ABSTRACT
Thymic function decreases progressively with age but may be boosted in certain circumstances. We questioned whether heart transplantation was such a situation and whether thymic function was related to the onset of rejection. Twenty-eight antithymocyte globulin-treated heart transplant recipients were included. Patients diagnosed for an antibody-mediated rejection on endomyocardial biopsy had a higher proportion of circulating recent thymic emigrant CD4+ T cells and T cell receptor excision circle levels than other transplanted subjects. Thymus volume and density, assessed by computed tomography in a subset of patients, was also higher in patients experiencing antibody-mediated rejection. We demonstrate that thymic function is a major determinant of onset of antibody-mediated rejection and question whether thymectomy could be a prophylactic strategy to prevent alloimmune humoral responses.
Subject(s)
Graft Rejection/etiology , Graft Survival/immunology , Heart Transplantation/adverse effects , Isoantibodies/adverse effects , T-Lymphocytes/immunology , Thymus Gland/physiopathology , Tissue Donors , Adult , Aged , Antilymphocyte Serum/administration & dosage , Female , Follow-Up Studies , Graft Rejection/pathology , HLA Antigens/immunology , Humans , Male , Middle Aged , Postoperative Complications , Prognosis , Risk Factors , T-Lymphocytes/pathology , Young AdultABSTRACT
Attempts to evaluate the protective effect of live attenuated SIV vaccine strains have yielded variable results depending on the route of immunization, the level of attenuation, the level of divergence between the vaccine candidate and the challenge. The protective mechanisms induced by these vaccines are still not well understood. In an effort to address whether the diversity of the CD4+ T cell repertoire in cynomolgus macaques plays a role in the immunological protection following SIVmacC8 infection, we have performed a longitudinal follow-up of the CD4 repertoire by heteroduplex tracking assay in macaques mock-infected or infected with either the attenuated SIVmacC8 or its homologous SIVmacJ5 and challenged with simian-human immunodeficiency virus (SHIV89.6P). Viral load and CD4 absolute counts were determined in these animals and the presence of SHIV89.6P virus in challenged animals was evaluated by PCR and serology. In all macaques that were protected against the challenging virus, we demonstrated a reduced diversity in the CD4+ TRBV repertoire and a few dominant CD4+ T cell clones during early primary infection. In contrast, CD4 TRBV repertoire in unprotected macaques remained highly diverse. Moreover, some of the CD4 T cell clones that were expanded during primary SIV infection re-emerged after challenge suggesting their role in protection against the challenging virus. These results underline the importance of maintaining the CD4 T cell repertoire developed during acute infection and point to the restriction of the CD4 response to the vaccine as a correlate of protection.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Immunity, Innate/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Animals , CD4-Positive T-Lymphocytes/physiology , CD4-Positive T-Lymphocytes/virology , Disease Susceptibility , Macaca , SAIDS Vaccines , Simian Immunodeficiency Virus/physiology , Viral LoadABSTRACT
Although the thymus constitutes a target organ for most protein and steroid hormones, it has been quite difficult to determine the precise control exerted in vivo by the endocrine system upon thymic function. The biological role of the thymus is to ensure the generation of a diversified population of peripheral T cells able to respond to non-self-antigens but nevertheless tolerant to self-antigens. For a long time, thymic function could not be monitored, as a consequence of the absence of adequate technology to differentiate recent thymic emigrants from naive T cells. The generation of T cell receptor (TCR) diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCR alpha and beta chains. During these processes, by-products of the rearrangements are generated in the form of TCR excision circles (TRECs). As these molecules are lost upon further cell division, their quantification is actually considered as a very valuable tool to estimate thymic function. The most appropriate TREC is deltaRec-Psi(J)alpha TREC or signal joint TREC resulting from deltaRec-Psi(J)alpha rearrangement (TCRD deletion) that occurs late during thymopoiesis, before V(alpha)-J(alpha) rearrangement. Here we describe how TREC quantification is a powerful and reliable method to evaluate the impact of hormones and endocrine disorders upon thymic function.
Subject(s)
Gene Rearrangement, T-Lymphocyte , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Thymus Gland/physiology , Aged , Aged, 80 and over , Aging/immunology , Antiretroviral Therapy, Highly Active , HIV Infections/therapy , Humans , Middle Aged , Self Tolerance , Thymus Gland/drug effects , Thymus Gland/immunologyABSTRACT
Despite vigorous cell-mediated immune responses to human and simian immunodeficiency viruses (HIV/SIV) the immune system is unable to clear latently infected resting T cells. These infected cells are reactivated by antigenic stimulation, leading to viral replication. By using the SIV/macaque model of HIV pathogenesis, the dynamics of T cell infiltration into delayed type hypersensitivity sites specific for the purified protein derivative of bacillus Calmette-Guérin have been studied. Early viral mRNA synthesis coincided with the infiltration of antigen-specific T cells. When the infiltration of anti-SIV-specific T cells was rapid compared with the kinetics of viral assembly, the sites were sterilized before the transition to late viral mRNA synthesis occurred. When their infiltration was slow, ephemeral foci of replication were identified. These findings were paralleled by plasma viremia; low viremia coincided with rapid sterilization of the delayed type hypersensitivity sites, whereas high load was found in association with local replication and delayed sterilization. These data suggest that although effective local control of SIV is possible once antiviral T lymphocytes have arrived on site, the slower deployment of these T cells may allow the virus to escape and thus to reseed the pool of memory T cells.
Subject(s)
Antigens, Viral/immunology , Simian Immunodeficiency Virus/immunology , T-Lymphocytes/immunology , Animals , Base Sequence , DNA Primers , Hypersensitivity, Delayed , In Situ Hybridization , Macaca mulatta , Reverse Transcriptase Polymerase Chain Reaction , Simian Immunodeficiency Virus/physiology , Virus ReplicationABSTRACT
Human immunodeficiency virus (HIV) infection is characterized by the massive infiltration of secondary lymphoid organs with activated CD8(+) T lymphocytes. While converging data indicated that these cells were HIV-specific cytotoxic T lymphocytes (CTLs) responsible for HIV spread limitation, direct evidence was lacking. Here, the presence of HIV-specific effector CTLs was demonstrated directly ex vivo in 15 of 24 microdissected splenic white pulps from an untreated patient and in 1 of 24 tonsil germinal centers from a second patient with incomplete viral suppression following bitherapy. These patients had plasma HIV RNA loads of 5900 and 820 copies per milliliter. The frequencies of HIV-1 DNA(+) cells in their lymphoid organs were more than 1 in 50 and 1 in 175, respectively. Spliced viral messenger RNA (a marker for ongoing viral replication) was present in most immunocompetent structures tested. Conversely, CTL activity was not found in spleens from 2 patients under highly active antiretroviral therapy, with undetectable plasma viral load. These patients had much lower spleen DNA(+) cell frequencies (1 in 2700 and 1 in 3800) and no white pulps containing spliced RNA. CTL effector activity as well as spliced viral messenger RNA were both concentrated in the white pulps and germinal centers. This colocalization indicates that viral replication in immunocompetent structures of secondary lymphoid organs triggers anti-HIV effector CTLs to these particular locations, providing clues to target therapeutic intervention.
Subject(s)
Germinal Center/immunology , HIV Infections/immunology , HIV-1/physiology , T-Lymphocytes, Cytotoxic/immunology , Adult , Germinal Center/virology , Humans , Male , Virus Replication/immunologyABSTRACT
The tremendous dynamics of HIV infection finds expression in the tempo of sequence diversification. Genetic diversity calculations require the clearance of a majority of infected cells, the obvious predator being anti-HIV immune responses. Indeed, infiltration of germinal centers (GCs) by HIV-specific CD8(+) cytotoxic T lymphocytes has been described. A corollary to this description would be limited diffusion of virus within lymphoid structures. HIV efficiently infects and replicates mainly in activated CD4(+) T lymphoblasts. These cells are found within GCs after their activation in the adjacent periarteriolar lymphoid sheath (PALS). Here GCs and PALS have been dissected from consecutive 10-micrometer sections through splenic tissue from three HIV-1-infected patients. Nested PCR amplification of the two first hypervariable regions of the env gene indicated that 38-78% of sections contained HIV-infected cells. Since there are several hundred CD4(+) T cells per GC section, approximately 0.09-0.64% harbor proviral DNA. Such a low frequency not only suggests that virions on the follicular dendritic cell surfaces do not readily infect adjacent T cells but also indicates highly restricted spread of HIV within GCs and the PALS. Sections were heavily infiltrated by CD8(+) cells, which, together with a large body of extant data, suggests that the majority of infected cells are destroyed by HIV-specific cytotoxic T lymphocytes before becoming productively infected. Finally, sequence analysis revealed that those HIV-positive cells were multiply infected, which helps explain widespread recombination despite a low overall frequency of infected cells.
Subject(s)
Germinal Center/virology , HIV Infections/virology , HIV-1/immunology , Spleen/virology , Amino Acid Sequence , Base Sequence , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/virology , DNA, Viral/metabolism , Genome, Viral , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Infections/immunology , HIV Infections/pathology , HIV-1/genetics , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Recombination, Genetic , Sequence Homology, Amino Acid , Spleen/cytologyABSTRACT
Somatic mutation of rearranged immunoglobulin V genes occurs in germinal centers (GC), resulting in affinity maturation of the immune response. Rearranged T cell receptor (TCR) genes were thought to be excluded from this process despite similarities in their gene structure. Somatic mutations were found among TCR V alpha (TCRAV) chains of antigen-specific T cells localized in GC of mice. Here, somatically mutated TCR V beta 3 (TCRBV) chains are identified among microdissected splenic white pulps from HIV-positive individuals. Both the frequency and the nature of the base substitutions were found to be similar to those of mutated immunoglobulin VH genes. This was true for intrinsic mutations in the TCR framework regions as well as for mutations underlying selective pressures in the TCRBV5 gene segment. The concentration of mutations and a preference for replacement mutations in complementarity determining regions of expanded clones were indicative of a positive selection process.
Subject(s)
Dissection , HIV Seropositivity/immunology , Mutation/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Spleen/metabolism , Base Sequence , Cell Line , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Germinal Center/immunology , Germinal Center/metabolism , Germinal Center/pathology , HIV Seropositivity/genetics , HIV Seropositivity/pathology , Humans , Molecular Sequence Data , Receptor-CD3 Complex, Antigen, T-Cell/genetics , Spleen/pathology , Spleen/surgery , Splenectomy , T-Lymphocytes, CytotoxicABSTRACT
The impact of antigenic stimulation on the dynamics of simian immunodeficiency virus (SIV) replication was studied following repeated intravenous BCG inoculation of a SIV infected macaque. At the site of a delayed type hypersensitivity reaction to purified protein derivative of M. tuberculosis, a distinctive SIV variant was noted, probably as a result of the infiltration of activated antigen-specific T cell clones as opposed to infection by blood borne virus in situ. The dynamics of SIV quasispecies in peripheral blood suggests sequential waves of viral replication, illustrating the role of antigenic stimulation as a driving force in viral dissemination and pathogenesis.
Subject(s)
BCG Vaccine/pharmacology , Gene Products, env/genetics , Hypersensitivity, Delayed , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/physiology , T-Lymphocytes/immunology , Virus Replication/immunology , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Eyelids , Gene Products, env/chemistry , Gene Rearrangement, T-Lymphocyte , Genetic Variation , Macaca mulatta , Molecular Sequence Data , Mycobacterium tuberculosis/immunology , Phylogeny , Polymerase Chain Reaction , Polymorphism, Genetic , Receptor-CD3 Complex, Antigen, T-Cell/genetics , Simian Immunodeficiency Virus/drug effects , Simian Immunodeficiency Virus/genetics , Virus Replication/drug effectsSubject(s)
Membrane Glycoproteins , Point Mutation , Polymerase Chain Reaction , Viral Envelope Proteins , Adenine , Amino Acid Sequence , Animals , Guanine , HIV Envelope Protein gp120/genetics , Lentivirus/genetics , Molecular Sequence Data , Nucleic Acid Heteroduplexes , Simian Immunodeficiency Virus/geneticsABSTRACT
Macrophages are often considered as a reservoir of latent infection in HIV+ patients, and their infection may indeed be very important functionally. However, some quantitative studies did not find high infection frequencies in peripheral blood monocytes. Since lymphoid organs are the major site of infection, macrophage infection was tested in spleens removed from HIV+ patients for treatment of different syndromes. Ten replicates of limiting dilutions from different cell populations were submitted to a nested PCR specific to conserved regions of HIV1 env DNA. On an average, 1/2,300 adherent cells carried HIV1 DNA (n = 7; range: 1/55,000 to 1/660). These adherent cells, obtained after two days of culture, comprised the whole macrophage population, with no biases introduced by surface molecule selection, but were not pure (41-78% macrophages). Only 1/37,000 CD14+ monocyte/macrophages were positive (n = 6; range: 1/130,000 to 1/22,000). Therefore, the infection frequency of the isolated splenic monocytes/macrophages from these patients could be estimated at between 1/37,000 and 1/2,300. In contrast, 1/60 CD4+ T lymphocytes were positive (n = 7; range: 1/190 to 1/17). Within the experimental limits, such as cell isolation, required for accurate quantification, this study in the spleen indicates, as have other studies on peripheral blood, that macrophages do not quantitatively constitute an important reservoir of HIV when compared to CD4+ T lymphocytes.
Subject(s)
HIV Infections/virology , HIV-1/growth & development , Macrophages/virology , Spleen/virology , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , DNA, Viral/analysis , Dendritic Cells/virology , Flow Cytometry , Humans , Lipopolysaccharide Receptors/biosynthesis , Polymerase Chain Reaction , Spleen/cytology , SplenectomySubject(s)
Macaca mulatta/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Animals , Base Sequence , Genes , Humans , Molecular Sequence Data , Polymorphism, Genetic , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Sequence Alignment , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
Dendritic cells (DC) are specialized antigen-presenting leukocytes that are responsible for the activation of naive as well as memory T lymphocytes. If infected by human immunodeficiency virus (HIV), DC may transfer virus to CD4+ lymphocytes. However, the question of whether DC are infected in vivo is controversial. As HIV infection is more active in secondary lymphoid organs than in blood, infection of splenic DC isolated from HIV-seropositive patients was investigated. Splenic DC were first enriched and characterized by flow cytometry from HIV- donors. After direct isolation, they were negative for monocyte and T- and B-lymphocyte markers, negative for CD1a, but positive for major histocompatibility complex class II and CD4. After in vitro maturation, major histocompatibility complex class II expression increased, while CD4 expression was lost. Extensive purification from the spleens of seven HIV+ patients was performed by fluorescence-activated cell sorting. The frequency of cells harboring HIV DNA in purified populations was quantified by limiting-dilution PCR. Directly isolated DC (average, 1/3,000; range, 1/720 to 1/18,000) were in each patient 10 to 100 times less infected than CD4+ T lymphocytes (average, 1/52; range, 1/17 to 1/190). On average, 1/1,450 (1/320 to 1/6,100) unseparated mononuclear splenocytes (containing 5% CD4+ lymphocytes) harbored HIV DNA. In conclusion, in these HIV+ patient spleens, DC seem to be infected, but HIV-DNA positive CD4+ T lymphocytes accounted for the vast majority of infected mononuclear splenocytes.
Subject(s)
CD4-Positive T-Lymphocytes/virology , Dendritic Cells/virology , HIV Seropositivity/virology , Spleen/virology , DNA, Viral/analysis , HIV/genetics , HIV/isolation & purification , HIV Seropositivity/pathology , Humans , Lymphocyte Culture Test, Mixed , Macrophages/virology , Phenotype , Polymerase Chain Reaction , Spleen/pathologyABSTRACT
Simian immunodeficiency virus (SIV) quasispecies development was followed in four monkeys (Macacca fascicularis) infected by intramuscular inoculation of phage lambda-SIVmac239 DNA. Rooted phylogenetic trees were reconstructed and used to interpret the data. The rate of fixation of base substitutions varied within and between animals reaching 3.3 x 10(-2) per site per year. These data suggest that the tempo of quasispecies development requires both massive viral replication and efficient clearance of SIV. Despite this, no significant difference was found between the observed and expected ratio of synonymous/nonsynonymous substitutions, suggesting that there was little or no selection of antigenic variants in the V1 and V2 hypervariable regions of envelope.
Subject(s)
Genetic Variation/genetics , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/physiology , Virus Replication , Amino Acid Sequence , Animals , Base Sequence , DNA, Viral/blood , Female , Macaca fascicularis , Male , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/growth & developmentABSTRACT
Human immunodeficiency virus (HIV) replication and T cell proliferation was investigated in situ by a PCR based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation, rather than the infection of CD4+ T lymphoblasts by blood borne virus or immune complexes. HIV infected white pulps could be infiltrated by HIV specific cytotoxic T lymphocytes, so implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen driven T cell recruitment and activation, HIV replication and spread, with consequent destruction of the newly infected cells.
Subject(s)
HIV Antibodies , HIV/physiology , Spleen/pathology , T-Lymphocytes, Cytotoxic , Virus Replication , Amino Acid Sequence , Antibody Specificity , Clone Cells , Dissection , Genotype , HIV/genetics , Humans , Micromanipulation , Molecular Sequence DataABSTRACT
Human immunodeficiency virus (HIV) replication and T cell proliferation were investigated in situ by a PCR-based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation rather than the infection of CD4+ T lymphoblasts by blood-borne virus or immune complexes. HIV-infected white pulps could be infiltrated by HIV-specific cytotoxic T lymphocytes, thereby implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen-driven T cell recruitment and activation, as well as HIV replication and spread, with consequent destruction of the newly infected cells.
Subject(s)
HIV Seropositivity/immunology , HIV/genetics , Spleen/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Antigen Presentation , Base Sequence , CD4-Positive T-Lymphocytes/immunology , Cell Movement , HIV/growth & development , Humans , Lymphocyte Activation , Molecular Sequence Data , Polymerase Chain Reaction , Proviruses/genetics , Proviruses/growth & development , Receptors, Antigen, T-Cell, alpha-beta/genetics , Sequence Homology, Amino Acid , Spleen/cytologyABSTRACT
A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 proviruses in an infected spleen was observed. Antigenic stimulation of infected cells might explain this partition.
Subject(s)
HIV Envelope Protein gp120/genetics , HIV Infections/microbiology , HIV-1/genetics , Proviruses/genetics , Spleen/microbiology , Amino Acid Sequence , Base Sequence , Genetic Variation , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Phylogeny , Proviruses/isolation & purification , Sequence Homology, Nucleic AcidABSTRACT
Cytotoxic T lymphocytes (CTL) are present at high activities in adult patients infected with the human immunodeficiency virus (HIV). In this report, CTL effectors were identified in peripheral blood mononuclear cells (PBMC) of children born to HIV-1-infected mothers. These CTL killed HLA-matched HIV-1-infected H9 target cells or doubly transfected P815-A2-env, gag or nef mouse tumor cells, which expressed the viral antigens in association with HLA-A1/A3 or HLA-A2, respectively. HIV-1-specific CTL were detected early after birth (less than 2 months) and remained present during the asymptomatic phase of the infection. As in HIV-1-infected adults, HIV-specific CTL declined with disease progression. Surprisingly, HIV-1-specific CTL were detected in the PBMC of three children who subsequently became seronegative.
Subject(s)
HIV Infections/immunology , HIV-1/immunology , Pregnancy Complications, Infectious/immunology , T-Lymphocytes, Cytotoxic/immunology , Base Sequence , Child , Child, Preschool , Cytotoxicity, Immunologic , Epitopes , Female , HLA-A Antigens/analysis , Humans , Infant , Infant, Newborn , Killer Cells, Natural/physiology , Molecular Sequence Data , PregnancyABSTRACT
The activity of the human immunodeficiency virus type 1 (HIV-1) transactivation protagonists tat and TAR has been analyzed from sequential primary material. The sequences were amplified from uncultured peripheral blood mononuclear cells. Despite fluctuations within the tat and TAR quasispecies there was no obvious selection for a variant encoding more powerful transactivation components either in vivo or ex vivo, indicating that this system is not exploited during disease progression. The basal levels of the natural promoters were, depending on the cell line, two- to fourfold higher than that of the reference promoter, itself derived from ex vivo adapted HIV-1 Lai.
Subject(s)
Acquired Immunodeficiency Syndrome/microbiology , Genes, tat , HIV Long Terminal Repeat , HIV-1/genetics , Amino Acid Sequence , Base Sequence , CD4-Positive T-Lymphocytes , Gene Expression Regulation, Viral , Genetic Variation , Humans , Leukocyte Count , Molecular Sequence Data , Promoter Regions, Genetic , Selection, Genetic , Time Factors , Transcription, Genetic , Transcriptional ActivationABSTRACT
Two of the first human immunodeficiency virus type-1 (HIV-1) strains isolated were authenticated by reanalyzing original cultured samples stored at the Collection Nationale de Culture des Microorganismes as well as uncultured primary material. Cloned polymerase chain reaction products were used to analyze coding sequences of the V3 loop in the gp120 glycoprotein. The original isolate HIV-1 Bru, formerly called LAV, was derived from patient BRU. HIV-1 Lai was derived from patient LAI and contaminated a HIV-1 Bru culture between 20 July and 3 August 1983. The culture became, in effect, HIV-1 Lai, identifiable by a unique motif in the V3 loop. Because of this contamination two, rather than one, HIV-1 isolates were sent to the Laboratory of Tumor Cell Biology at the National Cancer Institute on 23 September 1983. Original HIV-1 Bru was indeed present in the sample marked JBB/LAV. However the M2T-/B sample harbored HIV-1 Lai, a strain capable of growing on established cell lines. The striking similarity between HIV-1 Lai (formerly LAV-Bru) and HTLV-3B sequences remains.