Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.     

Rate of HIV-1 decline following antiretroviral therapy is related to viral load at baseline and drug regimen

OBJECTIVES AND DESIGN: The dynamics uf viral decline following the initiation of antiretroviral treatment were studied in 29 HIV-1-infected patients participating in a two-arm trial comparing immediate (group A: ritonavir, zidovudine and lamivudine) and delayed (group B: ritonavir supplemented by zidovudine and lamivudine on day 21) triple therapy. Parameters underlying viral dynamics were estimated using mathematical models tailored to these treatment protocols. RESULTS: The decline in plasma HIV-1 density between day 0 and 21 was steeper in group A (-2.27+/- 0.46 log10) than group B (-1.87+/-0.56 log10). In a subset of patients amenable to full mathematical analysis, a short-lived productively infected cell compartment (producing approximately 97% of total virions) decayed with a half-life of 1.0-2.5 days, whereas a long-lived infected cell compartment decayed with a half-life of 18.8-32.8 days. Estimates for the time for the elimination of virus from these two cell populations ranged from 474 to 802 days. The rate of loss of productively infected CD4+ T cells was positively correlated with baseline viral load in group A and in the combined dataset. CONCLUSIONS: These results suggest that HIV-infected cell populations may have a faster turnover in patients with higher viral loads due to higher infection rate parameters, higher rates of virus production, or lower virus clearance rates.     

Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.     

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen-driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.     

CD45RA(bright)/CD11a(bright) CD8+ T cells: effector T cells

An important aspect of peripheral T cell development is the differentiation from naive into memory cells. To distinguish naive from memory cells, CD45RA and CD11a are commonly used: CD45RA+ or CD11a(dim) T cells are regarded as naive, while CD45RA- or CD11a(bright) T cells are thought to be of memory type. There is, however, a CD8+ T cell subset which is CD45RA+ and at the same time CD11a(bright). It increases with age and in patients with systemic viral infections, though its functional role in the immune response is unknown. In the present study, we give evidence that this subset is related to memory-like T cells as it produces IFN-gamma and tumor necrosis factor-alpha, contains high levels of perforin, and expresses CD95 in the same way as memory-type CD45RA-/CD11a(bright) CD8+ T cells. Since it contains a high percentage of CD28- and CD57+ cells, is increased in size and granularity, and is transiently expressed following in vitro stimulation of naive CD8+ T cells, we speculate that this subset mainly represents recently activated effector T cells that are able to interact with CD80 and CD86 (B7-1 and B7-2 respectively) negative tissue cells.     

Rapid clearance of simian immunodeficiency virus particles from plasma of rhesus macaques

Perturbation of the equilibrium between human immunodeficiency virus type 1 (HIV-1) and the infected host by administering antiretroviral agents has revealed the rapid turnover of both viral particles and productively infected cells. In this study, we used the infusion of simian immunodeficiency virus (SIV) particles into rhesus macaques to obtain a more accurate estimate of viral clearance in vivo. Consistently, exogenously infused virions were cleared from plasma with an extremely short half-life, on the order of minutes (a mean of 3.3 min). This new estimate is approximately 100-fold lower than the upper bound of 6 h previously reported for HIV-1 in infected humans. In select animals, multiple tissues were collected at the completion of each experiment to track the potential sites of virion clearance. Detectable levels of SIV RNA were found in lymph nodes, spleen, lungs, and liver, but not in other tissues examined. However, only approximately 1 to 10% or less of the infused virions were accounted for by the thorough tissue sampling, indicating that the vast majority of the infused particles must have been degraded over a short period of time. Should the rapid clearance of virions described here be applicable to infected patients, then HIV-1 production and thus the number of productively infected CD4(+) T lymphocytes or the viral burst size must be proportionally higher than previous minimal estimates.     

Differential expression of CD40 ligand on T cell subsets. Implications for different roles of CD45RA+ and CD45RO+ cells in IgE production

Physical contact between human T lymphocytes and B lymphocytes is required for the induction of IgE production. In the present study, we examined the abilities of CD45RA+ and CD45RO+ human T cell subsets to provide help for IgE production by human peripheral blood B cells in the presence of IL-4. Purified peripheral CD45RA+ T cells are much better inducers of IgE synthesis than are CD45RO+ T cells. Activation of CD45RA+ T cells, but not CD45RO+ T cells, via the TCR/CD3 complex is sufficient to confer the ability to provide IgE help, suggesting that an inducible T cell surface molecule plays an important role in this system. The CD40 ligand, an inducible T cell surface molecule, is expressed at higher levels on CD45RA+ T cells as compared with CD45RO+ T cells following CD3-stimulation. Blocking of the CD40-CD40 ligand interaction in vitro by the addition of a soluble form of B cell CD40 Ag completely blocks IgE production induced by CD45RA+ T cells. Finally, the in vitro conversion of CD45RA+ T cells to the CD45RO+ phenotype is accompanied by a loss in the ability of these cells to express the CD40 ligand in response to anti-CD3 stimulation as well as a loss in their ability to provide IgE help. These results suggest that both CD45 subsets may play significant and distinct roles in the induction of IgE production under physiologic conditions: CD45RO+ T cells provide IL-4 and the CD45RA+ subset provides the second signal via the CD40 ligand.     

High prevalence of thymic tissue in adults with human immunodeficiency virus-1 infection

The thymus in adults infected with the HIV-1 is generally thought to be inactive, both because of age-related involution and viral destruction. We have revisited the question of thymic function in adults, using chest-computed tomography (CT) to measure thymic tissue in HIV-1-seropositive (n = 99) or HIV-1-seronegative (n = 32) subjects, and correlating these results with the level of circulating CD4(+) and CD8(+) T cells that are phenotypically described as naive thymic emigrants. Abundant thymic tissue was detectable in many (47/99) HIV-1-seropositive adults, aged 20-59. Independent of age, radiographic demonstration of thymic tissue was significantly associated with both a higher CD4(+) T cell count (P = 0.02) and a higher percentage and absolute number of circulating naive (CD45RA+CD62L+) CD4(+) T cells (P < 0.04). The prevalence of an abundant thymus was especially high in younger HIV-1-seropositive adults ( 40 yr) regardless of CD4 count (P = 0.03). These studies suggest that the thymus is functional in some but not all adults with HIV-1 disease.     

T cell responses to heat-shock protein 60: differential responses by CD4+ T cell subsets according to their expression of CD45 isotypes

We demonstrate that human T lymphocytes proliferate in vitro to highly purified human heat-shock protein 60 (Hu.hsp60). The response to this self Ag was confined to the CD45RA+ RO- T cell subset, with minimal responses by adult CD45RA- RO+ T cells. Experiments using keyhole limpet hemocyanin as a prototypic novel Ag, or tetanus toxoid as a recall Ag, were consistent with the notion that CD45RA+ RO- and CD45RA- RO+ T cell subsets can be designated as naive and memory cells, respectively; thus, responses to Hu.hsp60 were confined to the putative naive subset. In contrast, both CD45RA+ RO- and CD45RA- RO+ T cell populations proliferated to bacterial hsp60 from Mycobacterium leprae, Escherichia coli, or Chlamydia trachomatis. However, only CD45RA- RO+ (memory) T cells responded to a mycobacterial hsp60-derived peptide previously defined as a major bacteria-specific epitope. Experiments with cord blood T cells, which are CD45RA+ RO- and can be considered truly naive, showed that the peptide could elicit responses from naive T cells in vitro; cord blood cells also responded to Hu.hsp60. Since bacterial hsp60 Ags contain both conserved and nonconserved epitopes, we speculate that in vivo challenge with bacterial hsp60 will activate T cells capable of seeing either type of epitope, but only those that see nonconserved epitopes maintain the CD45RA- RO+ memory phenotype. However, T cells recognizing conserved epitopes, while not apparently being recruited to the memory pool, may nevertheless play a role in immunoregulation, particularly in the context of inflammation, when expression of Hu.hsp60 is increased.     

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor zeta chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.     

Productive infection of neonatal CD8+ T lymphocytes by HIV-1

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.     

Infection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy

Children affected with human immunodefficiency virus (HIV)-associated nephropathy (HIVAN) usually develop significant renal glomerular and tubular epithelial cell injury. The pathogenesis of these changes is not clearly understood. Human renal tubular epithelial cells (RTEc) do not express CD4 surface receptors, and it is not clear whether these cells can be infected by HIV-1. Certain strains of HIV-1, however, have been shown capable of infecting CD4-negative epithelial cell lines. We hypothesized that the inability of laboratory strains of HIV-1 to infect renal epithelial cells may be due to a limited tropism, as opposed to wild-type viruses derived from children with HIVAN, and that viruses derived from these children are capable of infecting RTEc from the same patient. Here, we have demonstrated that HIV-1 isolates from children with HIVAN can productively infect RTEc through a CD4 independent pathway, and that infected mononuclear cells can transfer the virus to human RTEc. Human RTEc sustained low levels of viral replication and HIV-1 inhibited the growth and survival of cultured human RTEc. Thus, HIV-1 may directly induce degenerative changes in RTEc of children with HIVAN. Infected macrophages may play a relevant role in this process by transferring viruses to RTEc.     

cDNA encoding a single-chain antibody to HIV p17 with cytoplasmic or nuclear retention signals inhibits HIV-1 replication

HIV-1 gag p17 protein is an attractive target for molecular intervention, because it is involved in the viral replication cycle at both the pre- and postintegration levels. In the present experiments, we targeted p17 by intracellularly expressing a cDNA encoding an Ab to p17. cDNA from a hybridoma-secreting Ab to p17 was cloned, sequenced, reconstructed as a single-chain Ab fragment (scFv), and expressed in the cytoplasm or nucleus with appropriate retention signals. The expressed scFvs had no effect on T cell growth or CD4 expression and bound specifically to HIV-1 p17. Human CD4+ Jurkat T cells that expressed scFvs and were infected with HIV-1 showed a marked reduction in virus replication compared with cells expressing vector alone. The inhibition of virus replication was more pronounced when scFvs were expressed in the cytoplasm rather than the nucleus. From these studies, we conclude that the intracellular expression of a single-chain Ab to p17 inhibits HIV replication; in addition, the degree of inhibition is related to the intracellular targeting site.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

In vitro responses of human CD45R0brightRA- and CD45R0-RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0brightRA- or CD45R0(-)-RAbright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0-RAbright T cells, which converted to CD45R0brightRA- after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0brightRA- T cells compared to the CD45R0-RAbright subset. The poor responses of CD45R0-RAbright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0-RAbright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0+RA+). Alternatively, if primed CD45R0-RAbright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0bright and CD45RAbright expression identify memory and naive CD4+ T cells, respectively.     

Structure of chain d of the gigantic hemoglobin of the earthworm

Phenotypic and functional properties of gammadelta T cells, which play an important role in mucocutaneous immunity, were examined to elucidate whether immunological abnormality in Behcet's disease may be related to a specific T cell population. We found that CD45RA+ Vgamma9+ Vdelta2+ gammadelta T cells, which constitute a minor population of gammadelta T cells in healthy individuals, were increased in number in Beh?et's disease irrespective of disease activity. This CD45RA+ subset of gammadelta T cells in the active, but not inactive, phase of this disease expressed IL-2Rbeta and HLA-DR, suggesting that they are activated in vivo in active Beh?et's disease. In addition, the CD45RA+ gammadelta T cells produced extreme amounts of tumour necrosis factor and contained perforin granules. These data indicate that a phenotypically distinct subset of gammadelta T cells, CD45RA+ CD45RO- Vgamma9+ Vdelta2+, may contribute to immunological abnormalities which may lead to complexity of pathophysiology in Beh?et's disease.     

CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue

The human chemokine receptors CCR5 and CXCR4 have emerged as the predominant cofactors, along with CD4, for cellular entry of HIV-1 in vivo whereas the contribution of other chemokine receptors to HIV disease has not been yet determined. CCR5-specific (R5) viruses predominate during primary HIV-1 infection whereas viruses with specificity for CXCR4 (R5/X4 or X4 viruses) often emerge in late stages of HIV disease. The evolution of X4 viruses is associated with a rapid decline in CD4+ T cells, although a causative relationship between viral tropism and CD4+ T cell depletion has not yet been proven. To rigorously test this relationship, we assessed CD4+ T cell depletion in suspensions of human peripheral blood mononuclear cells and in explants of human lymphoid tissue on exposure to paired viruses that are genetically identical (isogenic) except for select envelope determinants specifying reciprocal tropism for CXCR4 or CCR5. In both systems, X4 HIV-1 massively depleted CD4+ lymphocytes whereas matched R5 viruses depleted such cells only mildly despite comparable viral replication kinetics. These findings demonstrate that the coreceptor specificities of HIV-1 are a causal factor in CD4+ T cell depletion ex vivo and strongly support the hypothesis that the evolution of viral envelope leading to usage of CXCR4 in vivo accelerates loss of CD4+ T cells, causing immunodeficiency.