Expression of L-Selectin (CD62L), CD44, and CD25 on activated bovine T cells

Abstract

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.

FIG. 1.

Histogram overlays demonstrating CD25, CD44, and CD62L expression on bovine T-cell subsets. Mononuclear cells were isolated and cultured with no stimulation (dotted lines) or 5 μg of M. bovis PPD/ml (dark solid lines) for 5 days. After 5 days of culture, cells were harvested and stained for five-color analysis as described in Materials and Methods. Gates were set on live (i.e., Annexin V⁻ 7AAD⁻) and CD4⁺, CD8⁺, or γδ TCR⁺ cells. Histograms represent the log fluorescence (CY 5) of CD4⁺, CD8⁺, or γδ TCR⁺ cells stained with either anti-bovine CD25, CD44, or CD62L.

FIG. 2.

In vitro kinetics of CD25, CD44, and CD62L expression on bovine CD4⁺ T cells. Mononuclear cells were isolated and cultured with no stimulation (closed boxes) or with 5 μg of M. bovis PPD/ml (open boxes) and harvested at days 0, 3, 5, and 7 of culture for flow cytometric analysis. Live (i.e., Annexin V⁻ 7AAD⁻) CD4⁺ (PE⁺) cells were analyzed to determine the geometric mean fluorescence (CY 5) intensity of CD25, CD44, or CD62L expression. Data are presented as means (± SEM; n = 3) of the geometric CY 5 intensity. *, differs (P < 0.05) from nonstimulated samples at the same time point.

FIG. 3.

Mean fluorescence intensity (PerCP) of activation markers on nonstimulated and PPD-stimulated T-cell subsets from M. bovis-infected cattle. Isolated mononuclear cells were incubated with media alone (i.e., nonstimulated; open bars) or 5 μg of M. bovis PPD/ml (i.e., PPD-stimulated; closed bars) for 6 days and analyzed for CD4, CD8, or γδ TCR expression (PE) as well as CD25, CD44, or CD62L expression (PerCP). For analysis, gates were set on live (i.e., forward and side scatter properties of Annexin V⁻ 7AAD⁻ cells) and T-cell subset marker-positive (i.e., CD4⁺, CD8⁺, or γδ TCR⁺ cells) cells, and the geometric mean fluorescence intensity (PerCP) of CD25, CD44, or CD62L expression was determined. Data are presented as means (± SEM; n = 10). P values for differences from nonstimulated samples for the respective T-cell subset (i.e., comparisons between open and closed bars for each graph) were as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. “a” indicates differences (P < 0.01) from expression by PPD-stimulated CD8⁺ and γδ TCR⁺ cells for the respective activation marker (i.e., comparisons between closed bars for each graph).

FIG. 4.

Percent activation marker expression on nonstimulated and PPD-stimulated T-cell subsets from M. bovis-infected cattle. Isolated mononuclear cells were incubated with media alone (i.e., nonstimulated; open bats) or 5 μg of M. bovis PPD/ml (i.e., PPD stimulated; closed bars) for 6 days and analyzed for CD4, CD8, or γδ TCR expression (PE) as well as CD25, CD44, or CD62L expression (PerCP). The percent CD25⁺, CD44⁺, or CD62L⁺ cells was determined for live (i.e., forward and side scatter properties of Annexin V⁻ 7AAD⁻ cells) and T-cell subset marker-positive (i.e., CD4, CD8, or γδ T cells) cells. Data are presented as means (± SEM; n = 10). P values for differences from nonstimulated samples for the respective T-cell subset (i.e., comparisons between open and closed bars for each graph) were as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. “a” indicates differences (P < 0.01) from expression by PPD-stimulated CD8⁺ cells for the respective activation marker subset (i.e., comparisons between closed bars for each graph).

FIG. 5.

Mean fluorescence intensity (PerCP) of activation markers on PPD-stimulated, PKH67^bright (i.e., nonproliferative fraction; open bars) or PKH67^dim (i.e., proliferative fraction; closed bars) T-cell subsets from M. bovis-infected cattle. Isolated mononuclear cells were stained with PKH67 and incubated with 5 μg of M. bovis PPD/ml for 6 days and analyzed for PKH67 staining intensity (FL-1 channel); CD4, CD8, or γδ TCR expression (FL-2 channel; PE); and CD25, CD44, or CD62L expression (FL-3 channel; PerCP). The geometric mean fluorescence intensity (PerCP) of CD25, CD44, or CD62L expression was determined for individual T-cell subsets (i.e., CD4⁺, CD8⁺, or γδ TCR⁺ cells), with further discrimination based on PKH67 staining intensity (i.e., bright or dim). Only cells with light scatter properties typical of live cells (i.e., Annexin V⁻ 7AAD⁻) were evaluated. Data are presented as means (± SEM; n = 10). P values for differences from PKH67^bright fractions for the respective T-cell subset (i.e., comparisons between open and closed bars for each graph) were as follows: *, P < 0.05; **, P < 0.01; ***. “a” indicates difference (P < 0.05) from expression by PPD-stimulated CD8⁺ and γδ TCR⁺ cells; “b” indicates difference (P < 0.05) from expression by PPD-stimulated CD8⁺ cells for the respective activation marker (i.e., comparisons between closed bars for each graph).

FIG. 6.

Gating strategy for differentiation of live, apoptotic, and dead cells based upon Annexin V and 7AAD staining of M. bovis PPD-stimulated bovine PBMC. Mononuclear cells from M. bovis-infected (10⁵ CFU) cattle were isolated and cultured with no stimulation (A), 5 μg of PPD/ml (B), or 1 μg of PWM/ml (C) for 5 days. Gates were set based on Annexin V and 7AAD staining properties in order to discriminate between live (R1, Annexin V⁻ 7AAD⁻), early apoptotic (R2, Annexin V⁺ 7AAD^dim/−), and dead (R3, Annexin V⁺ 7AAD^bright) cells. Values on plots represent percentages of cells within individual gates. Dot plots of Annexin V and 7AAD staining for a representative animal are provided, with similar staining patterns detected for other animals and for both staining protocols used for five-color analysis (i.e., with either the streptavidin-RED613 or streptavidin-ECD conjugate).