Distinct chemokine triggers and in vivo migratory paths of fluorescein dye-labeled T Lymphocytes in acutely simian immunodeficiency virus SIVmac251-infected and uninfected macaques

Abstract

To define the possible impact of T-lymphocyte trafficking parameters on simian immunodeficiency virus (SIV) pathogenesis, we examined migratory profiles of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T lymphocytes in acutely SIVmac251-infected and uninfected macaques within 48 h after autologous transfer. Despite significant upregulation of homeostatic chemokine CCL19/macrophage inflammatory protein 3beta and proinflammatory chemokine CXCL9/monokine induced by gamma interferon in secondary lymphoid tissue in SIV infection, no differences in CFSE+ T-lymphocyte frequencies or cell compartmentalization in lymph nodes were identified between animal groups. By contrast, a higher frequency of CFSE+ T lymphocytes in the small intestine was detected in acute SIV infection. This result correlated with increased numbers of gut CD4 T lymphocytes expressing chemokine receptors CCR9, CCR7, and CXCR3 and high levels of their respective chemokine ligands in the small intestine. The changes in trafficking parameters in SIV-infected macaques occurred concomitantly with acute gut CD4 T-lymphocyte depletion. Here, we present the first in vivo T-lymphocyte trafficking study in SIV infection and a novel approach to delineate T-lymphocyte recruitment into tissues in the nonhuman primate animal model for AIDS. Such studies are likely to provide unique insights into T-lymphocyte sequestration in distinct tissue compartments and possible mechanisms of CD4 T-lymphocyte depletion and immune dysfunction in simian AIDS.

FIG. 1.

Quantitation of CCL19 and CXCL9 transcript levels. Relative transcript levels for homeostatic chemokine CCL19 and proinflammatory chemokine CXCL9 were determined for secondary lymphoid organs and gut tissues in a group of uninfected animals (numbers 30880, 30892, and 30893) and acutely SIV_mac251-infected animals (numbers 30019, 29677, and 32222) at necropsy time point (day 14 p.i. in the SIV-infected group), according to previously established procedures (7). Transcript levels were normalized based on GAPDH transcription; for each chemokine, values were calibrated based on the tissues expressing the lowest levels in the SIV-uninfected group. Transcription levels are depicted as n-fold induction or reduction (<1) compared to transcription in the tissues expressing the lowest levels in uninfected animals. Each symbol represents transcript levels in one experimental macaque in a given tissue (the SIV-infected group with solid black symbols and the SIV-uninfected group with open symbols). Levels for gut-derived cell suspensions for SIV-uninfected animal 30880 were not determined. Transcript levels were significantly different between SIV-infected and SIV-uninfected animals for secondary lymphoid tissue and gut tissues (P < 0.05). Lung Ln, lung-derived lymph nodes encompassing bronchiolar and mediastinal tissue; BM, bone marrow; Ax, axillary; Ln, lymph node; Ing, inguinal; Mes, mesenteric; IEL, gut-derived intestinal epithelial lymphocytes; LPL, gut-derived lamina propria lymphocytes.

FIG. 2.

Immunofluorescent detection of CFSE⁺ T lymphocytes in lymph node. For immunofluorescent detection of CFSE⁺ cells and T lymphocytes, axillary lymph node tissue sections from a representative SIV-uninfected animal (30893) (a) and a representative SIV-infected animal (32222) (b; enlarged view, panels c to e) were double labeled with antibodies directed against fluorescein and CD3. Secondary detection was performed with Alexa 488-conjugated (CFSE; green) and Alexa 568-conjugated antibodies (CD3; red). Nuclear dye labeling was performed using DAPI (blue). Most CFSE⁺ cells in the paracortex were CFSE and CD3 double positive. Examples of CFSE⁺ T lymphocytes in the paracortex of animal 32222 are shown, positive for CD3 (c, arrows) and CFSE (d, arrows), appearing yellow in a three-color overlay (e, arrows).

FIG. 3.

Flow cytometric analysis of CFSE⁺ T-lymphocyte frequencies in tissues. The representative gating strategy for flow cytometric analysis is illustrated, based on PBMC freshly isolated from animal 32222 on day 14 p.i. at the time of necropsy (A, top). A small lymphocyte gate was used, based on forward and side scatter parameters and excluding larger monocytes and dendritic cells. T lymphocytes were subsequently gated, based on CD3 expression and the presence of CFSE⁺ cells within this cell pool (A). PBMC and tissue cell suspensions from spleen; inguinal (Ing), axillary (Ax), and mesenteric (Mes) lymph nodes (Ln); bone marrow; liver; gut mucosal lamina propria (LPL); and intestinal epithelium (IEL) were labeled with antibodies directed against CD3 and CD4. The relative frequency of the CFSE⁺ subset is indicated within each graph (percentage of CFSE⁺ of total tissue CD3⁺ T lymphocytes). The relative frequencies of CFSE⁺ T lymphocytes in tissues of SIV-uninfected animals (numbers 30880, 30892, and 30893) and SIV-infected animals (numbers 30019, 32222, and 29677) at the time of necropsy were compared (from previously frozen samples) (B). Lymphocytes were gated based on forward and side scatter parameters, CD3 expression, and presence of CFSE (see the gating strategy described for panel A). No significant changes in the relative frequency of CFSE⁺ T lymphocytes (as a percentage of total CD3+ T lymphocytes) were observed (P > 0.05).

FIG. 3.

Flow cytometric analysis of CFSE⁺ T-lymphocyte frequencies in tissues. The representative gating strategy for flow cytometric analysis is illustrated, based on PBMC freshly isolated from animal 32222 on day 14 p.i. at the time of necropsy (A, top). A small lymphocyte gate was used, based on forward and side scatter parameters and excluding larger monocytes and dendritic cells. T lymphocytes were subsequently gated, based on CD3 expression and the presence of CFSE⁺ cells within this cell pool (A). PBMC and tissue cell suspensions from spleen; inguinal (Ing), axillary (Ax), and mesenteric (Mes) lymph nodes (Ln); bone marrow; liver; gut mucosal lamina propria (LPL); and intestinal epithelium (IEL) were labeled with antibodies directed against CD3 and CD4. The relative frequency of the CFSE⁺ subset is indicated within each graph (percentage of CFSE⁺ of total tissue CD3⁺ T lymphocytes). The relative frequencies of CFSE⁺ T lymphocytes in tissues of SIV-uninfected animals (numbers 30880, 30892, and 30893) and SIV-infected animals (numbers 30019, 32222, and 29677) at the time of necropsy were compared (from previously frozen samples) (B). Lymphocytes were gated based on forward and side scatter parameters, CD3 expression, and presence of CFSE (see the gating strategy described for panel A). No significant changes in the relative frequency of CFSE⁺ T lymphocytes (as a percentage of total CD3+ T lymphocytes) were observed (P > 0.05).

FIG. 4.

CFSE⁺ PBMC trafficking to gut lamina propria in SIV-infected and uninfected macaques. For immunohistochemical detection, tissue sections from the small intestines of representative SIV-uninfected macaque 30892 (a) and SIV-infected animal 30019 (b) were stained with an anti-fluorescein antibody and a biotinylated secondary antibody. CFSE⁺ cells were visualized using BCIP/NBT substrate (CFSE⁺ cells appear dark purple), and tissue sections were counterstained with methyl green. CFSE⁺ cells were observed inside follicles within the lamina propria in the uninfected animal 30892 (a, arrows) and at a higher frequency in SIV-infected animal 30019, dispersed throughout the lamina propria (b, arrows). For double-immunofluorescent labeling, tissue sections from the same animals were stained with primary antibodies directed against fluorescein and CD20 and detected with secondary antibodies conjugated to Alexa 488 (CFSE; green) or Alexa 568 (CD20; red). Nuclear dye labeling was performed using DAPI (blue). Similar to immunohistochemical results, CFSE⁺ cells from the representative uninfected macaque were predominantly observed inside follicles (c, arrows), while CFSE⁺ cells in the SIV-infected group were found throughout the lamina propria (d, arrows).

FIG. 5.

Flow cytometric detection of CFSE⁺ T lymphocytes in the gut lamina propria. Gut mucosal LPL derived from healthy animal 30892 and SIV-infected animal 30019 were stained with antibodies directed against CD3 (PerCP conjugated) and CD4 or CD8 (both APC conjugated). Cells were gated based on forward and side scatter parameters and CD3 expression (as shown in the gating strategy described for Fig. 3A). The relative frequency of CD4 and CD8 T lymphocytes (of total CD3⁺ T lymphocytes) is depicted (CFSE-negative population), in addition to the total frequency of CFSE⁺ T lymphocytes.

FIG. 6.

Frequency of gut lamina propria CD4 T lymphocytes and their expression of chemokine receptors CCR9, CXCR3, and CCR7. Gut lamina propria tissue suspensions from two SIV-uninfected animals (30892 and 30893) and three acutely SIV-infected animals (30019, 32222, and 29677) were stained with fluorochrome-conjugated antibodies directed against CD3 (PerCP), CD4 (APC), and chemokine receptors CCR9, CXCR3, or CCR7 (PE conjugated). Labeled cell suspensions were examined by flow cytometry, and cells were analyzed based on a small lymphocyte gate and CD3 expression. CD4 T-lymphocyte frequencies were then determined as a percentage of total CD3⁺ LPL (first column). Gated gut lamina propria CD4 T lymphocytes were examined for expression of chemokine receptors CCR9, CXCR3, and CCR7 by histogram analysis. The percentages in histogram plots represent the relative frequencies of chemokine receptor-positive subsets in the CD4 T-lymphocyte compartment in the gut mucosa.