Thomas W. McCloskey

CD8+ T Cells in HIV Disease Exhibit Cytokine Receptor Perturbation and Poor T Cell Receptor Activation but Are Responsive to γ-Chain Cytokine–Driven Proliferation

BACKGROUND Cytokines are important for inducing T cell maturation, proliferation, and survival. Despite the known dysregulation of cytokines in human immunodeficiency virus (HIV) infection, cytokine receptor expression is relatively unexplored. METHODS We examined maturation markers (naive, central memory, effector memory, and effector); the cytokine receptors interleukin (IL)-2R beta , common gamma (C gamma ) chain, IL-7R alpha , IL-15R alpha; and proliferative responses of T cells in a cohort of HIV-infected pediatric patients (median age, 14.82 years) receiving antiretroviral therapy, arbitrarily designated as immunologic responders (group I) and nonresponders (group II) on the basis of a CD4+ T cell count cutoff of 25%. RESULTS Patients had increased percentages of effector memory CD8+ T cells, in comparison with those in healthy control subjects, with reduced expression of IL-7R alpha in the central memory and effector memory subsets and of the C gamma chain in all maturation subsets of CD8+ T cells. IL-7R alpha +CD8+ T cell percentages were directly correlated with CD4+ T cell percentages. In immunologic nonresponders, anti-CD3+ or HIV Gag antigen-induced CD8+ T cell proliferation was impaired, but proliferation in response to the homeostatic cytokines IL-2 and IL-15 was preserved.Conclusions. Cytokine receptor deficiencies may contribute to immune deficiency in HIV-infected patients, and gamma -chain-utilizing cytokines may play an important role in vivo in maintaining the memory subsets of T cells in patients with CD4+ T cell deficiency.

Alterations in Apoptosis of Cord and Adult Peripheral Blood Mononuclear Cells Induced by In Vitro Infection with Respiratory Syncytial Virus

Respiratory syncytial virus (RSV), a major cause of morbidity in children, results in severe lower respiratory tract infections. With an in vitro infection system of isolated cord or adult peripheral blood mononuclear cells, addition of virus to cell cultures resulted in significant reductions in cell deaths, as measured by 2 independent assays: quantitation of cells with subdiploid levels of DNA and cells with DNA strand breaks. Decreased cell death was observed in lymphocytes and monocytes of cord and adult samples, with more dramatic effects evident in cells from cord blood. This may be linked to the increased virulence observed in infants with RSV infection. These data suggest that RSV may be equipped with some mechanism to prevent apoptosis, which is a major component of the host defense system used to eliminate virally infected cells.

Discordant expression of perforin and granzyme A in total and HIV-specific CD8 T lymphocytes of HIV infected children and adolescents

Objective: Perforin and granzyme are cytotoxic effector molecules that are believed to play essential roles in cytotoxic T cell (CTL) activity. We tested the hypothesis that dysregulation of these effector molecules contributes to defects of CD8 antiviral immune responses in pediatric subjects in chronic stages of perinatal HIV infection. Design/Method: Studies of CD8 T cells were conducted in 33 treatment experienced HIV+ patients (median age, 10.6 years) and in 14 age-matched healthy controls. CD8 T cells specific for HIV Gag and Pol peptides were identified in HLA-A2+ patients by tetramer binding assays. HIV-specific and total CD8 T cells were examined for perforin, granzyme and expression of CD27, a marker that is lost in terminally differentiated cells. Results: Three populations of CD8 T cells were identified: granzyme+ perforin+; granzyme+ perforin− and cells negative for both perforin and granzyme. In HIV infected patients, granzyme+ cells were increased in total CD8 T cells (39% versus 13% in controls) and were highest in HIV Gag-specific CD8 cells (42%). Perforin+ CD8 T cells were approximately fivefold fewer than granzyme+ CD8 T cells and were enriched in CD27 negative cells. Most HIV-specific CD8 cells were CD27+. Granzyme expression in CD8 T cells correlated negatively with CD4 percentage and positively with virus load. Conclusion: A disproportionate and generalized increase in CD27+, granzyme+, CD8 T cells is a hallmark of established pediatric HIV infection. These findings support the concept of skewed maturation, with failure of CD8 T cells to mature into perforin-enriched, CD27-negative, effector cells.

Alterations in T-Cell Receptor Vβ Repertoire of CD4 and CD8 T Lymphocytes in Human Immunodeficiency Virus-Infected Children

ABSTRACT Perturbations in the T-cell receptor (TCR) Vβ repertoire were assessed in the CD4 and CD8 T lymphocytes of human immunodeficiency virus (HIV)-infected children who were receiving therapy during the chronic phase of infection by flow cytometry (FC) and PCR analysis. By FC, representation of 21 TCR Vβ subfamilies was assessed for an increased or decreased percentage in CD4 and CD8 T cells, and by PCR, 22 TCR Vβ subfamilies of CD4 and CD8 T cells were analyzed by CDR3 spectratyping for perturbations and reduction in the number of peaks, loss of Gaussian distribution, or clonal dominance. The majority of the TCR Vβ subfamilies were examined by both methods and assessed for deviation from the norm by comparison with cord blood samples. The CD8-T-lymphocyte population exhibited more perturbations than the CD4 subset, and clonal dominance was present exclusively in CD8 T cells. Of the 55 total CD8-TCR Vβ families classified with clonal dominance by CDR3 spectratyping, only 18 of these exhibited increased expression by FC. Patients with high numbers of CD8-TCR Vβ families with decreased percentages had reduced percentages of total CD4 T cells. Increases in the number of CD4-TCR Vβ families with increased percentages showed a positive correlation with skewing. Overall, changes from normal were often discordant between the two methods. This study suggests that the assessment of HIV-induced alterations in TCR Vβ families at cellular and molecular levels yields different information and that our understanding of the immune response to HIV is still evolving.

Apoptosis in T-Lymphocyte Subsets in Human Immunodeficiency Virus-Infected Children Measured Immediately Ex Vivo and following In Vitro Activation

ABSTRACT Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.

Response to Superantigen Stimulation in Peripheral Blood Mononuclear Cells from Children Perinatally Infected with Human Immunodeficiency Virus and Receiving Highly Active Antiretroviral Therapy

ABSTRACT Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-γ) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-γ-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.

T cell receptor Vβ repertoire of the antigen specific CD8 T lymphocyte subset of HIV infected children

ObjectiveAnalysis of the T cell receptor Vβ repertoire during HIV infection reveals expansions in multiple Vβ families of CD8 T cells, but their antigenic specificity is ill-defined. We sought to determine the TCR Vβ repertoire of HIV specific CD8 T lymphocytes in infected children. Design/methodsWe performed flow cytometry to examine TCR Vβ families as identified by specific monoclonal antibodies and binding of HIV peptide loaded tetrameric MHC complexes in peripheral blood samples from a group of HIV infected children. ResultsSimultaneous assessment of 12 selected expanded Vβ families amongst nine HIV infected patients for tetramer binding revealed only one child in whom the expanded Vβ population bound HIV Gag or Pol tetramers. In four HIV infected children, percentage tetramer binding cells was determined in 21 TCR Vβ families. The tetramer binding cells of three children exhibited a widely distributed TCR Vβ repertoire while in the fourth patient they were preferentially localized within two TCR Vβ families. Repeat analysis revealed that the TCR Vβ repertoire of tetramer binding cells was stable. ConclusionsThese data provide evidence that the HIV-specific CD8 T cell response in children is usually distributed widely among many different TCR Vβ families. The heterogeneity of the TCR Vβ repertoire usage by the antigen specific CD8 T cells may reflect the dynamic interaction between host and pathogen during the course of HIV infection and may be influenced by the rate of viral mutation, CD4 T cell helper activity, or other factors.

Responses of Monocytes in Peripheral Blood Mononuclear Leukocytes (PBMLs) Exposed to Respiratory Syncytial Virus (RSV) In Vitro † 868

Responses of Monocytes in Peripheral Blood Mononuclear Leukocytes (PBMLs) Exposed to Respiratory Syncytial Virus (RSV) In Vitro † 868

TUNEL Assay Quantification of Apoptosis in Peripheral Blood Mononuclear Leukocytes Exposed to Respiratory Syncytial Virus (RSV)

TUNEL Assay Quantification of Apoptosis in Peripheral Blood Mononuclear Leukocytes Exposed to Respiratory Syncytial Virus (RSV)