Thymic function is a major determinant of onset of antibody-mediated rejection in heart transplantation.

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.

Antigenic stimulation by BCG vaccine as an in vivo driving force for SIV replication and dissemination.

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.

Long-term cultures of HTLV-III--infected T cells: a model of cytopathology of T-cell depletion in AIDS.

Long-term cultures were established of HTLV-III-infected T4 cells from patients with the acquired immune deficiency syndrome (AIDS) and of T4 cells from normal donors after infection of the cells in vitro. By initially reducing the number of cells per milliliter of culture medium it was possible to grow the infected cells for 50 to 60 days. As with uninfected T cells, immunologic activation of the HTLV-III-infected cells with phytohemagglutinin led to patterns of gene expression typical of T-cell differentiation, such as production of interleukin-2 and expression of interleukin-2 receptors, but in the infected cells immunologic activation also led to expression of HTLV-III, which was followed by cell death. The results revealed a cytopathogenic mechanism that may account for T4 cell depletion in AIDS patients and suggest how repeated antigenic stimulation by infectious agents, such as malaria in Africa, or by allogeneic blood or semen, may be important determinants of the latency period in AIDS.

LAV revisited: origins of the early HIV-1 isolates from Institut Pasteur.

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.

Evidence for HTLV-III in T-cells from semen of AIDS patients: expression in primary cell culture, long-term mitogen-stimulated cell cultures, and cocultures with a permissive T-cell line.

The development of acquired immunodeficiency syndrome or of acquired immunodeficiency syndrome-related complex by transmission of human T-lymphotropic retrovirus III by semen has previously been implicated by epidemiological studies. In vitro investigations were performed on mononuclear cells obtained from the semen of patients with acquired immunodeficiency syndrome to identify human T-lymphotropic retrovirus III or related retrovirus. The presence of human T-lymphotropic retrovirus III was demonstrated (a) in primary cell cultures, by the detection of the Mr 24,000 protein by indirect immunofluorescence assays by Day 6; (b) in activated long-term cell culture by reverse transcriptase activity, by indirect immunofluorescence (Mr 24,000 protein); and (c) in cocultures of T-cells from semen of AIDS patients and H9 cells by reverse transcriptase activity, indirect immunofluorescence, and the presence of virus particles by electron microscopy.

Quantification of T cell receptor rearrangement excision circles to estimate thymic function: an important new tool for endocrine-immune physiology.

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.

HIV-1 expression by T8 lymphocytes after transfection.

Acquired immune deficiency syndrome (AIDS) is an immunosuppressive disease associated with the depletion of T4 lymphocytes. Recently, an HIV-1 genome was molecularly cloned and shown to be fully infectious in vitro by transfection experiments. In this study, we show that HIV-1 can be transfected into T4 and T8 lymphocyte subpopulations and viral proteins and infectious virus particles are observed in both short- and long-term cultures. In addition, transfected T8 cells can be maintained in culture for long periods without apparent cytopathic effect.

Clonal expansion of T cells and HIV genotypes in microdissected splenic white pulps indicates viral replication in situ and infiltration of HIV-specific cytotoxic T lymphocytes.

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.

Persistence of restricted CD4 T cell expansions in SIV-infected macaques resistant to SHIV89.6P superinfection.

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.

Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro.

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A. Meyerhans, R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjö, and S. Wain-Hobson, Cell 58:901-910, 1989). Once again in vitro culture resulted in the selection of minor forms. Over a 4-year period in vivo, there was no obvious selection for, or outgrowth of, any particular nef or U3/R sequence. Few defective nef protein sequences were observed, which argues against nef acting as a negative regulatory factor. Although no functionally defective promoter/trans-activation-responsive elements were identified, the transactivation efficiencies varied between 0.2 and 2 times that of the control. The sequence encoding the most efficient trans-activation-responsive region did not outgrow others. The extreme genetic heterogeneity of the different samples of the locus, either in vivo or in vitro, indicates that there is no such thing as a single, distinct HIV sequence. It is suggested that different HIV-1 loci evolve independently, recombination being responsible for their uncoupling.

Nonhomogeneous distribution of human immunodeficiency virus type 1 proviruses in the spleen.

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.

Absence of selection of HIV-1 variants in vivo based on transcription/transactivation during progression to AIDS.

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.

Somatic hypermutation of the T cell receptor V beta gene in microdissected splenic white pulps from HIV-1-positive patients.

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.

Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers.

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.

The tempo and mode of SIV quasispecies development in vivo calls for massive viral replication and clearance.

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.

Genetic organization of a chimpanzee lentivirus related to HIV-1.

Simian immunodeficiency viruses have been isolated from four species of monkey, the 'captive' macaque and mangabey and the 'feral' African green monkey and mandrill. While none of these viruses is a replica of HIV-1, the macaque and mangabey viruses represent correct genetic models for HIV-2, possessing exactly the same complement of genes. Recently a lentivirus has been identified in two wild chimpanzees (Pan troglodytes troglodytes) in Gabon, west equatorial Africa, and isolated from one of them. This virus is referred to as SIVCPZ. Sera from these animals cross reacted with all the HIV-1 proteins including the envelope glycoproteins. Here, we describe the molecular cloning and sequencing of an infectious proviral clone of SIVCPZ. The overall genetic organization was the same as that of HIV-1, but phylogenetic analysis revealed that the sequence was more divergent than any HIV-1 sequence reported so far. The vpu gene product, found only in the type 1 viruses, was particularly different (64% divergent to HIV-1BRU) suggesting that the SIVCPZ represents a distinct subtype. These findings indicate that there is a larger pool of simian lentiviruses than previously suspected and revives debate as to the origins of HIV-1.

The infiltration kinetics of simian immunodeficiency virus-specific T cells drawn to sites of high antigenic stimulation determines local in vivo viral escape.

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.

Infection frequency of dendritic cells and CD4+ T lymphocytes in spleens of human immunodeficiency virus-positive patients.

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.