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Journal of Virology, November 2005, p. 13759-13768, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13759-13768.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Distinct Chemokine Triggers and In Vivo Migratory Paths of Fluorescein Dye-Labeled T Lymphocytes in Acutely Simian Immunodeficiency Virus SIV_mac251-Infected and Uninfected Macaques

Candice C. Clay,^1,2, Denise S. Rodrigues,^2,, Danielle J. Harvey,³ Christian M. Leutenegger,⁴ and Ursula Esser^2*

Immunology Graduate Program, University of California—Davis, Davis, California 95616,¹ Department of Pathology and Laboratory Medicine, University of California—Davis, School of Medicine, Sacramento, California 95817,² Department of Public Health Sciences, University of California—Davis, Davis, California 95616,³ Lucy Whittier Molecular and Diagnostic Core Facility, University of California—Davis, California 95616⁴

Received 11 November 2004/ Accepted 4 August 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 SIV_mac251-infected and uninfected macaques within 48 h after autologous transfer. Despite significant upregulation of homeostatic chemokine CCL19/macrophage inflammatory protein 3ß 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human immunodeficiency virus (HIV) infection has profound qualitative and quantitative effects on cellular components of the immune system, inducing a gradual decline of circulating peripheral blood CD4 T lymphocytes and CD8 T lymphocytes at late stages of infection (9, 14). The observed T-lymphocyte decline, as well as dysfunctional CD4 and CD8 T-lymphocyte responses (2, 4, 15, 30, 48), contribute to the overall deterioration of immune functions in HIV-infected individuals, ultimately leading to the general loss of immune competence and the development of AIDS (21, 31, 46). Nevertheless, the mechanisms contributing to the lack of antiviral immune control and absence of immune protection leading to AIDS are still poorly defined. The processes thought to directly contribute to CD4 T-lymphocyte decline in peripheral blood involve decreased thymic output to replenish peripheral CD4 T-lymphocyte pools, reduction of peripheral CD4 T-lymphocyte numbers by programmed cell death, and increased migration of circulating CD4 T lymphocytes to lymphatic tissues (1, 8, 9, 22, 27, 38, 42). The latter mechanism and parameters associated with CD4, as well as CD8, T-lymphocyte trafficking in the nonhuman primate model for AIDS will be the focus of the present study.

Migration of peripheral blood T lymphocytes into secondary lymphoid tissue and distal effector sites is considered to be a consequence of chemokine-mediated recruitment of both effector and virally susceptible target cells into lymphatic tissues where CD4 T lymphocytes fuel viral replication rates in HIV-positive patients and simian immunodeficiency virus (SIV)-infected rhesus macaques (35). In addition, during later stages of viral infection, recruitment into tissues likely contributes to the loss of peripheral CD4 (and CD8) T lymphocytes, when normal homeostatic mechanisms to maintain peripheral blood T-lymphocyte pools are disrupted (9). Direct evidence for increased T-lymphocyte homing into lymphatic tissue has been scarce, and the proposed mechanisms are difficult to examine in vivo. With humans, because of safety considerations and ethical issues, in vivo cell tracking studies using fluorescent dye-labeled populations are not feasible. In the nonhuman primate AIDS model, technology barriers have precluded in vivo T-lymphocyte trafficking experiments with SIV-infected rhesus macaques. Accordingly, the central questions which remain undefined encompass if enhanced T-lymphocyte trafficking to secondary lymphoid tissue and distal effector sites are paired with peripheral T-lymphocyte decline and if altered migratory parameters and ineffective compartmentalization within tissues are associated with dysfunctional T-lymphocyte responses in human or simian AIDS. Conversely, it remains undetermined if particular T-lymphocyte migratory patterns promote delayed disease onset and correlate with protective immune responses, as with long-term nonprogressors. Finally, it is unclear to what extent phenotypic markers such as chemokine receptor expression on peripheral blood- or tissue-derived T lymphocytes predict cell movement and positioning within chemokine gradients and are associated with site-specific homing profiles in HIV or SIV infection.

Our group has now overcome the existing technology barrier to examine in vivo cell migratory paths in rhesus macaques by conducting autologous transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE) dye-labeled populations. Carboxyfluorescein diacetate enters the cytoplasm and predominantly labels cytoplasmic proteins by forming fluorescent succinimidyl esters. CFSE remains stable over many weeks or even months and enables long-term cell tracking in vivo (10, 33, 47). Furthermore, CFSE divides equally between daughter cells upon cell division. It may therefore be used to visualize cell proliferation in vitro or in vivo, as the mean fluorescence intensity (MFI) is reduced by half with each successive cell division (25). It has been extensively used with various model systems (in vivo and in vitro) including mouse, rabbit, and sheep models, as well as with human cells in vitro (10, 11, 25, 33, 37, 39). Using this fluorescent dye, we recently conducted T-lymphocyte trafficking experiments of dye-labeled populations in vivo in rhesus macaques and delineated chemokine profiles in uninfected macaques (7). In the current study, we defined migratory characteristics of autologously transferred, CFSE⁺ peripheral blood T lymphocytes in acutely SIV-infected rhesus macaques to secondary lymphoid tissue and the intestinal mucosa and observed divergent T-lymphocyte migratory patterns, tissue compartmentalization, and chemokine triggers for gut-specific homing in SIV infection. The study presented here facilitates the delineation of cross talk between peripheral blood and the small intestine, which constitutes a major lymphoid compartment, principal viral reservoir during acute SIV infection, and site of acute and chronic CD4 T-lymphocyte depletion in SIV and HIV infections (5, 13, 22, 28, 40, 43).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. Three adult male macaques (numbers 29677, 30019, and 32222), ranging in weight from 8.0 to 11.1 kg, were recruited into this study; three SIV-uninfected animals (numbers 30880, 30892, and 30893) were used as controls (7). The animals were housed at the California National Primate Research Center. Experiments were approved by the Institutional Animal Use and Care Committee of the University of California at Davis. All six animals tested seronegative for simian type D retrovirus, simian T-cell leukemia virus type 1, and SIV. Animals in the SIV-infected group (numbers 30019, 29677, and 32222) were intravenously infected with pathogenic viral isolate SIV_mac251 (1,000 50% tissue culture infective doses in 1 ml). Maximal plasma viral RNA levels were reached at day 11 postinfection (p.i.), ranging from 2.0 to 5.1 x 10⁷ viral RNA copies/ml.

Autologous lymphocyte transfer and necropsy tissue collection. Lymphocytes from the acutely SIV-infected macaques were isolated at day 12 p.i., according to previously established procedures (7). In brief, animals were anesthetized and sequentially bled, and lymphocytes were purified by density gradient centrifugation on Ficoll-Hypaque (Amersham Biosciences, Piscataway, NJ). Peripheral blood mononuclear cells (PBMC) were labeled for 6 to 8 min with 5 µM CFSE (Molecular Probes, Eugene, OR) in serum-free phosphate-buffered saline at 16 x 10⁶ to 20 x 10⁶ cells/ml, according to established procedures (26, 29). CFSE-labeling reactions were terminated with autologous serum obtained 1 to 3 months prior to the procedure (stored at –20°C) for animals 29677 and 30019. A total of 5.1 x 10⁸ to 7.4 x 10⁸ PBMC were fluorescein dye labeled in vitro and intravenously transferred within 5 h of isolation, representing approximately 5.3 x10⁸ T lymphocytes in animal 30019, 5.5 x10⁸ in animal 29677, and 3.6 x10⁸ in animal 32222. Animals were euthanized within 40 to 48 h posttransfer (day 14 p.i). Similar to the SIV-infected group, three SIV-uninfected, gender-matched adult macaques (numbers 30880, 30892, and 30893) received autologous CFSE⁺ PBMC prior to necropsy and tissue collection within 48 h posttransfer, as described in detail previously (7). At the time of necropsy, maximal blood volumes were collected, and all six animals were whole-body saline perfused prior to tissue collection into RPMI (Invitrogen, Carlsbad, CA) and formalin.

Flow cytometry and immunohistochemistry. Complete blood cell counts were assessed by the California National Primate Research Center clinical laboratory staff. For examination of CFSE dye-labeled populations by four-color flow cytometry, three-color staining panels were prepared utilizing antibodies conjugated to phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC): CD3 (clone SP34-2-PerCP), CD4 (clone MT477-PE or MT477-APC) and CD8 (clone SK1-PE or SK1-APC) (Becton Dickinson, San Jose, CA), CCR9 (clone 112509-PE), CXCR3 (clone 49801-PE) (R&D Systems, Minneapolis, MN), and human CCL19-Tetramer-PE (34). The staining procedure was conducted with phosphate-buffered saline supplemented with 3 to 5% fetal calf serum for 30 min on ice with previously frozen tissue cell suspensions (unless otherwise noted). Sample data were acquired on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), and from 0.5 x 10⁶ to 1.25 x 10⁶ events were collected in the lymphocyte gate based on forward and side scatter parameters. Data files were analyzed utilizing FlowJo software (TreeStar, Inc., Ashland, OR).

For immunohistochemistry and immunofluorescence approaches, formalin-fixed tissues were embedded in paraffin, and 6-µm sections were prepared. For immunohistochemical staining, CFSE⁺ cells were labeled with a rabbit polyclonal antibody directed against fluorescein (Molecular Probes, Oregon) and a biotinylated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (Vector Laboratories, Bulingame, CA). Cells were then visualized with a standard Vectastain ABC-Alkaline Phosphatase kit (Vector Laboratories, Burlingame, CA) using final substrate 5-bromo-4-chloro-3-indoyl phosphate-nitroblue tetrazolium (BCIP/NBT) and tissue sections counterstained with methyl green (Vector Laboratories, Burlingame, CA). For immunofluorescence experiments, CFSE was detected indirectly. The rationale for indirect CFSE detection was threefold. (i) Carboxyfluorescein undergoes rapid photobleaching under illumination, and other dyes are more photostable (such as Alexa dyes). (ii) Potential light sensitivity of specimens can be ignored and samples can be preserved over the long term without signal loss, as antibody recognition only requires chemical stability. (iii) Signal-to-noise ratios can be improved by dye selection and/or signal amplification when autofluorescence in tissues is high (such as in the small intestine). Accordingly, tissue sections were incubated overnight with anti-fluorescein and either mouse anti-human CD20 (clone L26) (Dako, Carpinteria, CA) or rat anti-CD3 (clone NCL-CD3-12) (Novocastra, Newcastle, United Kingdom) antibodies. For secondary detection, tissue sections were subsequently labeled with Alexa 488-conjugated donkey anti-rabbit IgG antibodies for CFSE and Alexa 568-conjugated highly cross-adsorbed goat anti-mouse IgG (for CD20⁺ B lymphocyte detection) or Alexa 568-conjugated anti-rat (for detection of CD3⁺ T lymphocytes) secondary antibodies (Molecular Probes, Eugene, OR). For nuclear staining, 4',6'-diamidino-2'-phenylindole dihydrocholoride (DAPI) (Molecular Probes, Eugene, OR) was used.

Quantitation of viral RNA and chemokine transcription. The presence of plasma SIV viral RNA was measured by TaqMan analysis, according to established procedures (20). Chemokine transcript profiling was conducted based on previously described primer/probe pairs following total RNA isolation from tissue suspensions derived from distinct sites (7). In brief, chemokine transcription was reported as the n-fold difference relative to gene transcription in the lowest expressing tissue in SIV-uninfected controls for each individual chemokine, following normalization for presence of total RNA based on endogenous GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript levels.

Statistics. Differences between SIV-infected and SIV-uninfected animals across tissue samples for each chemokine (transformed to the natural logarithm scale) were assessed using repeated measures analyses through SAS Proc MIXED, assuming a compound symmetry structure to the correlation between repeated measures. Assumptions of the models were checked both graphically and numerically and were reasonably met by the data. A P value of <0.05 was considered statistically significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Presence of chemokine triggers CCL19 and CXCL9 in tissues in both animal groups. To examine alterations in chemokine signals that may impact T-lymphocyte migratory circuits to secondary lymphoid organs and to distal effector sites in acutely SIV-infected animals compared to results for uninfected macaques, we determined transcript levels for homeostatic lymph node homing chemokine CCL19/macrophage inflammatory protein 3ß and proinflammatory chemokine CXCL9/monokine induced by gamma interferon in various tissues in both animal groups at time of necropsy (day 14 p.i. in the SIV-infected group). Repeated measures analyses, examining all secondary lymphoid and gut tissues jointly, revealed significant differences between SIV-infected (30019, 29677, 32222) and uninfected (30880, 30892, and 30893) animals, as well as across tissue samples for each chemokine tested (P < 0.001) (Fig. 1). Both homeostatic chemokine CCL19, ligand for CCR7, and proinflammatory chemokine CXCL9, ligand for CXCR3, were previously reported to be upregulated in secondary lymphoid tissue in acute SIV infection by in situ hybridization approaches and microarray analysis (6, 36). This was similarly observed with these animals, where CCL19 levels in the infected animals were approximately 27 times higher in a given secondary lymphoid tissue sample, on average, than the levels in the uninfected animals (P value < 0.0001), while CXCL9 levels were about 18 times higher (P < 0.0001). Notably, we also observed elevated CXCL9 and CCL19 levels in the infected animals in the gut lamina propria (Fig. 1), indicating the presence of chemoattractants in the intestinal mucosa for both CXCR3⁺ and CCR7⁺ T lymphocytes.

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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 SIVmac251-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.

CFSE⁺ T-cell trafficking to secondary lymphoid organs. Based on increased levels of lymph node homing chemokine CCL19 and the presence of proinflammatory trigger CXCL9 in secondary lymphoid tissue (Fig. 1), we anticipated that a higher frequency of CFSE⁺ T lymphocytes had migrated into paracortical regions of lymph nodes within the 2-day in vivo migratory period during the acute SIV infection phase. To examine trafficking of CFSE⁺ T lymphocytes to these sites and reveal potential differences in CFSE⁺ cell frequency (and localization) in acutely SIV-infected versus uninfected macaques, double-immunofluorescence-labeling experiments of lymph node tissue sections were conducted. The predominant number of CFSE⁺ cells in both animal groups were CFSE and CD3 double positive, as shown for representative SIV-uninfected animal 30893 and SIV-infected animal 32222 (Fig. 2a and b, arrows; magnified views are shown in Fig. 2c to e). This unambiguously identified most infiltrating CFSE-labeled cells as T lymphocytes, consistent with their migration into the lymph node paracortex (and localization within the T-cell zone). The immunofluorescence findings were also consistent with flow cytometric analysis demonstrating that an average of 87% of CFSE⁺ cells in lymph nodes were CD3⁺ T lymphocytes (data not shown). However, despite significantly higher chemokine CCL19 and CXCL9 transcript levels in the lymph nodes of SIV-infected animals, there were, notably, no apparent changes in CFSE⁺ T-lymphocyte frequencies (or cell compartmentalization) in SIV-infected animals in response to these migratory triggers. These findings were subsequently independently confirmed by flow cytometry analyses of tissue suspensions derived from secondary lymphoid tissue and other tissues, as shown here for representative SIV-infected animal 32222 (Fig. 3A) and as summarized for both animal groups (Fig. 3B). The gating strategy of distinct T-lymphocyte subsets involved initial gating on small lymphocytes, followed by gating on CD3⁺ T lymphocytes and examination of CFSE⁺ T-lymphocyte frequencies (Fig. 3A). Similar to observations in uninfected macaques, dye-labeled T lymphocytes in the representative acutely SIV-infected macaque were detected in all organs examined, including peripheral blood, lymph nodes, spleen, liver, and bone marrow. Furthermore, CFSE⁺ T lymphocytes in the small intestine could be readily observed. The highest relative frequencies of CFSE⁺ T lymphocytes were detected in the peripheral blood and spleen (0.82 to 0.96%), followed by bone marrow (0.73%), lymph nodes (0.49 to 0.67%), gut lamina propria (0.52%), and intestinal epithelium (0.22%) (Fig. 3A). The CFSE⁺ T lymphocytes in the gut lamina propria and intestinal epithelium were less bright than those in other tissues, potentially reflecting one to two extra cell divisions within this compartment.

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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).

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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).

Examination of samples from all six animals by flow cytometry revealed similar T-lymphocyte frequencies in SIV-infected versus uninfected macaques in peripheral blood (0.82 to 1.25% versus 0.67 to 1.47%, respectively), spleen (0.96 to 1.5% versus 0.72 to 1.29%), and bone marrow (0.48 to 1.04% versus 0.43 to 0.97%) (Fig. 3B). Furthermore, no significant differences were identified in T-lymphocyte frequencies in both axillary and mesenteric lymph nodes between groups (Fig. 3B), in agreement with double immunofluorescence results (Fig. 2).

CFSE⁺ cell trafficking to the small intestine and chemokine and ligand receptor expression profiles. The small intestine represents a major lymphoid compartment and active site of viral replication during the acute infection phase. Accordingly, a further goal was to examine possible cross talk between T-lymphocyte pools in peripheral blood and those present in the intestinal mucosa that may impact gut tissue-specific trafficking parameters, viral replication rates, T-lymphocyte turnover, and T-lymphocyte immune functions. We directly compared the gut homing frequency of CFSE⁺ lymphocytes of uninfected and acutely SIV-infected macaques by immunohistochemistry and double immunofluorescence of gut mucosal tissues. Representative uninfected macaque 30892 was similar in weight to SIV-infected animal 30019 and received the highest number of CFSE⁺ cells in the SIV-uninfected group during autologous transfer (8 x 10⁸ total PBMC) and only a slightly higher number than acutely SIV-infected animal 30019 (which received 7.3 x 10⁸ total PBMC). No CFSE⁺ cells were observed in gut control tissue of CFSE-negative animal 29513 by either staining procedure (data not shown). In the representative experimental animals, infiltrating CFSE⁺ cells were readily identified in the intestinal mucosa (Fig. 4). However, a notably higher CFSE⁺ cell frequency was identified in SIV infection based on the dye-positive cells in this region (Fig. 4b and d). CFSE⁺ cells in the uninfected animal were almost exclusively localized within lamina propria lymphoid aggregates (Fig. 4a and c), whereas in SIV infection, generally fewer follicles were observed and the CFSE⁺ cells were found dispersed throughout the lamina propria (Fig. 4b and d) and excluded from follicles (when present) (Fig. 4d). The CFSE⁺ cells in both animal groups were identified as non-B lymphocytes by double-immunofluorescent detection of CFSE⁺ cells (green) and CD20⁺ B lymphocytes (red) (Fig. 4c and d), which identified most infiltrating cells as CFSE single positive.

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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).

To confirm these results and the higher CFSE⁺ cell frequency in animal 30019, flow cytometry analysis was conducted with gut lamina propria lymphocytes (LPL) of animals 30892 and 30019. The percentage of CFSE⁺ cells of total CD3⁺ T lymphocytes averaged 0.38% in animal 30892, while it reached 1.08% of total CD3⁺ T lymphocytes in animal 30019 (Fig. 5), in support of enhanced CFSE⁺ cell trafficking into the small intestine in this animal. However, in the SIV-infected macaque, CFSE⁺ T lymphocytes were less bright than CFSE⁺ T lymphocytes derived from peripheral blood or lymph nodes (from the same animal, number 30019), possibly a reflection of twofold CFSE MFI reduction with each cell division and cell expansion within this cell pool (prior to or following gut entry and likely exposure to various stimuli) (also shown for animal 32222) (Fig. 3A). Our data also indicate that the CFSE⁺ CD3⁺ T-lymphocyte subset included both CD4 and CD8 T lymphocytes (Fig. 5). However, the largest proportion of CFSE⁺ CD3⁺ T lymphocytes appeared negative for both cell surface CD8 and CD4 and may represent a CD4 T-lymphocyte subset with a downmodulated CD4 receptor following SIV infection, as had been demonstrated in vitro by an HIV or SIV viral Nef protein-dependent mechanism (3, 12, 23, 24, 41) and in vivo during HIV infection of SCID-hu mice (16).

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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.

Concomitant with a higher frequency of CFSE⁺ T lymphocytes in this site (Fig. 5), we observed CD4 T-lymphocyte depletion in this compartment in all three SIV-infected animals (Fig. 5 and Fig. 6), consistent with earlier reports of acute gut CD4 T-lymphocyte depletion during this infection phase (43). The frequency of CD4 T lymphocytes (of total CD3⁺ T lymphocytes) in this group of animals ranged from 3.5 to 10.7% (Fig. 5 and 6), while the CD4 T-lymphocyte frequencies were much higher in the uninfected macaques, ranging from 40.6 to 45.7%.

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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.

To define migratory parameters of gut lamina propria T lymphocytes and their ability to compartmentalize in response to intestinal chemokine signals, expression of gut homing chemokine receptor CCR9, which binds chemokine CCL25/thymus-expressed chemokine (18, 32), was examined on gut lamina propria CD4 T lymphocytes. In the uninfected group, CCR9 expression on CD4 T lymphocytes ranged from only 26.9 to 32.2% (Fig. 6). In contrast, the predominant number of CD4 T lymphocytes in the SIV-infected animals expressed this receptor at 2 weeks p.i., ranging from 73.9 to 94.9% (Fig. 6). We also defined the expression of chemokine receptors CXCR3 and CCR7, whose ligands were present at high levels in the gut lamina propria during acute SIV infection (Fig. 1) to examine their possible involvement in tissue homing, retention, and sequestration of CD4 T lymphocytes within the intestinal mucosa. Similar to CCR9 expression profiles, CXCR3 and CCR7 were expressed on a much higher number of CD4 T lymphocytes in SIV-infected animals, ranging between 77.2 to 96.2% in SIV infection, compared to 23.8 to 42.5% in uninfected macaques (Fig. 6). These drastic changes were not similarly observed within the intestinal CD8 T-lymphocyte pool during acute SIV infection (data not shown).

In summary, our findings for gut mucosal tissue demonstrate altered T-lymphocyte migratory characteristics, higher CFSE⁺ T-lymphocyte frequencies in gut tissue of the SIV-infected animals, and presence of homing determinants on CD4 T lymphocytes that may have facilitated their responsiveness to CCL19, CCL25, and CXCL9 ligand signals in the small intestine. Importantly, this occurred concomitant with massive CD4 T-lymphocyte depletion in this compartment.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study represents the first examination of in vivo T-lymphocyte migratory paths in the nonhuman primate model for AIDS, following autologous leukocyte transfer of CFSE dye-labeled populations. Based on the selected 48-h time course, our in vivo T-cell tracking studies of rhesus macaques suggest many differences (and commonalities) between trafficking pathways of dye-labeled T-lymphocyte populations in uninfected and acutely SIV_mac251-infected animals. Rather unexpectedly, the localization of dye-labeled T lymphocytes in lymph nodes and their frequency were remarkably similar between animal groups, despite significant upregulation of homeostatic secondary lymphoid chemokine CCL19 (P < 0.0001) and proinflammatory chemokine CXCL9 (P < 0.0001) in spleen and lymph nodes in SIV infection and possible chemokine-mediated recruitment of CCR7⁺ and CXCR3⁺ T-lymphocyte subsets. Previously, upregulation of chemokine CCL19 and CXCL9 mRNA in acute SIV infection was observed in the T-cell zone of lymph nodes by in situ hybridization approaches (6, 36), in agreement with findings with acutely SIV-infected animals in this study (Fig. 1). Consistent with chemokine transcript levels (Fig. 1) and corresponding chemokine receptor expression on dye-labeled T lymphocytes (reference 7 and data not shown), the autologously transferred CFSE⁺ T lymphocytes almost exclusively accumulated near high endothelial venules in the paracortex, with only few CFSE⁺ cells present in the follicular and mantle zones (Fig. 2). Nevertheless, the lack of marked differences in CFSE⁺ cell frequencies and localization within lymph nodes in SIV infection implies that other factors may impact migration to secondary lymphoid tissue, involving distinct tissue entry, retention, and exit parameters. Our findings also strongly suggest that chemokine levels and chemokine receptor expression may not be adequate to predict trafficking paths and T-lymphocyte sequestration in distinct tissue compartments but may require tracking T-cell migratory paths in vivo, as conducted in this study.

Although extensive similarities were observed in migratory patterns towards secondary lymphoid organs in the selected time course between animal groups, importantly, significant differences were revealed in T-lymphocyte homing towards effector sites, including to the gut mucosa (Fig. 4 to 6) and into the central nervous system in SIV infection (C. C. Clay and U. Esser, unpublished data). CFSE⁺ T lymphocytes in the lamina propria of the gut mucosa were readily detectable in the SIV-infected animals and within the gut mucosa, localized to distinct sites compared to uninfected macaques. In the latter group, rare CFSE⁺ T lymphocytes were identified in gut tissue, where infiltrating cells exclusively localized within lymphoid aggregates in inductive sites within the intestinal lamina propria (44, 45). In contrast, in SIV-infected animals, a higher frequency of CFSE⁺ T lymphocytes was identified (Fig. 4 and 5) and the CFSE⁺ cells were present throughout the gut lamina propria (Fig. 4), consistent with the diffuse localization of effector sites dispersed throughout this tissue (44, 45). The infiltrating T lymphocytes likely comprised both CD4 and CD8 T lymphocytes and included a CD3⁺ T-lymphocyte subset that was both CD8 and CD4 negative (CD3⁺ CD8^– CD4^–) (Fig. 5). The CD3 ⁺ CD8^– CD4^– infiltrating subset, in particular, will require further phenotyping and functional analysis in future studies, as it may represent an SIV-infected CD4 T-lymphocyte population with downmodulated CD4 cell surface expression. CD4 downmodulation mediated by HIV or SIV viral Nef protein has been demonstrated in vitro (3, 12, 23, 24, 41), as well as with HIV infection in the SCID-hu mouse model (16). The possible in vivo CD4 downregulation implies that the gut mucosa is not only a site of acute CD4 T-lymphocyte depletion of resident T lymphocytes during this infection phase, but may also be actively recruiting CD4 (CCR5⁺) T lymphocytes from the peripheral blood pool which represent additional targets for viral entry and virus replication in mucosal tissues (45). CD4 T-lymphocyte homing to the intestinal mucosa and subsequent gut CD4 T-lymphocyte depletion may significantly contribute to the gradual CD4 T-lymphocyte decline in peripheral blood in later stages of disease, when normal homeostatic T-lymphocyte maintenance mechanisms are disrupted (9).

Increased gut mucosal trafficking of CFSE⁺ T lymphocytes or, alternatively, the migration of dye-positive T lymphocytes to the small intestine (and proliferation within this site) occurred concomitantly with massive CD4 T-lymphocyte depletion in gut tissues in all three SIV-infected animals. Acute and chronic CD4 T-lymphocyte depletion in the gut mucosa was previously reported with both SIV and HIV infection and is thought to critically impact AIDS pathogenesis (5, 13, 28, 40, 43, 44). Possible redistribution of peripheral T-lymphocyte pools towards the intestinal mucosa in acute SIV infection may occur via CCR9-mediated trafficking in a CCL25-dependent manner (18, 32). Other signals such as adhesion molecules and alternate chemokine ligand-receptor interactions are likely to play an important role in migration and positioning within chemokine gradients in this compartment (17). Elevated chemokine CXCL9 and CCL19 levels in gut tissue of SIV-infected compared to that of uninfected animals (Fig. 1) may contribute to CFSE⁺ T-lymphocyte trafficking, retention, and compartmentalization within the lamina propria. Considering the markedly higher frequencies of ligand binding receptor CXCR3⁺, as well as CCR7⁺ gut mucosal T-lymphocyte subsets in the CD4 compartment, these data suggest possible recruitment of CXCR3⁺ and CCR7⁺ CD4 T-lymphocyte subsets into this site, which will be examined in future experiments.

Our studies may facilitate assessment of cell proliferation in vivo (and changes in the CFSE⁺ cell pool), based on twofold reduction in CFSE MFI with each cell division (25). Indeed, our initial experiments with acutely SIV-infected animals revealed differences in CFSE MFI levels between peripheral tissues (peripheral blood, spleen, and lymph nodes) and the small intestine. The observed two- to fourfold-lower fluorescence intensity in the gut mucosa, a site of active viral replication during acute SIV infection, may reflect one to two extra cell divisions. These findings are similar to the reported accumulation of highly proliferating CFSE-low CD8 T lymphocytes (unimodally distributed) in lung tissue during acute influenza virus infection (19). Absence of CFSE laddering may be related to a short in vivo migratory period (<48 h), recruitment of activated cells with a similar proliferative capacity (upon arrival and exposure to SIV antigens), and potential masking of smaller subsets with a distinct proliferative profile. Nevertheless, we cannot currently exclude the possibility that the loss in CFSE MFI is a consequence of events not associated with cell divisions and that the CFSE-low cells accumulating in the small intestine are nonrepresentative of the transferred PBMC (with lower CFSE MFI levels at the time of transfer). Alternatively, reduced fluorescence may indicate increased protein turnover in gut T lymphocytes or loss of membrane integrity in dying cells (and leakage of CFSE⁺ proteins from the cytoplasm). These possibilities will be examined in greater detail in future animal experiments and sample collections.

In this study, we presented a reference profile for chemokine levels, chemokine receptor expression, detection thresholds for CFSE⁺ T lymphocytes, and their trafficking paths into tissues for future adoptive transfer experiments in primates, by both our research group and other investigators. Importantly, we provided novel avenues to define cross talk between peripheral blood and the gut mucosa and demonstrated distinct gut mucosal trafficking parameters and CFSE⁺ T-lymphocyte migration to the small intestine in SIV infection that may affect antiviral immune functions, as well as the disease course. This work may have significant impact on the study of SIV pathogenesis and delineation of a possible mechanism(s) of peripheral T-lymphocyte decline in simian AIDS, based on disrupted T-lymphocyte trafficking circuits, altered chances for viral antigen encounter, and T-lymphocyte turnover in specific sites.

 
We thank the Animal Services, Veterinary Care, and Clinical Laboratory staff at the California National Primate Research Center for expert help and support in coordinating this study. We thank Chris Miller for providing SIV_mac251 virus stocks and John Altman and Eugene Ravkov for providing human CCL19 monomers. We also thank Holden Maecker, Mike McChesney, Todd Reinhart, and Stephen Stohlman for critical feedback and input.

This work was supported by IDEA award ID01-D-130 from the Universitywide AIDS Research Program of the State of California (to U.E.), partial salary support provided by a UC Davis Health Systems Research Award (to U.E.), a UC Davis Department of Pathology Graduate Student Research Award (to C.C.C.), and NIH training grant 5T32 AI060555 (to C.C.C.).

 
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Research III Building, Room 3400A, University of California—Davis Medical Center, 4645 2nd Ave., Sacramento, CA 95817. Phone: (916) 734-4789. Fax: (916) 734-2698. E-mail: uesser{at}ucdavis.edu.

Contributed equally to this work.

Present address: Division of Infectious Diseases, Universidade Federal de Sao Paulo, 04039-032 Sao Paulo, Brazil.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, November 2005, p. 13759-13768, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13759-13768.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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