ABSTRACT
The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4(+) and CD8(+) T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at -70 degrees C for < or =3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at -70 degrees C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.
Subject(s)
Blood/immunology , Freezing , Specimen Handling/methods , T-Lymphocytes/immunology , Candida/immunology , Cell Proliferation , Cell Survival , Flow Cytometry/methods , Human Experimentation , Humans , L-Selectin/analysis , Leukocyte Common Antigens/analysis , Lymphocyte Count , Lymphocyte Subsets/immunology , Pokeweed Mitogens/immunology , Tetanus/immunologyABSTRACT
BACKGROUND: Although the determinants of immune deficiency and immune restoration in chronic human immunodeficiency virus (HIV)-1 infection are not well understood, immune activation has been proposed as being central to the pathogenesis of HIV. METHODS: A randomized, controlled trial of cyclosporin A treatment for 2 weeks was performed in persons with chronic HIV-1 infection who were beginning a standardized antiretroviral therapy (ART) regimen. RESULTS: Treatment with cyclosporin A provided only a marginal and transient enhancement in circulating T cell restoration that was largely restricted to cells expressing the CCR7 chemokine receptor and that did not persist beyond 2 weeks. CONCLUSIONS: Cyclosporin A coadministered for 2 weeks with ART provided no sustained immunologic benefit to persons with chronic HIV-1 infection. If immune activation drives progressive immune deficiency in chronic HIV-1 infection, these activation pathways may not be sensitive to cyclosporin.
Subject(s)
Anti-Retroviral Agents/therapeutic use , Cyclosporine/therapeutic use , HIV Infections/drug therapy , HIV-1 , Immunosuppressive Agents/therapeutic use , Adult , Chronic Disease , Cyclosporine/administration & dosage , Drug Administration Schedule , Drug Therapy, Combination , Female , HIV Infections/immunology , Humans , Immunosuppressive Agents/administration & dosage , Lymphocyte Count , Male , Middle Aged , Receptors, Chemokine/biosynthesis , T-Lymphocytes/immunology , Treatment OutcomeABSTRACT
Older age is a strong predictor of accelerated human immunodeficiency virus (HIV) disease progression. We investigated the possible immunologic basis of this interaction by comparing older (>/=45 years) and younger (=30 years) HIV-infected adults with simultaneously enrolled, aged-matched, healthy volunteers. Cross-sectional comparisons suggested age-associated reductions in naive CD8(+) cells and in the expression of CD28(+) on CD8(+) cells among both HIV-infected subjects and control subjects. Opposite patterns of CD4(+) and CD8(+) cell differences were apparent between these subject groups. HIV infection, but not age, was associated with impairments in delayed-type hypersensitivity responses, lymphoproliferation, and spontaneous apoptosis and with alterations in expression of chemokine receptors CCR5 and CXCR4. Reduced thymic volumes were associated with age and with HIV infection among younger, but not older, subjects. Because of their common association with age and HIV disease, naive CD8(+) cell depletion, diminished CD28 expression on CD8(+) cells, and reduced thymic volumes are possible correlates of the interaction of age with HIV disease.
Subject(s)
Aging/immunology , CD28 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , HIV Infections/immunology , Thymus Gland/pathology , Adolescent , Adult , Aged , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cross-Sectional Studies , Disease Progression , Female , Gene Expression Regulation , HIV Infections/pathology , HIV-1 , Humans , Male , Middle Aged , Phenotype , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
To ascertain whether CD4(+) lymphocyte increases induced by interleukin (IL)-2 enhanced in vivo immune responses, 38 human immunodeficiency virus (HIV)-infected patients who had received highly active antiretroviral therapy (HAART) or HAART and IL-2 for at least 60 weeks were immunized with tetanus toxoid, inactivated glycoprotein 120-depleted HIV-1, and hepatitis A and B vaccines. Despite dramatic increases in CD4(+) lymphocyte counts, IL-2 did not enhance immunization responses.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , HIV Infections/immunology , Interleukin-2/pharmacology , Lymphocyte Activation/drug effects , AIDS Vaccines/immunology , Adjuvants, Immunologic/pharmacology , Adult , Anti-HIV Agents/therapeutic use , Antiretroviral Therapy, Highly Active , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , Drug Therapy, Combination , HIV Infections/drug therapy , Hepatitis A Vaccines/immunology , Hepatitis B Vaccines/immunology , Humans , Male , Tetanus Toxoid/immunologyABSTRACT
The chronically HIV-infected cellular reservoir in lymphoid tissue (LT) represents a formidable obstacle to the long-term success of antiretroviral therapy. Cytoreductive chemotherapy with cyclophosphamide (CTX) reduces cells in LT, and we hypothesized that coadministration of antiretroviral therapy with CTX may diminish the cellular reservoir over time. Ten antiretroviral treatment-naive subjects were recruited, and they received stavudine, lamivudine and nelfinavir (antiretroviral therapy, ART) until 2 consecutive plasma HIV RNA levels measured < 50 copies/ml (step 1). Five subjects then received ART alone, whereas five subjects received ART plus three escalating doses of CTX (step 2). Viral DNA was measured in LT obtained by excisional lymph node biopsy and peripheral blood mononuclear cells (PBMCs), using quantitative polymerase chain reaction at three time points in both groups (before steps 1 and 2, and after CTX). Viral DNA declined in both groups after the initiation of ART alone in step 1. During step 2 both groups experienced a modest decline compared with step 1. However, no significant differences were observed in viral DNA in LT or PBMCs between the ART alone and the ART plus CTX groups. Suppression of plasma HIV RNA levels < 50 copies/ml was not maintained in the ART plus CTX group, perhaps because of inadequate medication adherence. The group receiving ART plus CTX had lower CD4(+) lymphocyte counts and absolute total lymphocytes compared with the ART alone group. We conclude that the addition of CTX to ART did not diminish the cellular reservoir in HIV-infected persons.