Hydroxychloroquine sulfate: A novel treatment for lipin-1 deficiency?

BACKGROUND: Lipin-1 deficiency is a life-threatening disease that causes severe rhabdomyolysis (RM) and chronic symptoms associated with oxidative stress. In the absence of treatment, Hydroxychloroquine sulfate (HCQ) was administered to patients off label use on a compassionate basis in order to improve their physical conditions. METHODS: Eleven patients with LPIN1 mutations were treated with HCQ. Clinical and biological efficacy and tolerance were assessed, including pain and quality of life, physical capacities, cardiopulmonary parameters, creatine kinase levels and plasma proinflammatory cytokines. To explore a dose-dependent effect of HCQ, primary myoblasts from 4 patients were incubated with various HCQ concentrations in growth medium (GM) or during starvation (EBSS medium) to investigate autophagy and oxidative stress. FINDINGS: Under HCQ treatment, patient physical capacities improved. Abnormal cardiac function and peripheral muscle adaptation to exercise were normalized. However, two patients who had the highest mean blood HCQ concentrations experienced RM. We hypothesized that HCQ exerts deleterious effects at high concentrations by blocking autophagy, and beneficial effects on oxidative stress at low concentrations. We confirmed in primary myoblasts from 4 patients that high in vitro HCQ concentration (10 µM) but not low concentration (1 µM and 0.1 µM) induced autophagy blockage by modifying endolysosomal pH. Low HCQ concentration (1 µM) prevented reactive oxygen species (ROS) and oxidized DNA accumulation in myoblasts during starvation. INTERPRETATION: HCQ improves the condition of patients with lipin-1 deficiency, but at low concentrations. In vitro, 1 µM HCQ decreases oxidative stress in myoblasts whereas higher concentrations have a deleterious effect by blocking autophagy.

Systemic corticosteroids for the treatment of acute episodes of rhabdomyolysis in lipin-1-deficient patients.

Mutations in the LPIN1 gene constitute a major cause of severe rhabdomyolysis (RM). The TLR9 activation prompted us to treat patients with corticosteroids in acute conditions. In patients with LPIN1 mutations, RM and at-risk situations that can trigger RM have been treated in a uniform manner. Since 2015, these patients have also received intravenous corticosteroids. We retrospectively compared data on hospital stays by corticosteroid-treated patients vs. patients not treated with corticosteroids. Nineteen patients were hospitalized. The median number of admissions per patient was 21 overall and did not differ when comparing the 10 corticosteroid-treated patients with the 9 patients not treated with corticosteroids. Four patients in the non-corticosteroid group died during a RM (mean age at death: 5.6 years). There were no deaths in the corticosteroid group. The two groups did not differ significantly in the number of RM episodes. However, for the six patients who had RM and occasionally been treated with corticosteroids, the median number of RM episodes was significantly lower when intravenous steroids had been administered. The peak plasma creatine kinase level and the area under the curve were or tended to be higher in patients treated with corticosteroids-even after the exclusion of deceased patients or focusing on the period after 2015. The median length of stay (10 days overall) was significantly longer for corticosteroid-treated patients but was similar after the exclusion of deceased patients. The absence of deaths and the higher severity of RM observed among corticosteroid-treated patients could suggest that corticotherapy is associated with greater survival.

Bi-allelic loss-of-function OBSCN variants predispose individuals to severe recurrent rhabdomyolysis.

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

Clinical and biological characterization of 20 patients with TANGO2 deficiency indicates novel triggers of metabolic crises and no primary energetic defect.

TANGO2 disease is a severe inherited disorder associating multiple symptoms such as metabolic crises, encephalopathy, cardiac arrhythmias, and hypothyroidism. The mechanism of action of TANGO2 is currently unknown. Here, we describe a cohort of 20 French patients bearing mutations in the TANGO2 gene. We found that the main clinical presentation was the association of neurodevelopmental delay (n = 17), acute metabolic crises (n = 17) and hypothyroidism (n = 12), with a large intrafamilial clinical variability. Metabolic crises included rhabdomyolysis (15/17), neurological symptoms (14/17), and cardiac features (12/17; long QT (n = 10), Brugada pattern (n = 2), cardiac arrhythmia (n = 6)) that required intensive care. We show previously uncharacterized triggers of metabolic crises in TANGO2 patients, such as some anesthetics and possibly l-carnitine. Unexpectedly, plasma acylcarnitines, plasma FGF-21, muscle histology, and mitochondrial spectrometry were mostly normal. Moreover, in patients' primary myoblasts, palmitate and glutamine oxidation rates, and the mitochondrial network were also normal. Finally, we found variable mitochondrial respiration and defective clearance of oxidized DNA upon cycles of starvation and refeeding. We conclude that TANGO2 disease is a life-threatening disease that needs specific cardiac management and anesthesia protocol. Mechanistically, TANGO2 disease is unlikely to originate from a primary mitochondrial defect. Rather, we suggest that mitochondrial defects are secondary to strong extrinsic triggers in TANGO2 deficient patients.

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or ß hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Gene Therapy in Patients with Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ßA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-thalassemia. METHODS: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent ß-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. RESULTS: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-ß0/ß0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a ß0/ß0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe ß-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Gene Therapy in a Patient with Sickle Cell Disease.

Sickle cell disease results from a homozygous missense mutation in the ß-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling ß-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling ß-globin remained high (approximately 50% of ß-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions.

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes.

Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome.

IMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety.

Faster T-cell development following gene therapy compared with haploidentical HSCT in the treatment of SCID-X1.

During the last decade, gene therapy via ex vivo gene transfer into autologous hematopoietic stem cells has emerged as a convincing therapy for severe combined immunodeficiency caused by ILR2G mutation (SCID-X1) despite the occurrence of genotoxicity caused by the integration of first-generation retroviral vectors. However, the place of gene therapy among the therapeutic armamentarium remains to be defined. We retrospectively analyze and compare clinical outcomes and immune reconstitution in 13 consecutive SCID-X1 patients having undergone haploidentical hematopoietic stem cell transplantation (HSCT) and 14 SCID-X1 patients treated with gene therapy over the same period at a single center level: the Necker Children's Hospital (Paris, France). Our results show a clear advantage in terms of T-cell development of gene therapy over HSCT with a mismatched donor. Patients treated with gene therapy display a faster T-cell reconstitution and a better long-term thymic output. Interestingly, this advantage of gene therapy vs haploidentical HSCT seems to be independent of the existence of clinical graft-versus-host disease in the latter condition. If data of safety are confirmed over the long term, gene therapy for SCID-X1 appears to be an equal, if not superior, alternative to haploidentical HSCT.

A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).

Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Relationship between regulatory T cells and immune activation in human immunodeficiency virus-infected patients interrupting antiretroviral therapy.

UNLABELLED: Persistent immune activation plays a central role in driving Human Immunodeficiency Virus (HIV) disease progression. Whether CD4+CD25+ regulatory T cells (Tregs) are harmful by suppressing HIV-specific immune responses and/or beneficial through a decrease in immune activation remains debatable. We analysed the relationship between proportion and number of regulatory T cells (Tregs) and immune activation in HIV-infected patients interrupting an effective antiretroviral therapy (ART). Twenty-five patients were included in a substudy of a prospective multicenter trial of treatment interruption (TI) (ANRS 116). Proportions and numbers of Tregs and the proportion of activated CD4 and CD8 T cells were assessed at baseline and month 12 (M12) of TI. Specific anti-HIV CD4 and CD8 responses were investigated at baseline and M12. Non parametric univariate analyses and multivariate linear regression models were conducted. At baseline, the proportion of Tregs negatively correlated with the proportion of HLA-DR+CD8+T cells (r=-0.519). Following TI, the proportion of Tregs increased from 6.3% to 7.2% (p=0.029); absolute numbers of Tregs decreased. The increase in the proportion of HLA-DR+CD38+CD8+T cells was significantly related to the increase in proportion of Tregs (p=0.031). At M12, the proportion of Tregs did not negatively correlate with CD8 T-cell activation. Nevertheless, Tregs retain a suppressive function since depletion of Treg-containing CD4+CD25+ cells led to an increase in lymphoproliferative responses in most patients studied. Our data suggest that Tregs are efficient in controlling residual immune activation in patients with ART-mediated viral suppression. However, the insufficient increase in the proportion and/or the decrease in the absolute number of Tregs result in a failure to control immune activation following TI. TRIAL REGISTRATION: ClinicalTrials.gov NCT00118677.

Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We initiated a gene therapy trial in two ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then re-infused into the patients after they had received myeloablative treatment. Over a span of 24 to 30 months of follow-up, we detected polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggest that hematopoietic stem cells were transduced in the patients. Beginning 14 to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT. Thus, lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.

Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.

Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor gamma (IL2RG) gene to CD34+ BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31-68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain-only 2 (LMO2) proto-oncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene,CCND2. Additional genetic abnormalities in the patients' blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.

Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients.

The present study demonstrates that CD4(+)CD25(+) T cells, expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART), exhibit phenotypic, molecular, and functional characteristics of regulatory T cells. The majority of peripheral CD4(+)CD25(+) T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4(+)CD25(+) T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4(+)CD25(-) T cells, CD4(+)CD25(+) T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin, cytomegalovirus (CMV), and p24 significantly increased following depletion of CD4(+)CD25(+) T cells. Furthermore, addition of increasing numbers of CD4(+)CD25(+) T cells resulted in a dose-dependent inhibition of CD4(+)CD25(-) T-cell proliferation to tuberculin and p24. CD4(+)CD25(+) T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), thus indicating the presence of p24-specific CD4(+) T cells among the CD4(+)CD25(+) T-cell subset. Suppressive activity was not dependent on the secretion of TGF-beta or IL-10. Taken together, our results suggest that persistence of HIV antigens might trigger the expansion of CD4(+)CD25(+) regulatory T cells, which might induce a tolerance to HIV in vivo.

Regulatory CD8+ T cells control neonatal tolerance to a Th2-mediated autoimmunity.

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8(+) T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8(+) T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8(+) T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8(+)CD45RC(high) T cells and the emergence of a high percentage of IFN-gamma-synthesizing CD8(+) T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8(+) cell-mediated regulatory response.