Patterns of CD8 T cell clonal dominance in response to change in antiretroviral therapy in HIV-infected children

Kharbanda, Monica; Than, Soe; Chitnis, Vivek; Sun, Mingzhe; Chavan, Surendra; Bakshi, Saroj; Pahwa, Savita

doi:10.1097/00002030-200010200-00003

Cited by 10 publications

(13 citation statements)

References 32 publications

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“…In the HIV-infected children studied, marked distortions were observed in TCR V␤ repertoire in CD4 and CD8 T cells, with more perturbations in CD8 compared to CD4 T cells, which is in concordance with previous reports (9,13,21,22). While increases in the percentages of particular TCR V␤ families were detected by FC in CD4 and CD8 T cells, clonal dominance was exclusively present in the CD8-T-cell compartment (25), which has been previously shown to result from oligoclonal expansions by sequence analysis (12). A possible explanation lies in the robustness and kinetics of the response of each subpopulation subsequent to antigen encounter, as it has been demonstrated that the initial proliferation of CD8 cells is of greater magnitude than CD4 cells and of longer duration (13); a recent investigation of pediatric HIV infection found persistent high-level TCR V␤-specific expansions only within the CD8 subpopulation (22).…”

Section: Discussionsupporting

confidence: 91%

“…The term skewing was used for TCR V␤ families that did not show a Gaussian distribution or had a reduction in the number of peaks to Ͻ5 (28). The term clonal dominance was used for TCR V␤ families that contained a peak whose area was Ͼ50% of the total area for that family (6,12,25).…”

Section: Methodsmentioning

confidence: 99%

“…RNA (1 to 5 g) was reverse transcribed with Moloney murine leukemia virus reverse transcriptase enzyme (Gibco BRL, Grand Island, N.Y.) and TCR ␤-chain C region primer (C␤14). Multiplex PCR was performed with forward V␤ primers for TCR V␤ family 1 (V␤1), -2, -3, -4, -5.1, -5.2, -6, -7, -8, -9, -11, -12, -13.1, -13.2, -14, -15, -16, -17, -18, -20, -21, -22, -23, and -24 and 32 P-labeled C␤R reverse primer in the presence of AmpliTaq Gold DNA polymerase (PerkinElmer, Branchburg, N.J.) as described previously (12,25). V␤10 and -19 were not analyzed, as they are pseudogenes (3).…”

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confidence: 99%

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

Kharbanda

McCloskey

Pahwa

et al. 2003

Clin Vaccine Immunol

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Perturbations in the T-cell receptor (TCR) V␤ 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 V␤ subfamilies was assessed for an increased or decreased percentage in CD4 and CD8 T cells, and by PCR, 22 TCR V␤ 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 V␤ 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 V␤ families classified with clonal dominance by CDR3 spectratyping, only 18 of these exhibited increased expression by FC. Patients with high numbers of CD8-TCR V␤ families with decreased percentages had reduced percentages of total CD4 T cells. Increases in the number of CD4-TCR V␤ 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 V␤ families at cellular and molecular levels yields different information and that our understanding of the immune response to HIV is still evolving.The majority of peripheral blood CD4 and CD8 T cells express the ␣␤ T-cell receptor (TCR), with the ␤ chain represented by variable segments which are grouped into families based on sequence homology (16). A complete T-cell repertoire is indicative of an intact T-cell population with the potential to recognize a wide range of immunogens. Numerous reports have documented changes in the TCR V␤ repertoire during human immunodeficiency virus (HIV) infection in relation to disease progression and the effect of therapy (4,5,(8)(9)(10)(19)(20)(21)(22)(23). CDR3 length spectratyping (8,19,21) and flow cytometric (FC) analysis of TCR V␤ families labeled with specific monoclonal antibodies (MAbs) (4,7,26) are among the most frequently used assays for the analysis of TCR V␤ repertoire in HIV infection. The CDR3 spectratype is an indicator of the relative proportion of cells in each TCR V␤ family with CDR3 of particular lengths, while labeling of cells with TCR-V␤-specific MAbs provides a quantitative assessment of the percentages of particular TCR V␤ families in T cells. Thus, evaluation of the TCR V␤ repertoire with MAbs, in combination with CDR3 spectratyping, is expected to provide complementary information. In the present study, we analyzed the TCR V␤ repertoire in the CD4 and CD8 T cells of 22 HIV-infected children by CDR3 length analysis and by FC. MATERIALS AND METHODSPatient population. TCR V␤ repertoire analyses by PCR and FC were performed concurrently in 22 HIV-infected c...

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Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Kharbanda

McCloskey

Pahwa

et al. 2003

Clin Vaccine Immunol

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“…The cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cell samples were isolated by standard Ficoll-Hypaque gradient centrifugation. CD4 and CD8 T cells were separated from cord blood mononuclear cells and peripheral blood mononuclear cells by positive selection using immunomagnetic beads coated with human anti-CD4 and anti-CD8 monoclonal antibodies (Dynal Corporation, Great Neck, NY), as previously described (2,11,19).…”

Section: Methodsmentioning

confidence: 99%

“…In this previously described conventional method (2,11,19), 12 reaction tubes were used with two to three primers in each tube. Multiplex PCRs were carried out as before on a PTC-225 Peltier thermal cycler.…”

Section: Methodsmentioning

confidence: 99%

Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

Fernandes

Chavan

Chitnis

et al. 2005

Clin Vaccine Immunol

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Rationale: evaluation of the T-cell receptor (TCR) V␤-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V␤ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V␤ primers instead of the conventionally labeled downstream C␤ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C␤ primer and an Omniscript RT kit. The 24 V␤ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V␤ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C␤ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: V␤ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V␤ primers. The resolution was superior to that obtained with the labeled C␤ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C␤ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor V␤-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V␤ repertoire analyses in clinical trials.

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Dissociation of CD4+ cell counts from viral load and association with immune complexes in HIV+ hemophilia patients

et al. 2004

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Patterns of CD8 T cell clonal dominance in response to change in antiretroviral therapy in HIV-infected children

Abstract: Children with chronic HIV infection manifest CD8 T cell clonal dominance, which appears to be dependent upon the adequacy of the CD4 cells. With optimization of therapy, a gain in clonal dominance is the predominant response, except in situations of failure to contain viral replication.

Cited by 10 publications

References 32 publications

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

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

Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

Dissociation of CD4+ cell counts from viral load and association with immune complexes in HIV+ hemophilia patients

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