry and in situ immunofluorescence were used to study the surface phenotype and distribution of HLA-DR(+) CD3(-)14(-)16(-)19(-) human spleen DC. Spleens from organ donors with different clinical histories were used. Most (81% +/- 9%; n = 14) spleen DCs expressed high levels of the integrin CD11c. CD11c(+) DCs were distributed in 3 distinct regions-the peri-arteriolar T-cell zones, the B-cell zones, and the marginal zone, where they formed a ring of cells surrounding the white pulp, just inside a ring of CD14(+) red pulp macrophages, apparently more regularly organized than the previously described marginating DC population in the mouse spleen. The T-cell zones contained CD86(+) DCs, among which a subpopulation expressed CD83. These mature/activated CD86(+) DCs represented a minority (12% +/- 8%) of total spleen DCs in most organ donors: most spleen DCs are immature. In 3 of 18 (17%) donors, however, most (54%-81%) of spleen DCs were CD86(+), suggesting that in vivo DC activation had occurred. In one donor, a radical shift in DC distribution from the marginal zone to the T-cell zones was also observed. This activation of spleen DCs in vivo was reminiscent of the effects of experimental microbial product injection in mice, and it seemed to correlate with bacterial infection or multiple trauma. (Blood. 2001;97:3470-3477)