Natural CD8 Research Articles

Genetic ataxia telangiectasia porcine model phenocopies the multisystemic features of the human disease

Ataxia telangiectasia (AT) is a progressive multisystem autosomal recessive disorder caused by mutations in the AT-mutated (ATM) gene. Early onset AT in children is characterized by cerebellar degeneration, leading to motor impairment. Lung disease and cancer are the two most common causes of death in AT patients. Accelerated thymic involution may contribute to the cancer, and recurrent and/or chronic respiratory infections may be a contributing factor to lung disease in AT. AT patients have fertility issues, are highly sensitive to ionizing radiation and they present oculocutaneous telangiectasia. Current treatments only slightly ameliorate disease symptoms; therapy that alters or reverses the course of the disease has not yet been discovered. Previously, we have shown that ATM−/− pigs, a novel model of AT, present with a loss of Purkinje cells, altered cerebellar cytoarchitecture and motor coordination deficits. ATM−/− porcine model not only recapitulates the neurological phenotype, but also other multifaceted clinical features of the human disease. Our current study shows that ATM−/− female pigs are infertile, with anatomical and functional signs of an immature reproductive system. Both male and female ATM−/− pigs show abnormal thymus structure with decreased cell cycle and apoptosis markers in the gland. Moreover, ATM−/− pigs have an altered immune system with decreased CD8+ and increased natural killer and CD4+CD8+ double-positive cells. Nevertheless, ATM−/− pigs manifest a deficient IgG response after a viral infection. Based on the neurological and peripheral phenotypes, the ATM−/− pig is a novel genetic model that may be used for therapeutic assessments and to identify pathomechanisms of this disease.

Natural ThPOK− CD4 cytotoxic T lymphocytes : Thymic development of a new distinct self-specific CD4 T cell population.

Abstract The lineage commitment to either CD4 or CD8 T cells is made in the thymus, where thymocytes with specificity for MHC-II molecules differentiates into the CD4 “helper” linage whereas ones specific for MHC-I molecules commit to the CD8 “cytotoxic” lineage. The molecular regulation is controlled by the action of key transcription factors. Notably ThPOK, which promotes the CD4 fate and suppress the cytotoxic fate of CD4 T cells. Recently, we made a major breakthrough and showed that these lineages is not fixed and the mature peripheral CD4 T cells have the plasticity to lose ThPOK expression and reprogram to ThPOKneg CD4 CTL (cytotoxic T lymphocytes) marked by the expression of CD8a, Granzyme B and potent killer capacity. Investigating the origin of CD4 CTL, we observed in ThPOK fate maping mouse a strong proportion of CD4 CTL that never expressed ThPOK, suggesting that some CD4 CTL are natural cytotoxic cells that develop in the thymus. Based on a recent works allowing the analysis of mature CD4 cells in the thymus, we observed a substantial number of mature CD4 T cells that do not express ThPOK and display an active-like phenotype. Moreover, once injected, thymic ThPOKneg mature CD4 T cells remain ThPOKneg in the periphery and start to acquire CD8a expression demonstrating that natural CD4 CTL arise from the thymus. We also demonstrated that this population requires a strong TCR signal during their development, suggesting that natural CD4 CTL have a repertoire specific for self-antigens. We have identified a new distinct population of mature CD4 T cells that develop in the thymus: the natural cytotoxic CD4 T lymphocytes. We also established mechanism underlying these process by demonstrating that CD4 CTL needs a strong TCR signal for their differentiation.

Immunotherapy of Dual-Function Vector with Both Immunostimulatory and B-Cell Lymphoma 2 (Bcl-2)-Silencing Effects on Gastric Carcinoma.

BackgroundTumorigenesis is a kind of pathology marked by infinite proliferation and restrained apoptosis compared with normal cells. The abnormal expression of some proto-oncogenes and apoptosis inhibition are essential for tumor growth, which has been confirmed by molecular biologic and immunologic studies. The hypofunction of the host immune system also drives the development and metastasis of malignant tumors. Bcl-2, which has a critical role in regulating apoptosis, is overexpressed in several cancers.Material/MethodsIn this study, we constructed a dual-function small hairpin RNA (shRNA) vector containing an Bcl-2-silencing shRNA and a TLR7-stimulating ssRNA and examined it effect on tumor cell growth and proliferation.ResultsStimulation with this bi-functional vector in vitro promoted significant apoptosis of MFC cells by regulating the expression of apoptosis-related proteins and induced secretion of type I IFNs. Most importantly, this bi-functional vector more effectively inhibited subcutaneous MFC cell growth than did single shRNA and ssRNA treatment in vivo. Natural killer (NK) and CD4+ T cells were required for effective tumor suppression, and TLR7 was shown to play a helper role in the activation of NK cells and CD4+ T cells, possibly by regulating the expression of receptors or secretion of cytokines.ConclusionsThis bi-functional vector that contained ssRNA and shRNA may represent a promising approach for tumor therapy.

The Exportin-1 Inhibitor Selinexor Exerts Superior Antitumor Activity when Combined with T-Cell Checkpoint Inhibitors.

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth in vivo We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. In vitro, selinexor increased PDCD1 and CTLA4 gene expression in leukocytes and induced CD274 gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies (P < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells (P ≤ 0.050) and CD4+ T cells with a Th1 phenotype (P ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. Mol Cancer Ther; 16(3); 417-27. ©2017 AACRSee related article by Tyler et al., p. 428.

CD25+CD127+Foxp3- Cells Represent a Major Subpopulation of CD8+ T Cells in the Eye Chambers of Normal Mice.

The aim of this study has been to determine whether eye chambers constitute part of the normal migratory pathway of naive CD4+ and CD8+ T cells in mouse and if natural CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells are present within these eye compartments. To this aim, the cells obtained from aqueous humor (AH) of normal mice were phenotyped in terms of the expression CD4, CD8, CD25, CD127 and transcription factor Foxp3. The mean percentage of CD8+ T cells in the total AH lymphocyte population was as high as 28.69%; the mean percentage of CD8high and CD8low cells in this population was 34.09% and 65.91%, respectively. The presence of cells with the regulatory phenotype, i.e. CD25+Foxp3+ cells, constituted only 0.32% of CD8+ T cell subset. Regarding the expression of CD25, AH CD8+ T cells were an exceptional population in that nearly 85% of these cells expressed this molecule without concomitant Foxp3 expression. Despite having this phenotype, they should not be viewed as activated cells because most of them co-expressed CD127, which indicates that they are naive lymphocytes. With regard to the markers applied in the present research, CD8+CD25+CD127+Foxp3- T cells represent the most numerous subset of AH CD8+ cells. The results suggest that eye chambers in mice are an element in the normal migratory pathway of naive CD8+ T cells. The study presented herein demonstrated only trace presence of CD4+ cells in the eye chambers, as the mean percentage of these cells was just 0.56. Such selective and specific homing of CD8+ and CD4+ cells to the eye chambers is most clearly engaged in the induction and maintenance of ocular immune privilege.

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients

AbstractCancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called “damage-associated molecular patterns,” DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

Association of NKG2D gene variants with susceptibility and severity of rheumatoid arthritis.

NKG2D (KLRK1) is a C-type lectin receptor present on natural killer (NK) cells, γδ, CD8+ and CD4+ T cells. Upon ligand binding, NKG2D mediates activatory and co-stimulatory signals to NK cells and activated CD4+ T cells, respectively. Polymorphisms in NKG2D predispose to infectious diseases, cancer, transplantation and autoimmune disorders. We studied the influence of this NK receptor polymorphism on predisposition to and modification of the disease phenotype in patients with rheumatoid arthritis (RA). Eight different single nucleotide polymorphisms (SNP) in the NKG2 gene were genotyped in 236 patients with RA and 187 controls using Taqman 5' nuclease assays. NKG2D genotype/allele frequency did not differ between patients and controls. Subgroup analysis showed that the frequency of A allele of NKG2D9 and T allele of NKG2D10 was significantly higher in patients with deformities (a marker of severe disease) [11 versus 5%, Pc = 0·03, odds ratio (OR) = 2·44, 95% confidence interval (CI)=1·09-5·98 and 10 versus 4%, Pc = 0·04, OR = 2·45, 95% CI = 1·05-6·39, respectively], while the frequency of alleles G of NKG2D9 and A of NKG2D10 was greater in patients without deformities (Pc = 0·03, OR = 0·41, 95% CI = 0·17-0·91 and Pc = 0·04, OR = 0·41, 95% CI = 0·16-0·96). Similar trends of association were observed with deforming phenotype of RA in female patients and deforming young onset RA subgroups. Haplotype analysis revealed that the frequency of haplotype G-C-A-G-A-T-C-C was higher in patients than in controls (12 versus 8%, P = 0·04, OR = 1·61, 95% CI = 1·01-2·55), suggesting that it may predispose to RA. Our study suggests that the NKG2D gene polymorphisms may modify the risk of development and severity of RA.

Human CD39hi regulatory T cells present stronger stability and function under inflammatory conditions

Autoimmune diseases are characterized by an imbalance between regulatory T cells and effector T-cell subsets, such as Th1 and Th17 cells. Studies have confirmed that natural CD4+Foxp3+ Tregs were unstable and dysfunctional in the presence of pro-inflammatory cytokines. In the current study, human CD39hi Tregs and CD39low Tregs were sorted from Tregs in vitro after 7 days of expansion. The functions of both Treg subsets were investigated under inflammatory conditions in vitro and in vivo. In the presence of IL-1β and IL-6, cultured CD4+CD39hi Tregs maintained stable forkhead box protein 3 expression, whereas CD4+CD39low Tregs lost Foxp3 expression and trans-differentiated into Th1 or Th17 cells. Decreased IL-1βR and IL-6R expression on the CD39hi Tregs was the primary mechanism responsible for Treg stability. In addition, reduced activation of downstream molecules, such as STAT1 and STAT3, through the modulation of CpG demethylation played an important role. Finally, human CD4+CD39hi Tregs but not CD4+CD39low Tregs protected against xenograft versus host disease in model mice. These results strongly implied the physiological importance of CD39 expression and suggested that manipulation of CD39hi Tregs might represent a novel strategy for the treatment of autoimmune diseases.

Regulatory T-Cell Plasticity

During the past 2 decades, human and animals studies have clearly documented the crucial role of inflammation in the development and complications of atherosclerosis.1 Both innate immunity and adaptive immunity are involved in this process. The first evidence that suggested a role of adaptive immunity in atherosclerosis was the widespread detection of the major histocompatibility class II in human atherosclerotic plaques and the presence of a large amount of CD3+ lymphocytes in human and mouse atherosclerotic lesions. Most of the T cells in mouse and human atherosclerotic plaques are CD4+ T-helper (Th) cells expressing the αβ T-cell antigen receptor. Among CD4+ T cells, Th1 cells have been shown to exert proatherogenic effects, whereas regulatory T cells (Tregs) display atheroprotective properties. The role of Th2 and Th17 cells is still debated.1 Helper T-cell subsets are defined by the production of cytokines or the expression of characteristic lineage-defining transcription factors. Th1 cells are generated on priming in the presence of interleukin (IL)-12 that promotes the expression of the transcription factor T-bet and stimulates the production of the Th1 prototypical cytokine interferon-γ (IFN-γ).2 Tregs have a central role in the dominant control of immunologic tolerance and maintenance of immune homeostasis. They were first identified in mice and later in humans.3 The transcription factor FoxP3 is essential for the generation and the functions of Tregs.4 FoxP3 deficiency leads to a multiorgan autoimmune disease as can be observed in the scurfy mouse and in human immune dysregulation, polyendocrinopathy, enteropathy X–linked syndrome patients.5 Natural or thymic FoxP3+ Tregs acquire regulatory lineage commitment already on maturation in the thymus,3 whereas adaptive or peripheral FoxP3+ Tregs can be induced from mature CD4+ Th cells in the periphery under the influence of different stimulations, especially transforming …

Mobilization with cyclophosphamide reduces the number of lymphocyte subpopulations in the leukapheresis product and delays their reconstitution after autologous hematopoietic stem cell transplantation in patients with multiple myeloma.

BackgroundAutologous hematopoietic stem cell transplantation is considered the standard of care for younger patients with multiple myeloma. Several mobilization regimens are currently used, most commonly growth factors alone or in combination with chemotherapy. The aim of our study was to investigate the differences in lymphocyte subpopulation counts between three different mobilization regimens on collection day, in the leukapheresis product and on day 15 after autologous hematopoietic stem cell transplantation.Patients and methodsIn total 48 patients were prospectively enrolled in three different mobilization regimens; (i) filgrastim (20), (ii) pegfilgrastim (19) and (iii) cyclophosphamide + filgrastim (9). Lymphocytes, CD16+/56+ natural killer and CD4+/CD25high T regulatory cells were determined by flow cytometry.ResultsWe found a statistically significant difference between the mobilization regimens. Cyclophosphamide reduced lymphocyte and natural killer (NK) cell counts on collection day (lymphocytes 1.08 × 109/L; NK cells 0.07 × 109/L) compared to filgrastim (lymphocytes 3.08 × 109/L; NK cells 0.52 × 109/L) and pegfilgrastim (lymphocytes 3 × 109/L; NK cells 0.42 × 109/L). As a consequence lymphocyte and NK cell counts were also lower in the leukapheresis products following cyclophosphamide mobilization regimen (lymphocytes 50.1 × 109/L; NK cells 4.18 × 109/L) compared to filgrastim (lymphocytes 112 × 109/L; NK cells 17.5 × 109/L) and pegfilgrastim (lymphocytes 112 × 109/L; NK cells 14.3 × 109/L). In all mobilization regimens T regulatory cells increased 2-fold on collection day, regarding the base line value before mobilization. There was no difference in T regulatory cell counts between the regimens.ConclusionsMobilization with cyclophophamide reduces the number of mobilized and collected lymphocytes and NK cells as compared to mobilization with growth factors only and results in their delayed reconstitution following autologous hematopoietic stem cell transplantation. We found no difference between filgrastim and pegfilgrastim mobilization.

Thymic hyperplasia after chemotherapy in adults with mature B cell lymphoma and its influence on thymic output and CD4+ T cells repopulation

ABSTRACTTo investigate the thymic regenerative potential in adults accepting chemotherapy for lymphoma. The dynamics of thymic activity in 54 adults from baseline to 12 mo post-chemotherapy was analyzed by assessing thymic structural changes with serial computed tomography (CT) scans, and correlating these with measurements of thymic output by concurrent analysis of single-joint (sj) T-cell receptor excision circles (sjTREC) and CD31+ recent thymic emigrants (RTE) in peripheral blood. Furthermore, the consequence of thymic renewal on peripheral CD4+ T cell recovery after chemotherapy was evaluated. Time-dependent changes of thymic size and thymic output assessed by both sjTREC levels and CD31+ RTE counts in peripheral blood were observed during and after chemotherapy. Enlargement of thymus over baseline following chemotherapy regarded as rebound thymic hyperplasia (TH) was identified in 20 patients aged 18−53 y (median 33 y). By general linear models repeated measure analysis, it was found that, patients with TH (n = 20) had a faster recovery of sjTREC levels and CD31+ RTE counts after chemotherapy than patients with comparable age, gender, diagnosis, disease stage, thymic volume and output function at baseline but without TH (n = 18) (p = 0.035, 0.047); besides, patients with TH had a faster repopulation of both naïve CD4+ T cell and natural regulatory CD4+ T cell subsets than those without TH (p = 0.042, 0.038). These data suggested that adult thymus retains the capacity of regeneration after chemotherapy, especially in young adults. The presence of TH could contribute to the renewal of thymopoiesis and the replenishment of peripheral CD4+ T cell pool following chemotherapy in adults.

Interleukin 18 (IL-18) as a target for immune intervention.

Interleukin 18 (IL-18) is a pleiotropic cytokine involved in the regulation of innate and acquired immune response. In the milieu of IL-12 or IL-15, IL-18 is a potent inducer of IFN-gamma in natural killer (NK) cells and CD4 T helper (Th) 1 lymphocytes. However, IL-18 also modulates Th2 and Th17 cell responses, as well as the activity of CD8 cytotoxic cells and neutrophils, in a host microenvironment-dependent manner. It is produced by various hematopoietic and nonhematopoietic cells, including dendritic cells and macrophages. In an organism, bioactivity of the cytokine depends on the intensity of IL-18 production, the level of its natural inhibitory protein - IL-18BP (IL-18 binding protein) and the surface expression of IL-18 receptors (IL-18R) on the responding cells. This review summarizes the biology of the IL-18/IL-18BP/IL-18R system and its role in the host defense against infections. The prospects for IL-18 application in immunotherapeutic or prophylactic interventions in infectious and non-infectious diseases are discussed.

MicroRNAs in CD4+ T cell subsets are markers of disease risk and T cell dysfunction in individuals at risk for type 1 diabetes

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4+ FOXP3+ T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4+ T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4+ T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4+ T cell subsets are markers of risk and T cell dysfunction in T1D.

Regulation of Interleukin-12 Production in Antigen-Presenting Cells.

Interleukin-12 is a heterodimeric cytokine produced primarily by pathogen-activated antigen-presenting cells, particularly macrophages and dendritic cells, during encountering with intracellular microbes. IL-12 plays a key role in the activation of natural killer cells and CD4+ T helper cells in both innate and adaptive immune responses against infectious agents and immunosurveillance against endogenous malignancies. However, the potency of IL-12 makes it a target for stringent regulation. Indeed, the temporal, spatial, and quantitative expression of IL-12 during an immune response in a microenvironment contributes critically to the determination of the type, extent, and ultimate resolution of the reaction. Breaching of the delicate control and balance involving IL-12 frequently leads to autoimmune inflammatory disorders and pathogenesis. Thus, a better understanding of the regulatory mechanisms in the production and control of this cytokine is both scientifically significant and clinically beneficial. Here we provide an update on the research that has been conducted on this subject particularly in the last 10 years since the publication of a major thesis of this nature.

Immunological Derangement in Follicular Lymphoma Patients Developing Severe Side Effects during Treatment with PI3K Inhibitors

IntroductionPhosphoinositide-3-kinases (PI3Ks) are pivotal in various cellular functions including proliferation and survival, cell differentiation, intracellular trafficking and immunity. The PI3Kδ isoform is highly expressed in cells of hematopoietic origin, and is often dysregulated in various hematologic malignancies.New drugs targeting PI3Ks have emerged as promising treatment options for patients (pts) with CLL and indolent NHL, but a clear understanding of their effects on pts' immunity is lacking. Herein we performed detailed immunological analysis in two follicular lymphoma (FL) pts treated with PI3K inhibitors and developing severe immune-mediated side effects.Patients and MethodsTwo of 9 pts with relapsed FL treated with PI3Kδ (pt1) and PI3Kα/δ (pt2) inhibitors developed severe adverse events (SAE). Both pts were heavily pretreated including autologous transplantation and discontinued therapy due to SAE. Pt1 showed grade 3 transaminase elevation, esophagitis and diarrhea requiring hospitalization after 3 cycles of therapy. Diarrhea worsened with fever and weight loss, in spite of transient discontinuation and dose reduction of PI3k inhibitor. Colonoscopic biopsy showed GVHD-like colitis. He died 3 months later. Pt2 has developed, after the first cycle and over a period of one year, febrile neutropenia, erythroderma, ulcerative keratitis requiring corneal transplant, pure red cell aplasia, endocrine abnormalities, grade 3 diarrhea with weight loss, and inflammatory bowel disease-like colitis.Detailed T-cell subpopulations immunophenotyping and T-cell receptor (TCR) spectratyping were performed in samples obtained at 6 months and 1 year after the start of treatment.ResultsCompared to healthy controls (HC) both pts showed marked abnormalities in T-cell subsets including a very low percentage of CD4+CD45RA+CCR7+CD31+ recent T-emigrants (RTE) and of CD4+CD45RA+CCR7+ naïve T cells in both CD4+ and CD8+ populations; CD4+CD45RA-CCR7+ central memory were significantly decreased, while CD4+CD45RA-CCR7- effector memory T cells were increased. Natural CD4+CD25+CD127low/- regulatory T cell (Treg) were low in both pts and the defect equally involved naïve, central and effector memory Treg. On the contrary, inducible Treg (Tr1) did not differ between FL pts and HC.The predominance of effector T cells strongly biased the TCR repertoire. Cells expressing all TCR beta variable (BV) chains were clonally or oligoclonally expanded at the spectratyping analysis because their profiles deviated from the Gaussian patterns found in polyclonal repertoires of HC.DiscussionThe clinical picture of the two FL pts was reminiscent of congenital immunodeficiencies with defects of Treg activity (e.g. IPEX caused by FoxP3 gene mutation). Similar symptoms and T-cell abnormalities are also present in pts with inadequate thymic function and immunologic reconstitution due to severe chronic GVHD. The associated T-cell immunological derangement was characterized by: 1) a marked reduction of the thymic output; 2) a clear defect in natural Treg; 3) an increase of peripheral oligoclonal effector memory T cells. Preclinical data on the role of PI3kδ support its possible pathogenetic involvement: PI3Kδ is known to play critical roles during thymopoiesis and its deletion/inactivation results in a block at the CD4 CD8 double negative stage of T-cell development. In mice with a mutation of PI3Kδ subunit, Treg were increased in the thymus but decreased in all peripheral immune organs, suggesting an impaired emigration of these cells from the thymus. Moreover, mice expressing an inactive form of PI3Kδ develop inflammatory bowel disease.These preliminary data suggest that small molecule-mediated inhibition of PI3Kδ may cause severe alterations within the T-cell compartment in selected pts, which may derive from a defect in the thymic output, including that of Treg. The generation of mature, oligoclonal T cells can be either the result of an homeostatic T-cell proliferation or of a strong (auto)antigenic stimulation. To better elucidate such mechanisms, further studies are in progress on a larger number of pts showing different levels of PI3k inhibitor toxicity. Nevertheless, we suggest that a close follow-up of lymphocyte subpopulation changes should be worthwhile since it may anticipate the onset of these severe clinical manifestations. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prophylactic use of low-dose interleukin-2 early post-transplantation: a randomised study

BackgroundEvidence suggests that natural killer cells and regulatory CD4+CD25+FoxP3+ T (Treg) cells, which are expanded and stimulated by the application of interleukin-2, could mediate protection against graft-versus-host disease (GVHD) while maintaining graft anti-tumour activity. The aim of this study was to confirm that a low dose of interleukin-2 can prevent relapse while reducing chronic GVHD. This study has been presented in part as an oral presentation at the 56th American Society of Hematology Annual Meeting and Exposition (Dec 6, 2014) in San Francisco, CA, USA. MethodsIn this open-label, prospective, randomised controlled trial in a single centre in China, donor–recipient pairs were randomly assigned (1:1) into the interleukin-2 group and control group by the sub-investigator (treating physician of our transplant centre) according to a computer-generated randomisation system. We enrolled patients (aged ≥15 years) with standard-risk leukaemia (except Ph+ ALL and T-ALL) who had undergone unmanipulated allogeneic stem cell transplantation. The transplantation procedure for control group patients was the same as that of the interleukin-2 group, except for interleukin-2 treatment. Interleukin-2 treatment (daily 1×106 IU/m2 body-surface area) was repeated every 14 days for six courses until disease progression or unacceptable toxicity. The primary endpoint was the cumulative incidence of haematological relapse. The primary efficacy end point was a between-group comparison of the time from transplantation to relapse in the intention-to-treat population (all randomly assigned patients). 360 subjects were estimated to be needed to detect a 10% difference between groups in cumulative incidence of haematological relapse based on our previous results (from 20% to 10%) with a one-sided α of 0·05 and a power of 80%. Futility analyses after a quarter, half, and three-quarters of 360 cases were specified. This study was approved by the Institutional Review Board of Peking University, and written informed consent was obtained from all patients in accordance with the Declaration of Helsinki. This trial is registered with ClinicalTrials.gov, number NCT01517347. FindingsSince this study began in January, 2012, we have randomly assigned 43 patients to the interleukin-2 group and 47 patients to the control group. First interim analysis was done when 90 cases were enrolled. The cumulative incidence of haematological relapse of the interleukin-2 and control cohorts were 27% (95% CI 18–35%) and 9% (5–12%), respecitively; p=0·12). Low dose of interleukin-2 augmented reconstituted Treg cells and natural killer cells. The suppressive functions of interleukin-2-expanded Treg cells in inhibiting T-cell activation were comparable to healthy donors. Cytotoxicity of interleukin-2-expanded natural killer cells against K562 cells as well as against resting or activated conventional T cells (Tcon) was significantly enhanced compared to control group. Patients with severe chronic GVHD had a lower ratio of Treg to Tcon cells at 2 months and 3 months, and a lower ratio of CD56dim to CD56bri NK cells from 2 months to 5 months after allogeneic stem cell transplantation. Interleukin-2 therapy was well tolerated; the most common adverse event was fever. InterpretationLow dose of interleukin-2 given post-transplant did not decrease the cumulative incidence of haematological relapse and was associated with a higher frequency of an MRD-positive test and a lower frequency of chronic GVHD, which might be through expansion of Treg cells. Thus, the prophylactic application of interleukin-2 should be cautious and its effect on leukaemia relapse needs to be further explored. FundingThe National Natural Science Foundation of China, the Major State Basic Research Development Program of China, and Milstein Medical Asian American Partnership (MMAAP) Foundation Research Project Award in Hematology.

Onset of Graves' disease during long-term immunosuppressive therapy in a patient with membranous nephropathy.

SummaryA 67-year-old man was referred to our department for thyrotoxicosis with intermittent palpitation and 4-kg weight loss during the previous month. At the first visit, the patient was treated with cyclosporine A (CyA) for membranous nephropathy during the last 3 years and 8 months. Laboratory studies revealed that the serum TSH level was <0.005 μU/ml, and free thyroxine (fT4) and triiodothyronine (fT3) levels were elevated at 2.76 ng/dl and 5.96 pg/ml respectively. Anti-TSH receptor antibody (TRAb) level was increased at 26.4%. A clinical diagnosis of Graves' hyperthyroidism was given, and then thyrostatic treatment with thiamazole (MMI) at a dose of 10 mg daily was initiated after CyA withdrawal. After the initiation of MMI therapy, serum fT4 and fT3 attained the normal level within 1.5 months, with relief of symptoms followed by a remarkable decrease in urinary protein excretion from 2.0–5.2 g/day to ≤0.03 g/day. The patient maintained euthyroid with a low titre of TRAb for the succeeding 2 years and then MMI was finally stopped. Neither a relapse of hyperthyroidism nor a flare-up of nephrotic syndrome was observed for 3 years after MMI discontinuation. CyA has conflicting effects on immunologic self-tolerance by modulation of self-reactive T cells and natural CD4+CD25+Foxp3+ regulatory T cell (Treg) functions, and possibly becomes a triggering factor in the development of autoimmune disorders. This case may be interesting when considering the effect of each T cell subset on the development of Graves' disease.Learning points The balance between intrathyroidal self-reactive T cell and natural CD4+CD25+Foxp3+ Treg functions determine self-tolerance in the thyroid.CyA not only halts the expansion of self-reactive T cells but also impairs the function of Treg, which can provoke an unwanted immune response.A change in thyroid autoimmunity during treatment with CyA may result in the development of autoimmune thyroid diseases (AITD).Renal involvement in AITD frequently manifests as nephrotic syndrome, and thyrostatic treatment with thiamazole may be effective for excessive proteinuria.

Natural CD4+ T regulatory cell down-regulation of monocytic myeloid derived suppressor cell phenotypic and functional subpopulations in LP-BM5 murine retrovirus-induced immunodeficiency (VIR1P.1149)

Abstract Immunomodulatory cellular subsets, including myeloid derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumor microenvironment and have been targets of immunotherapy, but their role in retroviral-associated immunosuppression is less understood. Due to known crosstalk between Tregs and MDSCs in the tumor microenvironment, it is of interest to look at the interplay between these immune cells during LP-BM5-induced murine AIDS. Following in vivo natural Treg (nTreg) depletion, and subsequent LP-BM5 retroviral infection, enriched MDSCs from infected nTreg-depleted mice exhibit increased suppression of T cell, but not B cell, responses, as compared to MDSCs derived from non-depleted LP-BM5-infected controls. MDSCs from LP-BM5-infected nTreg-depleted mice also display altered phenotypic subsets, which correlate with functional differences. In addition, LP-BM5-induced MDSCs modulate in vitro Treg phenotype and function. These collective data highlight an antagonistic relationship between MDSCs and Tregs, and may provide insight into the immunotherapeutic targeting of these cells during retroviral infection.

HIV-1 Tat immunization restores immune homeostasis and attacks the HAART-resistant blood HIV DNA: results of a randomized phase II exploratory clinical trial.

BackgroundThe phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 μg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks.Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia <50 copies/mL in the last 6 months prior to enrollment, and CD4+ T-cell number ≥200 cells/μL. Subjects from a parallel observational study (ISS OBS T-002, Clinicaltrials.gov NCT0102455) enrolled at the same clinical sites with the same criteria constituted an external reference group to explore biomarkers of disease.ResultsThe vaccine was safe and well tolerated and induced anti-Tat Abs in most patients (79%), with the highest frequency and durability in the Tat 30 μg groups (89%) particularly when given 3 times (92%). Vaccination promoted a durable and significant restoration of T, B, natural killer (NK) cells, and CD4+ and CD8+ central memory subsets. Moreover, a significant reduction of blood proviral DNA was seen after week 72, particularly under PI-based regimens and with Tat 30 μg given 3 times (30 μg, 3x), reaching a predicted 70% decay after 3 years from vaccination with a half-life of 88 weeks. This decay was significantly associated with anti-Tat IgM and IgG Abs and neutralization of Tat-mediated entry of oligomeric Env in dendritic cells, which predicted HIV-1 DNA decay. Finally, the 30 μg, 3x group was the only one showing significant increases of NK cells and CD38+HLA-DR+/CD8+ T cells, a phenotype associated with increased killing activity in elite controllers.ConclusionsAnti-Tat immune responses are needed to restore immune homeostasis and effective anti-viral responses capable of attacking the virus reservoir. Thus, Tat immunization represents a promising pathogenesis-driven intervention to intensify HAART efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0151-y) contains supplementary material, which is available to authorized users.

Short Communication: Low Expression of Activation and Inhibitory Molecules on NK Cells and CD4(+) T Cells Is Associated with Viral Control.

Chronic HIV-1 infection induces severe immune alterations, including hyperactivation, exhaustion, and apoptosis. In fact, viral control has been associated with low frequencies of these processes. Here, we evaluated the expression of activation and inhibitory molecules on natural killer (NK) and CD4(+) T cells and plasma levels of proinflammatory cytokines in individuals exhibiting viral control: a cohort of HIV-1-exposed-seronegative individuals (HESN) and a cohort of HIV controllers. There was lower expression of CD69, LAG-3, PD-1, and TIM-3 in both cohorts when compared to a low-risk population or HIV progressors. In addition, HIV controllers exhibited lower plasma levels of proinflamatory molecules TNF-α and IP-10. These findings suggest that individuals exhibiting viral control have lower basal expression of markers associated with cellular activation and particularly immune exhaustion.