Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency.

Waldrop, Shar L.; Pitcher, Christine J.; Peterson, Dolores M.; Maino, Vernon C.; Picker, Louis J.

doi:10.1172/jci119338

Cited by 518 publications

(365 citation statements)

References 49 publications

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“…To test cell subsets for cytokines and chemokine production, a cytokine flow cytometry assay was employed to detect either CD3 + , CD4 + and/or CD8 + T lymphocytes that produced cytokines (IFN-c/TNF-a/MIP-1b) in response to mitogen stimulation according to methods described previously [48,49]. Briefly, fresh heparinized PBMC or jejunum LPL were resuspended at 1Â10 6 cells/mL in complete RPMI-10 and stimulated with PMA (50 ng/mL) and ionomycin (1 lg/mL).…”

Section: Cytokine Flow Cytometry Assaymentioning

confidence: 99%

Intestinal double‐positive CD4⁺CD8⁺ T cells are highly activated memory cells with an increased capacity to produce cytokines

2006

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Peripheral blood and intestinal CD4 + CD8 + double-positive (DP) T cells have been described in several species including humans, but their function and immunophenotypic characteristics are still not clearly understood. Here we demonstrate that DP T cells are abundant in the intestinal lamina propria of normal rhesus macaques (Macaca mulatta). Moreover, DP T cells have a memory phenotype and are capable of producing different and/or higher levels of cytokines and chemokines in response to mitogen stimulation compared to CD4 + single-positive T cells. Intestinal DP T cells are also highly activated and have higher expression of CCR5, which makes them preferred targets for simian immunodeficiency virus/HIV infection. Increased levels of CD69, CD25 and HLA-DR, and lower CD62L expression were found on intestinal DP T cells populations compared to CD4 + single-positive T cells. Collectively, these findings demonstrate that intestinal and peripheral blood DP T cells are effector cells and may be important in regulating immune responses, which distinguishes them from the immature DP cells found in the thymus. Finally, these intestinal DP T cells may be important target cells for HIV infection and replication due to their activation, memory phenotype and high expression of CCR5. IntroductionCD4 + CD8 + double positive (DP) T cells in the intestine have been shown to be a major early target in primary simian immunodeficiency virus (SIV) infection [1]. Rapid depletion of intestinal DP cells occurs faster and more profoundly than that of intestinal CD4 + singlepositive (SP) cells which we have previously correlated with increased levels of CCR5 expression on the former [1]. Although DP T cells have been described in the blood and intestine of humans, nonhuman primates, swine and rodents [2][3][4][5][6][7][8][9][10][11][12], little is known regarding their function or immunophenotypic (naive versus memory status etc.) characteristics. DP T cells are perhaps best known in the thymus, where they are T cell precursors in early states of T cell maturation. However, DP T cells in the peripheral blood have been shown to differ from those in the thymus [2].Several investigators have reported the presence of circulating blood DP T cells, in both healthy [13,14] and diseased individuals [15][16][17][18][19][20][21][22]. Existence of this unconventional and rare lymphocyte population in peripheral blood has been hypothesized to result from premature release of immature CD4 + CD8 + T cells from the thymus to the periphery, where their maturation into functionally competent SP cell continues [23]. However, in HIV and Epstein-Barr virus infections, the percentage of DP T cells can increase to 20% of circulating lymphocytes, suggesting they are effector cells responding to infection [14,15]. In humans and animals DP T cells have also been shown to have antiviral activity, further suggesting they are indeed antigen-specific effector cells rather than immature cells from the thymus [2, 4,24]. The intestine is the major target organ of HIV...

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Section: Cytokine Flow Cytometry Assaymentioning

confidence: 99%

Intestinal double‐positive CD4⁺CD8⁺ T cells are highly activated memory cells with an increased capacity to produce cytokines

2006

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“…However, to access this extremely small population of antigenspecific T cells within the large population of nonspecific T cells is still a major challenge. Current technologies employ MHC/peptide multimers [1][2][3][4] or methods to identify cytokine-secreting T cells upon in vitro re-stimulation with the antigen [5][6][7][8][9]. Both methods allow the analysis of live cells on the singlecell level but are limited by several restrictions: MHC multimers require defined antigenic peptide epitopes, and for many pathogens and autoimmune diseases no epitopes are known.…”

Section: Introductionmentioning

confidence: 99%

Identification and isolation of murine antigen‐reactive T cells according to CD154 expression

et al. 2007

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T helper (Th) cells are central regulators of adaptive immune responses. However, the detection of the small number of Th cells specific for a particular antigen or pathogen is still a major challenge. CD154 was recently introduced as a marker for antigen-specific Th cells. To date, this technology was not applicable for mice -arguably the most important immunological model system. CD154 is difficult to detect due to its rapid removal from the cell surface upon binding to CD40 during antigen-specific activation by APC. We present an efficient strategy to block the degradation of murine CD154 by combined use of antibodies against CD40 and CD154. This strategy makes CD154 easily accessible for surface staining, which allows isolation and expansion of rare antigen specific T cells. Importantly, CD154 identified all specific T cells in strongly Th1-or Th2-polarized immune responses against pathogens like Salmonella typhimurium and Heligmosomoides polygyrus, independent of their potential to produce cytokines. We demonstrate that CD154 can in fact be used as a reliable marker for antigen-specific CD4 T cells in mice, offering a unique option to analyze, isolate and rapidly expand the entire pool of Th-cells generated during a physiological T cell response in vivo.

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“…T helper activity can be evaluated by culturing PBMC in the presence of soluble viral proteins and then determining the lymphoproliferative response [5] or the intracellular cytokine production [6]. HCMV-specific CTL response determination is more complex.…”

Section: Introductionmentioning

confidence: 99%

“…HCMV-specific CTL response determination is more complex. The conventional approach used to determine virus-specific CD8 + CTL activity is CD8 + stimulation with autologous HCMV-infected cells for in vitro CTL expansion followed by measurement of CTL activity [3][4][5][6][7]. With the introduction of HLA-peptide tetramer technology [8], direct staining of circulating CD8 + T cells specific for a single peptide was enabled.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

et al. 2005

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Immature dendritic cells (DC) infected with an endotheliotropic (Huv + ) and leukotropic (Leuk + ) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4 + and CD8 + T cells. Infected DC were cocultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-c production. Efficient stimulation of HCMV-specific CD4 + and CD8 + T cell responses was achieved using DC productively infected with Huv + Leuk + VR1814 strain. On the contrary, a negligible CD8 + T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4 + and CD8 + T cell responses and may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.

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Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency.

Cited by 518 publications

References 49 publications

Intestinal double‐positive CD4⁺CD8⁺ T cells are highly activated memory cells with an increased capacity to produce cytokines

Intestinal double‐positive CD4⁺CD8⁺ T cells are highly activated memory cells with an increased capacity to produce cytokines

Identification and isolation of murine antigen‐reactive T cells according to CD154 expression

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

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Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency.

Cited by 518 publications

References 49 publications

Intestinal double‐positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines

Intestinal double‐positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines

Identification and isolation of murine antigen‐reactive T cells according to CD154 expression

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

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Intestinal double‐positive CD4⁺CD8⁺ T cells are highly activated memory cells with an increased capacity to produce cytokines

Intestinal double‐positive CD4⁺CD8⁺ T cells are highly activated memory cells with an increased capacity to produce cytokines