Antiretroviral Therapy Restores Diversity in the T-Cell Receptor Vβ Repertoire of CD4 T-Cell Subpopulations among Human Immunodeficiency Virus Type 1-Infected Children and Adolescents

Human immunodeficiency virus (HIV) type 1 infection perturbs the T-cell receptor (TCR) Vβ repertoire. The TCR CDR3 length diversity of individual Vβ families was examined within CD45RA and CD45RO CD4 T cells to assess the impact of the virus on clonality throughout CD4 T-cell activation and differentiation. A cross-sectional and longitudinal cohort study of 13 HIV-infected and 8 age-matched healthy children and adolescents examined the Vβ CDR3 length profiles within CD4 T-cell subsets by the use of spectratyping. HIV-infected subjects demonstrated higher numbers of perturbations in CD4 CD45RA T cells (5.8 ± 4.9 Vβ families) than healthy individuals (1.6 ± 1.8 Vβ families) (P = 0.04). Surprisingly, CD4 CD45RO central memory T cells from infected subjects showed no increased perturbations compared to the perturbations for the same cells from healthy subjects (2.9 ± 3.1 and 1.1 ± 1.8 Vβ families, respectively; P = 0.11). CD4 CD45RA TCR perturbations were higher among infected subjects with >25% CD4 cells than healthy subjects (mean number of perturbed Vβ families, 6.6 ± 5.4; P = 0.04). No correlations between perturbations in CD4 subsets and pretherapy age or viral load were evident. In contrast to CD8 T cells, HIV induces TCR disruptions within CD45RA but not CD45RO CD4 T cells. Therapy-induced viral suppression resulted in increases in thymic output and the normalization of the diversity of TCR within CD45RA CD4 T cells after 2 months of treatment. Perturbations occur prior to CD4 T-cell attrition and normalize with effective antiretroviral therapy. The impact of HIV on the diversity of TCR within naïve, central memory, and effector memory CD4 T cells is distinctly different from that in CD8 T cells.Human immunodeficiency virus type 1 (HIV-1) infection alters T-cell homeostasis by both impairing thymic output and inducing chronic T-cell activation. These disruptions are manifest by the increased level of expression of T-cell activation markers and decreased numbers of naïve T cells from the thymus (10, 12, 51). Oligoclonal T-cell expansion results in perturbations of the T-cell receptor (TCR) Vβ repertoire within both CD4 and CD8 T cells, with CD8 T cells being affected to a greater extent than CD4 T cells (7, 12, 16, 29, 50). Many of these abnormalities occur prior to CD4 T-cell attrition and are not fully reconstituted when viral replication is controlled by antiretroviral therapy (6, 17, 30). Multiple mechanisms have been postulated to contribute to this processes of aberrant T-cell activation and clonal expansion, including microbial translocation across the gastrointestinal tract as a result of virus-induced intestinal fibrosis (4, 5, 40) and the loss of immune regulation due to chronic HIV-induced antigenemia (8, 22).CD4 and CD8 T cells are heterogeneous populations that differ functionally and in their expression of activation and differentiation markers, forming the basis of their classification as naïve, central memory (CM), or effector memory (EM) T cells (42). Isoforms of CD45 (CD45RA and CD45RO) are frequently used to subdivide CD4 and CD8 T cells into functional subsets (1, 13, 25, 44, 45). Oligoclonal expansions and deletions within T-cell subpopulations can be measured by analysis of the hypervariable CDR3 region of the TCR (37). CDR3 length variation reflects changes within the TCR Vβ repertoire during antigen-induced T-cell activation (24, 25, 34). Differences in CDR3 length diversity within the CD4 or the CD8 CD45RA or CD45RO subset enable assessments of disruptions of the TCR repertoire and the detection of oligoclonal expansion that would have been missed if the analysis were limited to unfractionated T cells (25, 26). While optimal control of viral replication by antiretroviral therapy (ART) corrects many T-cell abnormalities and slows the progression to AIDS, it is not clear if therapy completely restores the TCR repertoire or fully diminishes T-cell activation (7, 14, 27, 51). In the present study, we examined the relationship of TCR diversity, thymic output, and the expression of T-cell activation markers within the CD45RA and CD45RO subpopulations of CD4 and CD8 T cells before and after the initiation of ART to determine the extent to which the control of viral replication restores the TCR repertoire.     

Disorders in Restructuring of T-Cell Receptor γ-Chain in Malignant Skin Lymphomas

We found an alternative form of mRNA with spliced second exon (IL-4delta2 mRNA) in mouse bone marrow and splenic cells. At rest, the amount of IL-4 mRNA markedly surpassed that of IL-4delta2 mRNA. Stimulation increased the content of both mRNA forms, but the alternative variant is accumulated more intensively and rapidly. We did not detect predominance of IL-4delta2 mRNA over full-length mRNA variant in the studied mouse tissues.     

T-Cell subpopulation shifts in human cord blood high- and low-affinity E·RFC

T-Cell subpopulation in human cord blood were enumerated by sheep erythrocytes (E) rosette formation. High-affinity E-RFC lymphocytes were identified by E-rosetting at 29°C and low-affinity E-RFC at 4°C. Human peripheral blood contains 51.4% ± 8.5 (SD) high-affinity E-RFC and 20.6% ± 10 (SD) low-affinity E-RFC. In contrast human cord blood had predominantly high-affinity E-RFC 33.7% ± 6.6 (SD) and few low-affinity E-RFC 2.8% ± 2.4 (SD). A subpopulation of T cells found in adult blood appeared to be lacking in cord blood.     

T-Cell Activation Leads to Reduced Collagen Maturation in Atherosclerotic Plaques of Apoe−/− Mice

Rupture of the collagenous, fibrous cap of an atherosclerotic plaque commonly causes thrombosis. Activated immune cells can secrete mediators that jeopardize the integrity of the fibrous cap. This study aimed to determine the relationship between T-cell-mediated inflammation and collagen turnover in a mouse model of experimental atherosclerosis. Both Apoe^−/− × CD4dnTβRII mice with defective transforming growth factor-β receptors in T cells (and hence released from tonic suppression of T-cell activation) and lesion size-matched Apoe^−/− mice were used. Picrosirius red staining showed a lower content of thick mature collagen fibers in lesions of Apoe^−/− × CD4dnTβRII mice, although both groups had similar levels of procollagen type I or III mRNA and total collagen content in lesions. Analysis of both gene expression and protein content showed a significant decrease of lysyl oxidase, the extracellular enzyme needed for collagen cross-linking, in aortas of Apoe^−/− − CD4dnTβRII mice. T-cell-driven inflammation provoked a selective and limited increase in the expression of proteinases that catabolize the extracellular matrix. Atheromata of Apoe^−/− − CD4dnTβRII mice had increased levels of matrix metalloproteinase-13 and cathepsin S mRNAs and of the active form of cathepsin S protein but no increase was detected in collagen fragmentation. Our results suggest that exaggerated T-cell-driven inflammation limits collagen maturation in the atherosclerotic plaque while having little effect on collagen degradation.A physical disruption of atherosclerotic plaques causes many acute thrombotic complications such as myocardial infarction and stroke.^1,2 Pathologists have identified a number of characteristics of vulnerable atherosclerotic plaques that have ruptured³ including large lipid cores and thin fibrous caps harboring activated macrophages and T cells.^1,4 The resistance of the atherosclerotic plaque to disruption depends in part on the integrity of its fibrous cap, which prevents contact between the highly thrombogenic lipid core and the circulating blood.^1,2 The fibrous cap is composed of smooth muscle cells (SMCs) and a collagen-rich extracellular matrix. The fibrillar collagens types I and III synthesized by SMCs primarily determine the tensile strength of the cap. Sites of plaque rupture characteristically display signs of inflammatory cell activation accompanied by dissolution of matrix.Inflammation may impair plaque stability because macrophages and mast cells release a set of collagen-degrading matrix metalloproteinases (MMPs) and cysteine proteases.^5,6,7 Additional possible mechanisms include inhibited expression of procollagen genes and death or reduced renewal of the collagen-producing SMC population, both phenomena promoted by T-cell-derived interferon (IFN)-γ.⁸ Several studies suggest that T cells may modulate plaque stability. Activated T cells accumulate in vulnerable plaques.⁴ Th1 cells, which dominate in plaques, secrete IFN-γ, a powerful inhibitor of endothelial and SMC proliferation.⁹ IFN-γ also inhibits differentiation and collagen gene expression in SMCs^10,11 and modulates expression of several MMPs and cathepsins.^12,13 In vivo treatment with IFN-γ increases atherosclerosis and transplant arteriosclerosis,^14,15 whereas targeted gene deletion in the IFN-γ receptor leads to reduced atherosclerosis in hypercholesterolemic mice.¹⁶ Mice with defective control of T-cell activity show modulation of atherogenesis: interleukin-10 targeted mice display increased plaque formation with reduced collagen accumulation,¹⁷ and Apoe^−/− mice lacking transforming growth factor (TGF)-β inhibition of T cells rapidly develop large, hyperinflammatory lesions.¹⁸ These data point to a proatherosclerotic and possibly destabilizing role for activated T cells.The present study aimed to assess the effect of inflammation on the fibrous component of the atherosclerotic plaque. We used Apoe^−/− × CD4dnTβRII mice, which lack functional TGF-β receptors on T cells. Therefore, uncontrolled T-cell activation leads to rampant inflammation and accelerated atherosclerosis in hypercholesterolemic animals.¹⁸ Therefore, Apoe^−/− × CD4dnTβRII mice provide an opportunity to study the effect of inflammation on atherosclerotic lesions. Our data show reduced enzyme-dependent collagen maturation in hyperinflamed lesions, whereas effects on procollagen expression and collagenolytic enzymes were modest. These results suggest a novel mechanism by which adaptive immunity can modulate plaque stability—impairment of collagen maturation by T-cell-dependent inflammation.     

Does the HBZ Gene Represent a New Potential Target for the Treatment of Adult T-Cell Leukemia?

Links between human T-cell leukemia virus type 1 and adult T-cell leukemia (ATL) were first suspected in 1980. Provirus integration has since been found in all ATL cells. Although the viral Tax protein is involved in the proliferation of the infected cells during the preleukemic stage, Tax expression is not systematically detected in primary leukemic cells. Recent studies found that the viral HBZ gene was always expressed in leukemic cells, suggesting its involvement in the progression of the infected cells toward malignancy. How could this new discovery be translated into possible new avenues for the prevention or treatment of ATL?     

Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8^＋ T-Cell Epitopes

Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.     

Flow Cytometry Evaluation of the T-Cell ReceptorVβ Repertoire Among HIV-1 Infected Individuals Before and After Antiretroviral Therapy

HIV-1 infection leads to serious impairment of the immune system and perturbations in the T cell receptor V repertoire are also described. Immune reconstitution can be potentially achieved in response to HAART. In the present study 10 patients were investigated for the V pattern expression before and after six months of HAART. TCR were analyzed for T CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 24 different V chains. Compared to eight Brazilian healthy controls, no differences in V pattern of expression was observed for patients before or on antiretroviral therapy. Some chains such as V 3, 14, 16, 20 and 21.3 were over utilized by both T subsets, independently of HIV infection and/or antiretroviral treatment, differing from the ones described for individuals of other nationalities. However, when each patient was taken individually, particular alterations were detected for the V gene usage, compared to controls, for all individuals. After treatment, significant V usage changes were observed for seven patients. One or more chains on both T subsets were engaged in this process, defining a preferential oligoclonal profile for TCR repertoire distribution, after HAART. Although no pattern of specific V changes was detected in the circulating T cells, we cannot exclude that differential immune responses to HIV or other important antigens are being focused by these cells.     

Current Topics in Prevention of Human T-Cell Leukemia Virus Type I Infection: NF-κ B Inhibitors and APOBEC3

Human T-cell leukemia virus type I (HTLV-I) is the first human retrovirus and causes adult T-cell leukemia/lymphoma (ATL). Constitutive activation of nuclear factor-κ B (NF-κ B) in the leukemic cells is essential for their growth and survival. Thus, NF-κ B inhibitors have been attracting attention as a potential strategy to treat ATL. Recently, the field of retrovirus research has been stimulated by the discovery of an innate host defense factor, APOBEC3, against the retroviruses. HTLV-I is relatively resistant to the antiviral effects of APOBEC3. To clarify the resistance of HTLV-I against APOBEC3 might contribute to the design of effective therapeutic approaches.     

Cocaine Enhances HIV-1–Induced CD4+ T-Cell Apoptosis: Implications in Disease Progression in Cocaine-Abusing HIV-1 Patients

Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1–associated CD4⁺ T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4⁺ T cells from HIV-1–negative and HIV-1–positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4⁺ T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4⁺ T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4⁺ T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4⁺ T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1–infected drug abusers.The HIV/AIDS pandemic has claimed the lives of an estimated 35 million people (http://www.who.int/mediacentre/factsheets/fs360/en/index.html, last updated October 2013). Although anti-retroviral therapy (ART) has dramatically reduced HIV/AIDS-related mortality,¹ substance use is a major barrier for combating the HIV/AIDS pandemic because it is associated with transmission, delayed diagnosis, delayed initiation of therapy, and poor adherence to therapy.² Cocaine is a commonly abused drug among HIV-1 patients,^3–5 and studies suggest that cocaine abuse may accelerate HIV-1 disease progression. For example, Vittinghoff et al⁶ documented an increased risk of HIV-1 disease progression among frequent cocaine users. Arnsten et al^7,8 have reported that active cocaine use strongly predicts failure to viral suppression. Similarly, Webber et al⁹ suggested that use of cocaine, along with alcohol, might accelerate HIV-1 disease progression. In addition, Lucas and colleagues^10–12 found that cocaine users have inferior virological and immunological responses to ART. The effects of cocaine on disease progression in these studies can be attributed, in part, to nonadherence to ART, because substance use is often associated with reduced adherence and/or access to ART.¹³ There are also reports that did not find significant association between cocaine abuse and HIV-1 disease progression.^14,15 However, Baum et al³ found that cocaine users have higher viral load and were twice as likely to progress to AIDS when controlled for ART use. Notably, Palepu et al¹⁶ reported that HIV-1–positive drug users, while taking ART, were less likely to suppress the viral load. Recently, Rasbach et al¹⁷ have suggested that active cocaine use among HIV-1 patients is associated with lack of virological suppression, independent of ART adherence. Although in vitro studies suggest that increased HIV-1 replication by cocaine^18–21 may play a role, the mechanism by which cocaine accelerates HIV-1 disease progression remains unclear. Therefore, we evaluated whether cocaine could potentiate HIV-1–induced CD4⁺ T-cell apoptosis because CD4⁺ T-cell decline is an important predictor of HIV-1 disease progression. Our data suggest a synergy between cocaine and HIV-1 on CD4⁺ T-cell apoptosis and highlight the molecular interplay between cocaine abuse and HIV-1 disease progression.     

Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

A major limitation to the application of adoptive immuno-therapy for cancer is the difficulty in isolating and expand-ing tumor specific T cells in human subjects. We are test-ing the feasibility of using gene transfer technology to ge-netically engraft a…     

Post-Natal Ontogenesis of the T-Cell Receptor CD4 and CD8 Vβ Repertoire and Immune Function in Children with DiGeorge Syndrome

DiGeorge syndrome (DGS) is a congenital disorder characterized by typical facial features, hypoparatyroidism, conotruncal cardiac defects and thymic hypoplasia. Although there are some reports addressing lymphocytes counts and function in DGS children over time, few data have been reported on the T-cell receptor Vβ (TCRBV) repertoire in relation to disease progression. The aim of this study was to evaluate the degree and nature of immunodeficiency and to investigate a possible correlation to clinical findings.We used third complementary region (CDR3) size spectratyping as a tool for monitoring T-cell repertoire diversity in 7 DGS’s children. The rate of thymic output, the phenotype and function of peripheral T-cells and the humoral immunity were also investigated. At baseline a profound alteration of the TCR repertoire was noted, mainly in the CD8+ T-cells, in DGS patients when compared to a control group. Furthermore, analysis of thymic output showed a significant decrease in TCR rearrangement excision circles (TRECs) levels in the patient group. Immunoglobulin abnormalities were also detected. The observed TCR repertoire alterations, although not statistically significant, may suggest an increased susceptibility to infections. A parallel increase in the TCR repertoire diversity and clinical improvement occurred during the follow-up. Our results confirm that the extent of immunodeficiency is highly variable and could improve through childhood, and indicate that TCR repertoire may be a useful marker to clinically monitor thymic function in this primary immunodeficiency.C. Cancrini and M.L. Romiti contributed equally to this work.     

T-Cell Activation and Differentiation Are Regulated by TNF During Murine DBA/2 → B6D2F1 Intestinal Graft-Versus-Host Disease

Administration of a tumor necrosis factor (TNF) inhibitor-encoding adenoviral vector decreases the severity of colonic inflammation in a DBA/2B6D2F1 murine model of colonic graft-versus-host disease (GVHD). The present studies evaluated the effect of TNF blockade on the splenic and colonic T-cell responses. cDNA encoding an artificial fusion protein consisting of the extracellular domain of the human 55-kDa receptor for TNF fused to a mouse IgG heavy chain was subcloned into an E1a-deficient adenoviral vector. Following transfer of DBA/2 T cells and bone marrow cells into irradiated B6D2F1 mice, the mice then received either the control adenovirus or the TNF inhibitor-encoding adenovirus. Splenic and colonic lymphocytes were isolated, stained with anti-H-2^b, anti-H-2^d, anti-CD3, anti-CD4, anti-CD8, and anti-CD45RB antibodies, and analyzed by flow cytometry. Splenic and colonic lymphocyte cytokine profiles also were assessed. More colonic T cells of donor origin were isolated from the control adenovirus recipients than from recipients of the TNF inhibitor encoding adenovirus (P = .027). Fewer CD4⁺ and CD8⁺ T cells were observed in colon but not in the spleen in the TNF inhibitor recipients. Fewer CD45RB^low(memory) T cells were observed in the CD4⁺ colonic lymphocytes isolated from the TNF inhibitor recipients than from controls. Importantly, lower levels of interleukin-2(IL-2) and interferon-gamma (INF-) but not of IL-4 were observed in the lamina propria lymphocyte RNA isolated from the TNF inhibitor recipients. Infiltration and expansion of donor T cells and T-cell activation in the colon appear to be regulated by TNF during murine DBA/2 B6D2F1 gut GVHD.     

Oligoclonal CD8+ T-Cell Expansion in Patients with Chronic Hepatitis C Is Associated with Liver Pathology and Poor Response to Interferon-α Therapy

The role of CD8(+) T lymphocytes in chronic hepatitis C virus (HCV) infection and in liver injury with subsequent development of fibrosis and cirrhosis is poorly understood. To address this question, we performed a follow-up study including 27 chronically HCV-infected individuals. We determined clonality and phenotypes of circulating CD8(+) T cells employing TCRBV spectratyping. Antigen specificity was tested by rMHC-peptide tetramer staining and stimulation with recombinant HCV antigens. In addition, T-cell clonality and phenotypes were followed during the variable clinical response of interferon- (IFN) alpha treatment. We could demonstrate that CD8(+) T-cell expansions were significantly associated with liver fibrosis and cirrhosis. Likewise, increased oligoclonality of circulating CD8(+) T cells in chronic HCV infection was identified as an indicator for poor clinical response to IFN-alpha therapy. Moreover, we also found that IFN-alpha therapy enhanced the differentiation of CD8(+) T cells towards a late differentiation phenotype (CD28(-) CD57(+)). In cases of virus elimination the disappearance of expanded terminally differentiated CD8(+) cells was observed. Thus, this study identifies an association of clonal expansions of circulating CD8(+) T cells with liver pathology and provides a possible explanation for the fact that response to IFN-alpha therapy diminishes with the duration of infection.     

IFN-α Acts on T-Cell Receptor-Triggered Human Peripheral Leukocytes to Up-Regulate CCR5 Expression on CD4+ and CD8+ T Cells

Interleukin-12 (IL-12) as well as IL-2 was recently shown to up-regulate CCR5 expression on T-cell receptor (TCR)-triggered human T cells. Because of the functional similarity between interferon-alpha (IFN-) and IL-12, the present study investigated whether IFN- also up-regulates T cell CCR5 expression. CCR5 was marginally detected on T cells from unstimulated human peripheral blood leukocytes (PBLs) and only slightly induced on PBL T cells following stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs). When anti-CD3/anti-CD28-triggered PBLs were exposed to IFN-, T cells expressed high levels of CCR5. The levels of CCR5 expression were comparable to those induced by IL-12. However, when purified T cells instead of unfractionated PBL were stimulated with anti-CD3/CD28 and then exposed to IL-12 or IFN-, CCR5 expression was induced by IL-12 but not by IFN-. IFN- was found to act on anti-CD3/anti-CD28-stimulated PBL to promote their IL-12 production. Moreover, addition of anti-IL-12 mAb to IFN--stimulated cultures of anti-CD3/CD28-pretreated PBL resulted in considerable inhibition of CCR5 expression. Together, these results indicate that IFN- as well as IL-12 up-regulates CCR5 expression on TCR-triggered T cells and that IFN- functions not by acting directly on T cells but via enhancing IL-12 production by PBL.