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Development and Homeostasis of T Cell Memory in Rhesus Macaque¹

Christine J. Pitcher^2,*, Shoko I. Hagen^2,*, Joshua M. Walker^*, Richard Lum^*, Bridget L. Mitchell^*, Vernon C. Maino, Michael K. Axthelm^* and Louis J. Picker^3,*

^* Vaccine and Gene Therapy Institute, Oregon Regional Primate Research Center, Oregon Health and Science University, West Campus, Beaverton, OR 97006; and BD Biosciences, San Jose, CA 95131

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The rhesus macaque (RM) is a critical animal model for studies of viral pathogenesis and immunity, yet fundamental aspects of their cellular immune response remain poorly defined. One such deficiency is the lack of validated phenotypic signatures for their naive and memory T cell subsets, and the resultant unavailability of accurate information on their memory T cell development, homeostasis, and function. In this study, we report a phenotypic paradigm allowing definitive characterization of these subsets and their comprehensive functional analysis. Naive T cells are optimally delineated by their homogeneous CD95^lowCD28^high₇ integrin^int (CD4⁺) or CD95^lowCD28^intCD11a^low (CD8⁺) phenotypes. This subset 1) was present in blood and secondary lymph tissues, but not effector sites; 2) vastly predominated in the fetal/neonatal immune system, but rapidly diminished with postnatal age; 3) lacked IFN- production capability, and specific responses to RM CMV; and 4) demonstrated low in vivo proliferative activity. CD4⁺ and CD8⁺ memory subsets were CD95^high, but otherwise phenotypically heterogeneous and included all IFN- production, RM CMV-specific responses, effector site T cells, and demonstrated high in vivo proliferative activity (10 times the naive subset). These analyses also revealed the RM "effector memory" subset within the overall memory population. This population, best defined by lack of CD28 expression, contained the majority of RM CMV-specific cells, was highly enriched in extralymphoid effector sites, and comprised an increasing proportion of total memory cells with age. The effector memory subset demonstrated similar in vivo proliferative activity and survival as CD28⁺ "central memory" T cells, consistent with independent homeostatic regulation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The evolution of phenotypic criteria for the delineation of human naive and memory/effector T cells over the past 12 years has revolutionized understanding of the cellular basis of immunologic memory and has greatly facilitated the investigation of T cell function and homeostasis in healthy and diseased individuals (1, 2, 3, 4, 5, 6, 7). The now well-established memory/naive paradigm holds that both CD4⁺ and CD8⁺ peripheral T cells can be readily divided into two major subsets: 1) an Ag inexperienced (naive) subset with a broad thymic-selected TCR repertoire, low immediate effector potential, high costimulatory requirements, low turnover, and almost exclusive recirculation through secondary lymphoid tissues; and 2) an Ag-experienced (memory/effector) subset with an Ag-selected TCR repertoire; heterogeneous, but highly differentiated, functional potential; heterogeneous, but on average low, costimulatory requirements; relatively high turnover; and the ability to broadly or selectively recirculate through the various extralymphoid tissues of the body (effector sites or potential effector sites) (4, 5, 7, 8, 9, 10). Although in unusual circumstances memory differentiation might occur in the absence of Ag (e.g., severe lymphodepletion) (11), the vast majority of memory cells are thought to undergo the naive to memory differentiation process immediately following their first contact with and activation by Ag in secondary lymphoid tissues coincident with rapid clonal expansion (5). The functional potential of the resultant memory/effector cells is thought to be initially determined by microenvironmental conditions during the naive to memory/effector transition, but then potentially modified during subsequent encounters with Ag in diverse microenvironments (5, 12, 13, 14).

This memory/naive T cell paradigm in humans was developed by painstaking, functional analysis of phenotypically defined T cell subsets and, not surprisingly, many of the phenotypic criteria used to discriminate naive and memory subsets ultimately involve cell surface molecules whose functions reflect this paradigm. Naive T cells homogeneously express moderate to high levels of cell surface molecules involved in secondary lymphoid tissue migration (CD62L, ₄₇ integrin, CCR7) and costimulation (CD27, CD28), low levels of general adhesion molecules (CD11a, CD49d, CD58, CD2) and apoptosis-associated molecules (CD95), lack expression of molecules involved in migration to extralymphoid effector sites (cutaneous lymphocyte-associated Ag, CCR5), and express high and low levels of the RA and RO CD45 isoforms, respectively (2, 4, 15, 16, 17, 18, 19). In contrast, consistent with their marked functional diversity, memory cells lack a precise identifying phenotype; instead, they express complex, but stereotyped, patterns of these markers that allow clear-cut differentiation from the naive subset only with strategic multiparameter analysis (2, 4). The CD4 and CD8 lineages differ in detail, but both conform to these general principles.

The increasingly sophisticated and validated phenotypic criteria for delineating naive and memory T cell populations in humans have been effectively applied to numerous investigations of immune pathobiology, most notably in HIV disease (1, 3, 6, 20, 21). However, much less rigor has been applied to the study of T cell memory in the major animal model for HIV infection, the rhesus macaque (RM).⁴ Cross-reacting, human-specific reagents have been used to investigate peripheral T cell populations in these animals, but without the optimization, validation, and functional correlation that would be required for the development of accurate analytic strategies. The availability of such strategies would greatly enhance the quality of data obtained from numerous ongoing studies of SIV immuno- and pathobiology, as well as other RM infectious disease models. Moreover, this human-like animal model would appear to offer a unique, heretofore underexploited opportunity to delineate aspects of in vivo T cell physiology that, due to practical or ethical considerations, are not readily approachable in the human system. Thus, we have undertaken the task of 1) determining optimal reagents and developing validated criteria for the delineation of naive and memory T cells in RM, 2) developing analytical approaches to ascertain the function and turnover of these cells, and 3) using these criteria and tools to explore fundamental aspects of RM memory T cell development and homeostasis in vivo. We were also interested in defining those components of memory directly involved in host defense against chronic viral infection—the so-called "effector memory" subset (8, 18, 22, 23). Our results establish a strikingly rapid, coordinate development of CD4⁺ and CD8⁺ T cell memory in normal RMs and characterize a remarkable steady-state dynamism in the turnover of these memory populations. Moreover, these animals quickly establish and maintain high frequencies of differentiated, effector memory CD8⁺ T cells, consistent with a requirement to maintain high levels of immune activity directed at persistent pathogens. These features are much more pronounced and time contracted than in the (western society) human, suggesting the RM model as an invaluable resource in the delineation of the fundamental mechanisms controlling T cell memory in primates.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

All animals used in this study were colony-bred RM (Macaca mulatta) of Indian origin maintained and used in accordance with guidelines of the Animal Care and Use Committee at the Oregon Regional Primate Research Center and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Healthy animals of either sex were selected to represent the life span of the RM for peripheral blood analyses. Tissues were obtained from necropsied animals through the Oregon Regional Primate Research Center’s Tissue Distribution Program. Unless otherwise indicated, these animals were free of known infectious or immunologic disease. Animals were anesthetized with ketamine hydrochloride for blood drawing and 5-bromo-2'-deoxyuridine (BrdU) injection. BrdU was given i.v. or i.p. at schedules indicated in the figure legends.

Cell preparation and Ag stimulation

PBMC were isolated from heparinized or citrated venous blood by density gradient sedimentation using Ficoll-Hypaque (Histopaque-1077; Sigma-Aldrich, St. Louis, MO). Peripheral lymph node (PLN) and spleen cells were obtained by gentle mincing of tissues in complete medium (RPMI 1640 medium; HyClone Laboratories, Logan, UT) supplemented with 10% heat-inactivated FCS (HyClone Laboratories), 2 mM L-glutamine (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), and 50 µM 2-ME (Sigma-Aldrich) with splenocytes also subjected to Ficoll-Hypaque centrifugation to remove RBCs. Small intestinal lamina propria cells were obtained as previously described (24) from segments of small intestinal mucosa with as few grossly recognizable Peyer’s patches as possible. Bronchoalveolar lavage cells were obtained by instilling and aspirating 150 ml of PBS into the main bronchus of each lung at necropsy. All cells were washed twice in HBSS (Ca²⁺/Mg²⁺-free, Cellgro/Mediatech; Fisher Scientific, Federal Way, WA) and resuspended in complete medium.

Ag stimulations for intracellular cytokine staining were performed as follows: PBMC or other cell preparations were placed in polypropylene tissue culture tubes (BD Biosciences, Franklin Lakes, NJ) at 1 x 10⁶ cells/ml complete medium (1–10 ml/tube) with appropriately titered "whole" RM CMV viral preparations (50 µl of preparation/ml), CMV immediate early-1 (IE-1) 15-mer peptide mixes (Ref. 25 and see Ags and Abs), the superantigen staphylococcal enterotoxin B (SEB, 200 ng/ml; Toxin Technology, Sarasota, FL), or no Ag as a negative control (previously shown to be equivalent to mock virus preparations), with or without one or both of the costimulatory mAbs CD28 and CD49d (0.5 µg/ml each; these mAbs provide exogenous costimulation so as to allow the total cohort of Ag-specific cells to respond in this assay; Refs 26, 27). The cultures were routinely incubated at a 5^o slant at 37°C in a humidified 5% CO₂ atmosphere for 6 h, with the final 5 h including 10 µg/ml brefeldin A (Sigma-Aldrich). After incubation, cells were harvested by washing in cold (4°C) Dulbecco’s PBS (dPBS; Life Technologies, Rockville, MD) with 0.1% BSA (Roche Biochemicals, Indianapolis, IN) and were kept at 4°C until processed for staining.

Immunofluorescent staining and flow cytometric analysis

For cell surface staining, 0.25–1.0 x 10⁶ cells were incubated with appropriately titered directly conjugated mAbs for 25 min at room temperature, followed by washing at 4°C, and resuspension in 1% paraformaldehyde in dPBS. Stained cells were then kept protected from light at 4°C until analysis on the flow cytometer. For intracellular cytokine analysis, stimulated cells were first stained on the cell surface with directly conjugated mAbs to CD3, CD4, CD8, and other phenotyping markers (25 min each at room temperature), washed once with cold dPBS/BSA before resuspension in fixation/permeabilization solution (BD Biosciences, San Jose, CA; used at 2x recommended concentration) at 2 x 10⁶ cells/ml, and incubated for 10 min at room temperature in the dark. Fixed and permeabilized cells were washed twice with cold dPBS/BSA, and then incubated on ice (protected from light) with directly conjugated anti-cytokine and CD69 mAbs for 25 min. Intracellular staining of Ki-67 vs cell surface markers was similar to cytokine analysis except that permeabilization was performed in 1x concentration fixation/permeabilization solution. For BrdU analysis (vs cell surface markers, Ki-67, or intracellular cytokine), cells were stained for surface markers first, fixed for 10 min at room temperature with FASCLyse solution (BD Biosciences), and then permeabilized for 12–14 h in 2x concentration fixation/permeabilization solution at 4°C. After washing twice with cold dPBS/BSA, cells were incubated on ice (protected from light) with directly conjugated mAbs specific for BrdU, Ki-67, anti-cytokine, and/or CD69 mAbs in the presence of 0.28 mg of bovine pancreas-derived DNase 1 (Sigma-Aldrich catalogue no. D4513) for 30 min. After staining, cells were resuspended in 1% paraformaldehyde in dPBS and stored in the dark at 4°C.

Six-parameter flow cytometric analysis was performed on a two-laser FACSCalibur instrument (BD Biosciences) using FITC, PE, peridinin chlorophyll protein-Cy5.5 (True Red), and allophycocyanin as the four- fluorescent parameters. List mode multiparameter data files (each file with forward scatter, orthogonal scatter, and four-fluorescent parameters) were analyzed by "cluster" analysis using the PAINT-A-GATE^Plus software program (BD Biosciences) (28). Special mention should be made of clustering CD8⁺ T cells in RM. Whereas CD4⁺ T cells can be accurately delineated by CD4 expression and small lymphocyte light scatter signals, this is not possible for CD8⁺ T cells using conventional CD8-specific reagents due to high expression of CD8 homodimers by RM NK cells and to low to moderate expression of these homodimers by CD4⁺CD28^- (effector memory) T cells (data not shown). Accurate clustering of conventional CD8⁺ T cells (which express CD8 heterodimers; data not shown) with a single fluorescence parameter therefore requires use of a CD8-specific reagent (see below). The specificity of staining and criteria for determining positive staining for Ki-67 and BrdU were determined using isotype-matched negative control mAbs, and, in the case of BrdU, analysis of control (non-BrdU-pulsed) animals. The procedures and criteria for delineating and quantifying responding (CD69 positive/cytokine positive) vs nonresponding T cells have been previously described in detail (26, 27).

Ags and Abs

RM CMV (Cercopithecine Herpesvirus 8) Ag preparations were made from 68.1 strain (ATCC VR-677)-infected monolayers of primary RM fibroblasts after a 90–100% cytopathic effect was reached. Infected cells were scraped off the flasks, and then cells and media were clarified by centrifugation at 3840 x g for 10 min. Cell pellets were resuspended in media, freeze-thawed three times, sonicated for 10 cycles (30 s/cycle), and then clarified by centrifugation at 3840 x g for 10 min. Supernatants from both original culture media and lysed cell pellets were combined and the virus was pelleted at 12,400 x g for 1 h. Viral pellets were resuspended in medium (2% of original culture volume) and titered in the cytokine flow cytometry response assay. RM CMV IE-1 peptides (consecutive 15-mers overlapping by 11 aa) were custom synthesized by Dr. D. Stoll (Natural and Medical Sciences Institute of the University of Tübingen, Tübingen, Germany) based on the IE-1 sequence of RM CMV strain 68.1 (GenBank accession no. M93360). Peptide sequences were confirmed by electrospray mass spectroscopy. To prepare total IE-1 mixes, the 137 overlapping peptides were individually solubilized in DMSO (Sigma-Aldrich) at 100 mg/ml and mixed together so that the final concentration of each individual peptide was 0.72 mg/ml. Two microliters of this mix was used per milliliter of cell stimulation medium (1.45 µg/ml final concentration of each peptide).

mAbs L200 (CD4; True Red, allophycocyanin conjugated), SP34 (CD3; FITC, PE, True Red, allophycocyanin), SK1 (CD8; FITC, allophycocyanin), L78 (CD69; PE, allophycocyanin), L293 (CD28; unconjugated PE), CD28.2 (CD28; FITC), L25.3 (CD49d; unconjugated PE), L48 (Leu45RA; FITC), M-T271 (CD27; PE), G25.2 (CD11a; FITC), H111 (CD11a; PE), FIB504 (₇ integrin; PE), SK11 (CD62L; PE), DX2 (CD95; allophycocyanin), B56 (Ki-67; FITC, PE), B44 (anti-BrdU; FITC), B27 (anti-IFN-; FITC, allophycocyanin), 11 (anti-TNF-; FITC, allophycocyanin), and IgG1 and IgG2 isotype-matched controls were obtained from BD Biosciences. mAbs 2H4 (CD45RA; PE) and 2ST8.5h7 (CD8; PE unconjugated) were obtained from Beckman Coulter (Fullerton, CA). Purified 2ST8.5h7 was custom conjugated to True Red by BD Biosciences.

Statistical analysis

Statistical analysis was conducted with the program Statview (Abacus Concepts, Berkeley, CA). The significance of differences between paired groups was analyzed with the Wilcoxon signed rank test. The relationships between variables were analyzed with the Spearman rank correlation test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Development of phenotypic criteria for naive/memory cell discrimination in RM

We adopted the following stepwise approach for the definition of naive and memory T cell subsets in RM: 1) assessment of mAbs known to delineate appropriate T cell populations in humans for (high-level) cross-reactivity with RM T cells, 2) selection of reagents and optimization of staining combinations so as to definitively separate the putative naive and memory populations and memory subsets of interest (e.g., the putative effector memory subset); and 3) validation of subset identity by functional analysis and studies of population dynamics. Our initial screening identified one or more mAbs against human CD11a, CD28, CD62L, CD45RA, CD49d, CD95, and ₇ integrin that were usefully cross-reactive with RM T cells and when assessed by multiparameter (four-color) flow cytometry in various combinations appeared to be capable of delineating putative naive and memory subsets (based on previous described criteria for human T cells (2, 4, 5, 18, 19)). Optimal separation of putative CD4⁺ naive and memory T cells was achieved by the combination of CD95, ₇ integrin, and/or CD28 in which putative naive cells were apparent as a uniform CD95^lowCD28^high, ₇ integrin^int population (Fig. 1A). For CD8⁺ T cells, CD95, CD28, and CD11a mAbs were the most discriminatory with the putative naive subset again showing the expected homogeneous phenotype—CD95^lowCD28^intCD11a^low (Fig. 1B).

View larger version (54K): [in this window] [in a new window]   FIGURE 1. Coordinate expression of putative memory/naive markers on RM CD4+ and CD8+ T cells. RM PBMC (6.8-year-old animal) were examined for their correlated expression of cell surface CD4 or CD8 vs CD95 vs various paired combinations of CD28, CD45RA, CD11a, 7 integrin, CD62L, and CD49d (4 integrin). Five thousand events gated on CD4+ (A) or CD8+ (B) small lymphocytes are shown. The putative naive population is colored blue based upon characteristic homogeneous expression of these markers (2 4 ), with the remaining cells (putative memory cells) colored red. Optimal discrimination of the naive cluster was observed with CD95 vs 7 integrin vs CD28 for CD4+ T cells and with CD95 vs CD28 vs CD11a for CD8+ T cells, and the frequencies provided in the light scatter profiles reflect these combinations. However, the equivalency of the colored populations in each profile was tested by extensive crossover analysis, and the coefficient of variation of the percent naive was <3% in these different analyses. The designations ++, +, and - reflect relative intensity levels of the indicated marker (using naive cell staining intensity as internal control) that define memory subsets referred to throughout this report. Arrowheads in A designate a distinct cluster of CD28-CD11ahighCD45RA+CD62L- cells, consistent with the CD4+ T cell effector memory population (see text); arrowheads in B designate a unique subset of likely gut-associated CD28-CD8+ T cell effector memory cells characterized by bright 7 integrin and dim CD11a.

Validation of criteria for naive/memory cell discrimination in RM by subset localization and development

Extensive data in both rodents and humans have established that recirculation of naive T cells is largely, if not exclusively, restricted to secondary lymphoid tissues (PLN, spleen, Peyer’s patch, tonsil), whereas memory cells are normally localized in both of these sites and extralymphoid "tertiary" sites (effector sites or potential effector sites) (5, 7, 30). In keeping with this paradigm, putative RM naive T cells, both CD4⁺ and CD8⁺, were essentially absent from representative tertiary sites (lung bronchoalveolar and small intestinal lamina propria T cells; Fig. 2), but formed a significant component of the T cell populations within PLN and spleen (Fig. 2 and Table I). As has been previously shown in other systems, small intestinal lamina propria memory cells (particularly the CD8⁺ subset) were almost exclusively ₇ integrin positive, likely due to the role of ₄₇ integrin in mediating T cell homing to this site and to in situ up-regulation of _e7 integrin (7). ₄₇ integrin has not been implicated in lung T cell homing (31), and in this site the subset of ₇ integrin^int cells apparent in Fig. 2 is predominantly due to in situ up-regulation of _e7 integrin (data not shown).

View larger version (51K): [in this window] [in a new window]   FIGURE 2. RM T cells bearing the putative naive phenotype are present in blood and secondary lymphoid tissue, but not extralymphoid effector sites. RM PBMC, and cells from PLN, small intestinal lamina propria, and bronchoalveolar lavage preparations (10.9-year-old animal) were examined for their correlated expression of cell surface CD4 or CD8 vs CD95 vs CD28 or 7 integrin. Two thousand events gated on CD4+ (A) or CD8+ (B) small lymphocytes are shown, with arrows designating the putative naive population in blood and PLN. Note the absence of a distinct naive T cell cluster in small intestine and lung.

A second fundamental precept of the naive/memory paradigm is the dominance of the naive subset in the newborn and the subsequent development of the memory subset with postnatal Ag exposure (5). Again, the delineation of naive and memory subsets by the above criteria conforms well to this paradigm. As shown in Fig. 3, CD4⁺ and CD8⁺ lineage cells with a putative naive phenotype dominate in neonatal blood, but cells bearing a putative memory phenotype rapidly increase with postnatal age. Interestingly, unlike our published experience in humans (32, 33), small populations of memory T cells, particularly CD8⁺ memory cells, are present in neonatal blood. Importantly, however, the frequency of these cells always increased with postnatal time, in keeping with our interpretation of these phenotypically defined subsets. It is also important to note that while memory T cells display defined ₇ integrin^{high, int, and low} subsets at birth, few CD28^- T cells are present in the neonatal memory population. This subset increases in relative frequency with postnatal time, consistent with a maturation process after the initial naive to memory transition (see below).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Peripheral blood T cells with a putative memory phenotype are rare at birth in RM, but rapidly accumulate in the first few months of postnatal life. PBMC from blood obtained in the first 24 h of life and at postnatal day 72 from the same RM were examined for their correlated expression of CD4 or CD8 vs CD95 vs CD45RA vs either CD28 or 7 integrin and analyzed as described in Fig. 1 legend. Five thousand events gated on CD4+ (A) or CD8+ (B) small lymphocytes are shown, with the overall percent naive (blue)/memory (red) provided in the top left profiles in each figure, and the percentage of the memory population with the designated phenotypes (CD28- or CD45RA++) shown in association with defining lines and arrows. Similar analyses on sequential neonatal and 2- to 5-mo postnatal specimens were performed on seven additional RM, and the frequencies of CD4+ (C) or CD8+ (D) T cells with the putative memory phenotype for all eight animals studied are shown.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. RM memory phenotype CD4+ and CD8+ T cells coordinately accumulate with age. This figure demonstrates a cross-sectional analysis of the frequencies of memory phenotype T cells throughout the RM life span. Memory T cell frequencies were determined using patterns of 7 integrin, CD28, and CD95 expression, as shown in Figs. 1 and 3. Spearman rank correlation coefficients () and p values are shown for percent CD4+ and percent CD8+ memory T cell frequencies vs age and vs each other.

Perhaps the most fundamental differences between naive and memory T cells concern functional capabilities and TCR repertoire. With respect to the former, naive T cells are largely incapable of (immediate) synthesis of certain effector cytokines such as IFN-, even with optimal TCR stimulation and costimulation, whereas this capability is developed by memory T cells upon receiving appropriate differentiation signals during the naive to memory transition (34). With respect to repertoire, the naive subset manifests the broad thymic selected repertoire with clonal frequencies to any given (potential) Ag too low to be recognized by current functional assays without prior proliferative expansion (e.g., 1:10⁶); in contrast, the memory subset manifests an Ag-selected repertoire and may include Ag-specific frequencies well above 1%, especially responses directed against persistent viruses like CMV (5). To test the functional and repertoire characteristics of our phenotypically defined subsets, we used cytokine flow cytometry, a technique that allows precise quantification and phenotypic characterization of TCR-triggered T cells (25, 26, 27). In this assay PBMC are incubated with Ag or other stimuli (and costimulatory Abs to lower response thresholds) for 6 h with the secretion inhibitor brefeldin A present the last 5 h. It is important to note that brefeldin A not only retains secreted products such as cytokines within the cell cytoplasm (in a modified Golgi), but also any induced cell surface molecules (27). Thus, even with potent polyclonal T cell stimulation, no change in surface phenotype (i.e., stained before fixation and permeabilization) is observed for most Ags during the course of this assay, including all those used as primary phenotyping markers in this study (data not shown).

To assess IFN- synthesis capabilities of putative naive vs memory subsets, we utilized stimulation with the superantigen SEB, which stimulates on the basis of TCR V chain recognition (26), and thus stimulates both naive and memory subsets. To ensure suprathreshold stimulation of naive cells, we also included costimulatory mAbs (either CD28 and CD49d or CD49d alone). As illustrated in Fig. 5 (representative of five independent experiments with different animals), this stimulation protocol elicited substantial frequencies of IFN--producing cells in both the CD4⁺ and CD8⁺ subsets—responses that were restricted almost entirely to the phenotypically defined memory population. This restriction was similar regardless of the level of exogenous costimulation provided (none, CD49d alone, or CD28 plus CD49d; data not shown). Importantly, IFN- producers were well represented in all ₇ integrin-, CD28-, and CD45RA-defined memory subsets, although the frequency of such responders in these subsets varied among different animals (likely due to different distributions of T cells with appropriate TCR-V).

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Memory, but not naive, phenotype T cells produce IFN- after superantigen stimulation. RM PBMC (8.4-year-old animal) were stimulated with the superantigen SEB plus CD49d for 6 h in the presence of the secretion inhibitor brefeldin A for the final 5 h, and then examined for their correlated expression of cell surface CD4 or CD8 vs CD95 vs either 7 integrin, CD28, CD45RA, or CD11a (stained before fixation and permeabilization) vs intracellular IFN- (stained after fixation and permeabilization), or cell surface CD4 or CD8 vs intracellular IFN- and CD69. Ten thousand events gated on CD4+ (A) or CD8+ (B) small lymphocytes are shown, with the events within the IFN-+ (SEB-responding) population enlarged and colored black (percent responding provided in the lower right corner of the CD69 vs IFN- profile). Parallel cultures treated with CD49 mAbs alone and analyzed in similar fashion revealed no IFN- response (data not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 6. T cells responding to the chronic virus RM CMV display a memory phenotype. RM PBMC (9.5- and 8.4-year-old animals for A and B, respectively) were stimulated with the whole CMV Ag preparations (CD4+ T cells; A) or IE-1 peptide mixes (consecutive 15-mers overlapping by 11 aa; CD8+ T cells, B) plus CD49d for 6 h in the presence of the secretion inhibitor brefeldin A for the final 5 h, and then examined for their correlated expression of cell surface CD4 or CD8 vs CD95 vs either 7 integrin, CD28, CD45RA, or CD11a (stained before fixation and permeabilization) vs intracellular TNF- (stained after fixation and permeabilization), or cell surface CD4 or CD8 vs intracellular IFN- and CD69. Fifteen thousand events gated on CD4+ (A) or CD8+ (B) small lymphocytes are shown, with the events within the TNF- plus (Ag-responding) population enlarged and colored black (percent responding provided in the lower right corner of the CD69 vs IFN- profile; see also Table II). Parallel cultures treated with CD49 mAbs alone and analyzed in similar fashion revealed no IFN- response (data not shown).

View this table: [in this window] [in a new window]   Table II. Distribution of CMV-specific T cells within the memory subsets defined by CD28, CD45RA, and 7 integrin

Although the concept of specific functional specialization among memory T cells goes back many years, there has been recent appreciation for a broad dichotomy in memory T cell differentiation based on the ability of a memory cell to manifest immediate (i.e., without further maturation), direct anti-pathogen effector activities in tertiary (extralymphoid) sites (8, 18, 22, 23, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44). So-called effector memory T cells are thought to be fully, perhaps terminally, differentiated cells with high potential for immediate cytotoxic function, polarized cytokine synthesis function (e.g., Th1 vs Th2), predominant homing to and localization within extralymphoid tissues, and reduced costimulatory requirements. This population also includes the majority of memory T cells specific for chronic pathogens, and accumulates with age. Effector memory T cells are thought to derive from so-called "central" memory cells which have little immediate cytotoxic potential, nonpolarized cytokine synthesis function, predominant homing to and localization within secondary lymphoid tissues, and higher costimulatory requirements. Although the phenotypic correlates used to delineate these populations vary among investigators, CD27, CD28, CD45RA, CCR7, and CD62L have been the most widely used in humans, with the most differentiated effector memory population characterized as CD27^-, CD28^-, CD45RA⁺, CCR7^-, and CD62L^-, and the "archetype" central memory population having the reciprocal phenotype.

As indicated above, analogous CD28^- and CD45RA^high memory subsets were recognizable, indeed quite prominent, in the RM, and included most of the RM CMV-specific memory T cells in both the CD4⁺ and CD8⁺ lineages. To further evaluate the significance of these phenotypes, we quantified the frequencies of the CD28^- and CD45RA^high subsets within the overall population of peripheral blood memory cells in our cross-sectional cohort of 87 animals and correlated these frequencies with age and with each other. As shown in Fig. 7, there was a striking correlation between the size of the CD28^- subset and age for both the CD4⁺ and CD8⁺ populations, although the pattern of this relationship was different for the two lineages. The fraction of memory CD8⁺ T cells with a CD28^- phenotype rapidly increased in the first 3 years of life and then leveled off; whereas for CD4⁺ memory cells, the accumulation of CD28^- cells was more gradual and continued through adulthood. For the CD4⁺ lineage, the CD45RA^high subset showed a similar correlation with age and, in fact, demonstrated a strong, direct correlation with the CD28^- subset (in keeping with our general impression on multiparameter flow cytometric analysis that in peripheral blood, these two subsets were largely, although not completely, overlapping; Fig. 1A and data not shown). In contrast, the CD8⁺/CD45RA^high memory subset showed no correlation with age or with the CD8⁺CD28^- subset. Indeed, multiparameter analysis of these two markers on CD8⁺ memory cells revealed multiple nonoverlapping patterns of expression (Fig. 8A). Furthermore, whereas CD8⁺CD28^- memory cells were highly enriched in effector sites (lung and gut lamina propria) as compared with lymph node, CD8⁺CD45RA^high memory cells were highest in peripheral blood, next highest in lymph node, and almost absent in the effector sites examined (Fig. 8B). This same relative tissue distribution was observed for CD4⁺CD28^- and CD4⁺CD45RA^high T cells as well (data not shown), suggesting that the correlation of the CD28^- and CD45RA^high phenotypes observed in the CD4⁺ memory T cell subset in peripheral blood did not extend to CD4⁺ memory T cells in tissues. Taken together, these data support the conclusion that the CD28^- phenotype consistently delineates RM effector memory differentiation for both CD4⁺ and CD8⁺ T cells, whereas the CD45RA^high phenotype does not.

View larger version (50K): [in this window] [in a new window]   FIGURE 7. Frequencies of CD28- T cells in peripheral blood increase with age within both the CD4+ and CD8+ memory T cell subsets, whereas CD45RA++ cells accumulate with age only within the CD4+ memory subset. This figure demonstrates a cross-sectional analysis of the frequencies of CD28-, CD45RA++, CD4+, or CD8+ T cells within the overall memory phenotype T cell population vs age of the animal and each other. CD28- and CD45RA++ memory T cell frequencies were determined as shown in Fig. 1. Spearman rank correlation coefficients () and p values are shown for each analysis.

View larger version (36K): [in this window] [in a new window]   FIGURE 8. The CD8+CD28- and CD8+CD45RA++ memory subsets are divergent with only the CD28- subset showing enrichment in effector sites. A, RM PBMC from four different animals (0.4, 3.4, 5.0, and 9.8 years old from left to right, respectively) were examined for their correlated expression of cell surface CD8 vs CD95 vs CD28 vs CD45RA. Five thousand events gated on CD8+CD95high (memory) small lymphocytes are shown. These profiles represent the spectrum of patterns observed for expression of CD28 and CD45RA among CD8+ memory T cells; note the lack of correspondence of the CD28- and CD45RA++ subsets. B, CD8+CD28- and CD8+CD45RA++ memory subsets were defined as indicated in Figs. 1B and 8A and quantified in four different animals (, 7.0 years old; , 12.3 years old; , 9.1 years old; •, 10.9 years old) in PBMC, PLN, MLN, bronchoalveolar lavage cells (lung), and small intestinal lamina propria cells (gut).

Data in both rodent models and humans have indicated that homeostasis of naive and memory cells is achieved by fundamentally different mechanisms: naive T cells are thought to be long-lived, with only low-level homeostatic turnover, whereas memory T cell homeostasis is associated with high turnover (9, 20, 45). To investigate the in vivo turnover of the phenotypically defined subsets described above, we used two complementary approaches: 1) in vivo pulsing with the thymidine analog BrdU followed by quantification of BrdU⁺ T cells, and 2) quantification of T cells expressing the cell cycle-associated nuclear Ag Ki-67. BrdU incorporation allows precise determination of cells in S phase during the pulse, and labeled cells can be followed over time. Staining is very discrete, and thus it is precisely quantitative. The main liability of this approach is the potential toxicity of BrdU (Ref. 46 ; although doses used here had no discernible toxic effects) and the impracticality of its use in large RM cohorts.

In contrast, Ki-67 reactivity, which implies a cell is cycling (i.e., non-G_o phase; Ref. 47), requires no pretreatment of the animals, and thus can readily be applied to large cohorts. However, Ki-67 expression is less discrete than that of BrdU, and its expression may miss cells proliferating in tissues that return to G_o phase before returning to peripheral blood. Moreover, the temporal significance of Ki-67 expression in vivo is poorly characterized; i.e., it is unclear whether Ki-67 reactivity suggests that a cell that has undergone DNA synthesis in the last few hours, the last few days, or even more remotely. To better interpret the in vivo significance of Ki-67 reactivity in RM, we focused our initial experiments on the coordinate study of Ki-67 and BrdU expression by T cells harvested immediately after varying periods of in vivo BrdU exposure in different animals. As shown in Fig. 9A for CD4⁺ T cells, 1 h after a single i.v. dose of BrdU, few Ki-67⁺ cells are also BrdU⁺. After three i.v. doses of BrdU over 24 h, only 31% of Ki-67⁺ cells are BrdU⁺. However, after two i.v. doses a day of BrdU for 3 consecutive days, 75% of total Ki-67⁺ and essentially all Ki-67^bright cells are BrdU⁺; in addition, there are few BrdU⁺ cells that lack Ki-67. After administering single high i.p. doses of BrdU for 4 consecutive days (given i.p. to prolong drug availability), essentially all Ki-67⁺ are BrdU⁺, but in this situation many of the BrdU⁺ cells (30%) are clearly beginning to lose Ki-67 reactivity. These observations suggest that Ki-67 expression delineates cells that have undergone S phase in the prior 3–4 days, and that few, if any, T cells in these normal animals remain arrested in the G₁ phase of the cell cycle (e.g., remain Ki-67⁺ without going through S phase and becoming BrdU⁺) for longer than this time period.

View larger version (46K): [in this window] [in a new window]   FIGURE 9. Ki-67+ reactivity delineates T cells that have undergone S phase in the preceding 3–4 days, and when analyzed in conjunction with BrdU pulsing allow estimation of T cell turnover. A, Different RM were i.v. pulsed with BrdU for 1 h (100-mg single i.v. dose, PBMC collected 1 h later), 1 day (three 100-mg i.v. doses, given 8 h apart with PBMC collection 2 h after the last dose), 3 days (two 150-mg i.v. doses/day for 3 days with PBMC collection 14 h after last dose), or 4 days (single 700-mg i.p. dose/day for 4 days with PBMC collection 21 h after last dose). PBMC were examined for their correlated expression of cell surface CD4 vs CD8 vs intracellular BrdU and Ki-67. Ten thousand events gated on CD4+ small lymphocytes are shown. B, The 3-day pulsed animal shown in A was followed at the designated intervals with the same analysis. Again, 10,000 events, gated on CD4+ small lymphocytes, are shown. Identical patterns were seen for CD8+ T cells in this animal and for both CD4+ and CD8+ T cells in an additional animal pulsed with BrdU at 150 mg i.v. for 6 days and studied at the same time intervals.

We next determined the recent proliferative history of putative naive and memory RM T cells by assessment of the correlated expression of either CD4 or CD8, Ki-67, CD95, and in separate analyses ₇ integrin or CD28 by multiparameter flow cytometry. As illustrated in Fig. 10, A–C, and shown for 25 animals in Fig. 10D, frequencies of Ki-67⁺ cells were 10-fold higher in the memory than in the naive subset. Interestingly, for both the memory and naive subsets, Ki-67⁺ frequencies within the CD4⁺ and CD8⁺ were highly correlated (Fig. 10D), indicating that within individual animals, homeostatic mechanisms, or in the case of memory cells, Ag encounters tend to coordinately impact both of these subsets. It was also of particular interest to assess the relative proliferative activity of memory subsets defined by CD28. As shown in Fig. 11, no significant difference in Ki-67 frequencies between the CD28^high and CD28^low memory subsets were observed for either the CD4⁺ or CD8⁺ populations. Thus, recent proliferative activity does not appear to distinguish effector and central memory populations.

View larger version (33K): [in this window] [in a new window]   FIGURE 10. RM memory phenotype CD4+ and CD8+ T cells display markedly higher recent proliferative activity than their naive phenotype counterparts. RM PBMC (3.8-year-old animal) were examined for their correlated expression of cell surface CD3 vs CD4 vs CD8 vs intracellular Ki-67 (A), cell surface CD4 vs CD95 vs either 7 integrin or CD28 vs intracellular Ki-67 (B), and cell surface CD8 vs CD95 vs either 7 integrin or CD28 vs intracellular Ki-67 (C). Five thousand events gated on CD3+ (A), CD4+ (B), or CD8+ (C) small lymphocytes are shown, with the events within the control+ (A, left panel) or Ki-67+ population (A, right panel; B and C) enlarged and colored black (percent positive provided in each profile for the designated subsets). In A, it should be noted that the CD3+CD4- subset shown is equivalent to the CD3+CD8+ population. D, Cross-sectional analysis of the frequencies of Ki-67+ cells (shown on a log scale) within the naive () and memory () subsets of the CD4+ vs CD8+ T cell populations in the peripheral blood of 25 RM (age range, 1.4–12.6 years). Ki-67+ cell frequencies within these subsets were determined on the CD95 vs 7 integrin profiles for the CD4+ subset and the CD95 vs CD28 profiles for the CD8+ subset. Spearman rank correlation coefficients () and p values for CD4+ vs CD8+ naive and CD4+ vs CD8+ memory Ki-67+ frequencies are = 0.82; p < 0.0001 and = 0.92, p < 0.0001, respectively.

View larger version (33K): [in this window] [in a new window]   FIGURE 11. CD28- effector memory and CD28++ central memory T cell subsets cannot be distinguished on the basis of recent proliferative activity. This figure demonstrates a cross-sectional analysis of the frequencies of Ki-67+ cells within the CD28-defined memory subsets (see Fig. 1) of the CD4+ vs CD8+ T cell populations in the peripheral blood of the same cohort used in Fig. 10D. Statistical analysis (Wilcoxon signed rank test) demonstrated no significant difference between Ki-67+ cell frequencies of the CD28++ and CD28- memory subsets for either the CD4+ or CD8+ T cell populations (p = 0.67 and 0.39, respectively).

View larger version (43K): [in this window] [in a new window]   FIGURE 12. BrdU-labeled memory T cell populations defined by high or low expression of CD28 and by specificity for CMV determinants show a similar rate of decay after BrdU washout. A, RM 15449 (13.0 years old) and RM 15463 (13.2 years old) were i.v. pulsed with BrdU for 6 days (single 150-mg dose of BrdU/day) and 3 days (150-mg of BrdU twice a day), respectively, and PBMC were harvested at 14 h and 7, 14, 21, 35, 49, and 63 days after the last dose. Fresh PBMC were examined for their correlated expression of cell surface CD4 or CD8 vs CD95 vs CD28 vs intracellular BrdU. Separate aliquots were stimulated with CMV Ag or IE-1 peptide mix as described in Fig. 5 legend and were then stained for cell surface CD4 and CD3 and intracellular TNF-, and BrdU (RM 15449; the CD3+CD4- phenotype was used to define the CD8+ T cell response to IE-1), or surface CD4 and intracellular CD69, TNF-, and BrdU (RM 15463). Frequencies of BrdU+ were determined in the total, CD28++CD28-, CMV/IE-1-responsive (TNF-+) CD4+ and CD8+ memory subsets. B, Representative flow cytometric profiles of the analysis in A (animal 15449). Ten thousand events gated on CD4+CD95high or CD8+CD95high (memory) small lymphocytes are shown. BrdU+ events are enlarged and colored black, with the percent BrdU+ for the CD28++, CD28+, and CD28- memory subsets indicated in each panel (except for the CD4+CD28+ subset, where cells were too few for accurate analysis). Note that although the frequency of BrdU+ T cells diminished over the 35-day period shown (most profoundly for the CD28++ subset), there is no discernible difference in the spectrum of BrdU staining intensities between the 14-h and 35-day samples for any of the subsets shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

With regard to validating phenotypic signatures of naive vs memory status, we have developed an analysis paradigm that clearly delineates, for both the CD4⁺ and CD8⁺ T cells, distinct populations with the expected physiologic characteristics of these subsets (4, 5, 7, 8, 9, 10). As summarized in Table III, the homogenous naive cell cluster, best delineated as CD95^low, B₇ integrin^int, and CD28^high among CD4⁺ T cells and by CD95^low, CD28^int, and CD11a^low among CD8⁺ T cells, was 1) present in blood and secondary lymph tissues (PLN, spleen), but not effector sites (lung, intestinal lamina propria); 2) vastly predominated in the fetal/neonatal immune system, but rapidly diminished with postnatal age; 3) lacked IFN- production capability and specific responses to RM CMV; and 4) demonstrated low in vivo proliferative activity/turnover. Most previous studies analyzing naive T cells in RM have relied upon CD45RA expression alone or the combination of CD45RA expression and CD62L expression to delineate this subset (48, 49, 50, 51). When other markers (CD11a, CD28, CD49d, CD95) have been used for phenotyping RM T cells (52, 53), they have not been precisely interpreted in the context of the naive/memory paradigm. Although CD45RA expression (and/or lack of expression of its largely reciprocal RO isoform) did indeed comprise the first reported criteria for naive T cell delineation in the human (15, 17), this phenotype has been subsequently shown to include a subset of Ag-experienced T cells in this species, especially among CD8⁺ T cells (Refs. 22, 35, 54, 55, 56 and see below), clearly invalidating the usefulness of this marker as a single criterion of naive/memory status in the human system. High expression of CD45RA on Ag-experienced cells is even more common in RM (up to 60% of memory cells with bright expression), including both CD8⁺ and CD4⁺ T cells, and in addition the CD45RA^low memory subset overlaps the naive subset with respect to CD45RA intensity (see Fig. 1). The tandem use of CD62L reactivity with CD45RA also fails to reliably distinguish the naive subset: we have observed significant contamination (>30% in older animals) of the CD45RA⁺CD62L⁺ population with CD95^high memory cells (data not shown). It is also important to note that surface CD62L is highly sensitive to proteolytic cleavage (29), often resulting in loss of expression on true naive cells (especially on stored or cryopreserved specimens) and thus, their misclassification. In contrast, the criteria developed here provide reliable separation of naive cells in both blood and secondary lymphoid tissues and are equally applicable to fresh, stored, and cryopreserved specimens.

The RM memory population was characterized, as expected, by phenotypic diversity, but there were discernible patterns in this diversity, patterns with important functional correlates. Both CD4⁺ and CD8⁺ T memory cells demonstrated a subset primarily characterized by loss of CD28 expression with stereotyped phenotypic and functional features. CD4⁺CD28^- T cells were generally CD11a^high, CD49d(₄ integrin)^high, CD62L^-, and ₇ integrin^{- to low}. CD8⁺CD28^- T cells were generally similar, except that this population also included a discrete ₇ integrin^bright, CD11a^low subset. These phenotypic features suggest a population with enhanced ability to home to extralymphoid (effector) sites, either generally or, in the case of the ₇ integrin^brightCD11a^low subset, specifically directed to intestinal lamina propria and related sites (5, 7). In keeping with this, the CD28^- subset was directly shown to be significantly enriched in representative RM effector sites, lung and intestinal lamina propria (Fig. 8), in a homing receptor appropriate manner (e.g., ₇ integrin^bright cells predominately in gut lamina propria; Fig. 2). We also demonstrated that CD28^-/dim T cells accounted for the majority of T cells specific for epitopes encoded by the chronic pathogen RM CMV (Fig. 5 and Table II), and that this population appears with a delay in early memory development and progressively accumulates within the memory subset thereafter (Figs. 3 and 7).

These features are characteristic of the effector memory subset described in humans and rodents (8, 18, 22, 23, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44), which has been envisioned as a terminally differentiated memory subset "trained" for direct anti-pathogen effector activity in extralymphoid sites. In humans, effector memory cells have been characterized by the largely overlapping CD28^- and CD27^- phenotypes and by "reversed" CD45 isoform expression (e.g., CD45RA^highRO^low). In RM, the CD27 and CD45RA criteria do not appear to apply. RM CD27 can be visualized by cross-reacting human CD27-specific Abs, but a definitive CD27^- memory subset is not observed, and the variability of expression intensity that is observed does not correlate with CD28 expression or any of the other phenotypic correlates of the effector memory subset (data not shown). With regard to CD45RA, correlation with the CD28^- subset is only observed for peripheral blood CD4⁺ T memory cells, not CD8⁺ memory cells or CD4⁺ cells in effector sites. Moreover, the CD8⁺CD45RA⁺⁺ subset does not accumulate with age. Taken together, these data are consistent with 1) the correlation of CD28 loss with a linear differentiation pathway among memory cells, and 2) the independent regulation of CD45RA (the expression of which may switch both on and off memory T cells in response to unknown signals).

The current paradigm for the homeostasis of the CD28^- effector memory subset is that this subset, as a terminally differentiated population, manifests limited replicative capacity and derives from and is maintained by the differentiation of the secondary lymphoid tissue-based, replication competent, CD28⁺ central memory subset (18, 22, 36, 37, 38, 57, 58). The stability of the CD28^- subset has been somewhat controversial, with some studies suggesting it is relatively resistant to apoptosis, and others suggesting it is relatively sensitive (37, 40, 57). These hypotheses have largely been based on in vitro experimentation, and the in vivo physiology of this subset must be considered unknown. In this study, we provide some insight into this issue by the analysis of Ki-67 expression and BrdU uptake among RM T cells in general and these memory subsets in specific.

First, as indicated above, memory cells constitute the vast majority of cycling cells, with rates of Ki-67 expression or BrdU uptake 10 times higher than the naive subset. This finding is in general agreement with several previous studies in RM (48, 50, 52), but the naive/memory delineation in these prior studies was based on CD45RA alone or CD45RA/CD62L and thus may not have reliably separated these subsets (as discussed above). Second, simultaneous analysis of Ki-67 and BrdU incorporation after varying BrdU pulse periods suggested complete labeling of Ki-67⁺ cells with 3- to 4-day pulses, and then loss of Ki-67 reactivity on the vast majority of labeled cells within 1 wk after the pulse. These observations indicate that the vast majority of Ki-67⁺ T cells in peripheral blood have undergone S phase in the preceding 3–4 days and few, if any, of these Ki-67⁺ cells appear to have undergone cell cycle arrest. Moreover, they suggest that the proliferative activity of the majority of T cells is episodic, with cells entering cell cycle, undergoing a limited number of divisions, and then returning to G_o phase (i.e., most proliferating cells are not continuously cycling). Third, frequencies of labeled cells decline slowly over the washout period with little discernible change in BrdU staining, indicating that a substantial fraction of postdivision, recirculating T cells revert to the nonproliferative state and survive for many weeks. Re-entry of labeled cells into the cell cycle appeared to be stochastic, occurring at a frequency similar to that of unlabeled cells. Fourth, Ki-67 reactivity and BrdU incorporation following 1-, 3-, or 6-day BrdU pulses were not significantly different between the CD28^- effector memory and the CD28⁺ central memory subsets. Significant Ki-67 reactivity within the CD28^- T cell subset has also been reported by Kaur et al. (52). In vitro maturation of CD28^- T cells from CD28⁺ precursors in humans takes many days to weeks (57, 58), and the delayed development of the CD28^- T cell subset relative to overall memory population in RM suggests this differentiation process is not immediate in vivo either. Thus, the Ki-67 reactivity and BrdU uptake (after as little as 24 h of pulsing) exhibited by the CD28^- effector memory subset is likely due to proliferation within this subset and not proliferation of CD28⁺ central memory T cells with subsequent differentiation to a CD28^- phenotype. Finally, decay rates of BrdU-labeled cells and BrdU staining intensities were similar among the CD28^- and CD28⁺ subsets, suggesting that either survival of these subsets was equivalent or that loss of CD28^- memory cells is precisely compensated by differentiating (but nonproliferating) CD28⁺ cells. Against this later interpretation was the observation that the decay of a steady-state IE-1-specific CD8⁺ memory subset, which is predominantly CD28^- and therefore lacks a significant CD28⁺ central memory reservoir, was similar to the overall CD28^- effector memory subset.

Overall, these observations strongly suggest that in vivo the CD28^- effector memory subset is as replication capable and as intrinsically stable as the CD28⁺ central memory subset, and therefore most likely constitutes a population capable of independent homeostasis. These data do not contradict the role of the CD28⁺ central memory subset as a precursor of the effector memory subset, but do suggest that after this differentiation, maintenance of the effector memory population is not completely dependent on this differentiation. Given the vastly different tissue-homing preferences of central and effector memory subsets and the consequent differences in their microenvironmental "niches" (59), it is highly likely the homeostasis of these subsets is controlled by distinct regulatory influences. Thus, sequential linear differentiation of memory T cells—central memory to effector memory—may be accompanied not only by changes in function, but also by the development of independent mechanisms of population homeostasis, analogous to the independent homeostasis of naive vs total memory populations (59).

In summary, this report has defined and validated a new analytic paradigm for the definition of naive and memory T cells in RM and has delineated optimized approaches for the study of the function and homeostasis of these cells. Preliminary data indicate that all of the technical and analytic approaches defined here for RM work equivalently in cynomologous monkeys (Macaca fascicularis) as well (data not shown). These nonhuman primates provide a manipulable, time-contracted model of memory development that much more closely resembles the human system than rodent models. Moreover, the application of these analytic tools to nonhuman primate infectious disease and vaccine models will facilitate determination of protective thresholds for T cell immunity and their maintenance over time.

	Footnotes

 
¹ This study was supported by National Institutes of Health Grants P51RR00163 and R21AI44758.

² C.J.P. and S.I.H. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Louis J. Picker, Vaccine and Gene Therapy Institute, Oregon Health and Science University, West Campus, 505 NW 185th Avenue, Beaverton, OR 97006. E-mail address: pickerl{at}ohsu.edu

⁴ Abbreviations used in this paper: RM, rhesus macaque; BrdU, 5-bromo-2'-deoxyuridine; dPBS, Dulbecco’s PBS; IE-1, immediate early-1; PLN, peripheral lymph node; MLN, mesenteric lymph node; SEB, staphylococcal enterotoxin B; int, intermediate.

Received for publication August 29, 2001. Accepted for publication October 24, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Notermans, D. W., N. G. Pakker, D. Hamann, N. A. Foudraine, R. H. Kauffmann, P. L. Meenhorst, J. Goudsmit, M. T. Roos, P. T. Schellekens, F. Miedema, S. A. Danner. 1999. Immune reconstitution after 2 years of successful potent antiretroviral therapy in previously untreated human immunodeficiency virus type 1- infected adults. J. Infect. Dis. 180:1050.[Medline]
2. Jr Collins, R. H., M. Sackler, C. J. Pitcher, S. L. Waldrop, G. B. Klintmalm, R. Jenkins, L. J. Picker. 1997. Immune reconstitution with donor-derived memory/effector T cells after orthotopic liver transplantation. Exp. Hematol. 25:147.[Medline]
3. Rabin, R. L., M. Roederer, Y. Maldonado, A. Petru, L. A. Herzenberg. 1995. Altered representation of naive and memory CD8 T cell subsets in HIV-infected children. J. Clin. Invest. 95:2054.
4. De Rosa, S. C., L. A. Herzenberg, M. Roederer. 2001. 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat. Med. 7:245.[Medline]
5. Picker, L. J., and M. H. Siegelman. 1999. Lymphoid organs and tissues. In Fundamental Immunology, 4th Ed., Chapter 14. W. E. Paul, ed. Lippincott-Raven, Philadelphia, p. 479.
6. Pakker, N. G., E. D. Kroon, M. T. Roos, S. A. Otto, D. Hall, F. W. Wit, D. Hamann, M. E. van der Ende, F. A. Claessen, R. H. Kauffmann, et al 1999. Immune restoration does not invariably occur following long-term HIV-1 suppression during antiretroviral therapy: INCAS Study Group. AIDS 13:203.[Medline]
7. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
8. Lanzavecchia, A., F. Sallusto. 2000. Dynamics of T lymphocyte responses: intermediates, effectors, and memory cells. Science 290:92.[Abstract/Free Full Text]
9. Tough, D. F., J. Sprent. 1994. Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179:1127.[Abstract/Free Full Text]
10. Michie, C. A., A. McLean, C. Alcock, P. C. L. Beverley. 1992. Lifespan of human lymphocyte subsets defined by CD45 isoforms. Nature 360:264.[Medline]
11. Tanchot, C., A. Le Campion, S. Leaument, N. Dautigny, B. Lucas. 2001. Naive CD4+ lymphocytes convert to anergic or memory-like cells in T cell-deprived recipients. Eur. J. Immunol. 31:2256.[Medline]
12. Picker, L. J., J. R. Treer, B. Ferguson-Darnell, P. A. Collins, P. R. Bergstresser, L. W. Terstappen. 1993. Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyte-associated antigen, a tissue-selective homing receptor for skin-homing T cells. J. Immunol. 150:1122.[Abstract]
13. Picker, L. J., J. R. Treer, B. Ferguson-Darnell, P. A. Collins, D. Buck, L. W. Terstappen. 1993. Control of lymphocyte recirculation in man. I. Differential regulation of the peripheral lymph node homing receptor L-selection on T cells during the virgin to memory cell transition. J. Immunol. 150:1105.[Abstract]
14. Teraki, Y., L. J. Picker. 1997. Independent regulation of cutaneous lymphocyte-associated antigen expression and cytokine synthesis phenotype during human CD4⁺ memory T cell differentiation. J. Immunol. 159:6018.[Abstract]
15. Sanders, M. E., M. W. Makagoba, S. O. Sharrow, D. Stephany, T. A. Springer, H. A. Young, S. Shaw. 1988. Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN- production. J. Immunol. 140:1401.[Abstract]
16. Bleul, C. C., L. Wu, J. A. Hoxie, T. A. Springer, C. R. Mackay. 1997. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc. Natl. Acad. Sci. USA 94:1925.[Abstract/Free Full Text]
17. Akbar, A. N., L. Terry, A. Timms, P. C. L. Beverely, G. Janossy. 1988. Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol. 140:2171.[Abstract]
18. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708.[Medline]
19. Picker, L. J., L. W. Terstappen, L. S. Rott, P. R. Streeter, H. Stein, E. C. Butcher. 1990. Differential expression of homing-associated adhesion molecules by T cell subsets in man. J. Immunol. 145:3247.[Abstract]
20. McCune, J. M., M. B. Hanley, D. Cesar, R. Halvorsen, R. Hoh, D. Schmidt, E. Wieder, S. Deeks, S. Siler, R. Neese, M. Hellerstein. 2000. Factors influencing T-cell turnover in HIV-1-seropositive patients. J. Clin. Invest. 105:R1.
21. Pakker, N. G., D. W. Notermans, R. J. de Boer, M. T. Roos, F. de Wolf, A. Hill, J. M. Leonard, S. A. Danner, F. Miedema, P. T. Schellekens. 1998. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. Nat. Med. 4:208.[Medline]
22. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, R. A. van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8⁺ T cells. J. Exp. Med. 186:1407.[Abstract/Free Full Text]
23. Masopust, D., V. Vezys, A. L. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413.[Abstract/Free Full Text]
24. Veazey, R. S., M. Rosenzweig, D. E. Shvetz, D. R. Pauley, M. DeMaria, L. V. Chalifoux, R. P. Johnson, A. A. Lackner. 1997. Characterization of gut-associated lymphoid tissue (GALT) of normal rhesus macaques. Clin. Immunol. Immunopathol. 82:230.[Medline]
25. Maecker, H. T., H. S. Dunn, M. A. Suni, E. Khatamzas, C. J. Pitcher, T. Bunde, N. Persaud, W. Trigona, T.-M. Fu, E. Sinclair, et al 2001. Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J. Immunol. Methods 255:27.[Medline]
26. Waldrop, S. L., K. A. Davis, V. C. Maino, L. J. Picker. 1998. Normal human CD4⁺ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis. J. Immunol. 161:5284.[Abstract/Free Full Text]
27. Waldrop, S. L., C. J. Pitcher, D. M. Peterson, V. C. Maino, L. J. Picker. 1997. 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. J. Clin. Invest. 99:1739.[Medline]
28. Terstappen, L. W., S. Huang, L. J. Picker. 1992. Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. Blood 79:666.[Abstract/Free Full Text]
29. Kishimoto, T. K., M. A. Jutila, E. C. Butcher. 1990. Identification of a human peripheral lymph node homing receptor: a rapidly down-regulated adhesion molecule. Proc. Natl. Acad. Sci. USA 87:2244.[Abstract/Free Full Text]
30. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101.[Medline]
31. Picker, L. J., R. J. Martin, A. Trumble, L. S. Newman, P. A. Collins, P. R. Bergstresser, D. Y. Leung. 1994. Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites. Eur. J. Immunol. 24:1269.[Medline]
32. Douek, D. C., R. D. McFarland, P. H. Keiser, E. A. Gage, J. M. Massey, B. F. Haynes, M. A. Polis, A. T. Haase, M. B. Feinberg, J. L. Sullivan, et al 1998. Changes in thymic function with age and during the treatment of HIV infection. Nature 396:690.[Medline]
33. McFarland, R. D., D. C. Douek, R. A. Koup, L. J. Picker. 2000. Identification of a human recent thymic emigrant phenotype. Proc. Natl. Acad. Sci. USA 97:4215.[Abstract/Free Full Text]
34. Picker, L. J., M. K. Singh, Z. Zdraveski, J. R. Treer, S. L. Waldrop, P. R. Bergstresser, V. C. Maino. 1995. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86:1408.[Abstract/Free Full Text]
35. Kern, F., E. Khatamzas, I. Surel, C. Frommel, P. Reinke, S. L. Waldrop, L. J. Picker, H. D. Volk. 1999. Distribution of human CMV-specific memory T cells among the CD8^pos. subsets defined by CD57, CD27, and CD45 isoforms. Eur. J. Immunol. 29:2908.[Medline]
36. Champagne, P., G. S. Ogg, A. S. King, C. Knabenhans, K. Ellefsen, M. Nobile, V. Appay, G. P. Rizzardi, S. Fleury, M. Lipp, et al 2001. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 410:106.[Medline]
37. Posnett, D. N., J. W. Edinger, J. S. Manavalan, C. Irwin, G. Marodon. 1999. Differentiation of human CD8 T cells: implications for in vivo persistence of CD8⁺ CD28^- cytotoxic effector clones. Int. Immunol. 11:229.[Abstract/Free Full Text]
38. Nociari, M. M., W. Telford, C. Russo. 1999. Postthymic development of CD28^-CD8⁺ T cell subset: age-associated expansion and shift from memory to naive phenotype. J. Immunol. 162:3327.[Abstract/Free Full Text]
39. Weekes, M. P., A. J. Carmichael, M. R. Wills, K. Mynard, J. G. Sissons. 1999. Human CD28^-CD8⁺ T cells contain greatly expanded functional virus-specific memory CTL clones. J. Immunol. 162:7569.[Abstract/Free Full Text]
40. Vallejo, A. N., M. Schirmer, C. M. Weyand, J. J. Goronzy. 2000. Clonality and longevity of CD4⁺CD28^null T cells are associated with defects in apoptotic pathways. J. Immunol. 165:6301.[Abstract/Free Full Text]
41. Hol, B. E., R. Q. Hintzen, R. A. Van Lier, C. Alberts, T. A. Out, H. M. Jansen. 1993. Soluble and cellular markers of T cell activation in patients with pulmonary sarcoidosis. Am. Rev. Respir. Dis. 148:643.[Medline]
42. Hintzen, R. Q., R. de Jong, S. M. Lens, M. Brouwer, P. Baars, R. A. van Lier. 1993. Regulation of CD27 expression on subsets of mature T-lymphocytes. J. Immunol. 151:2426.[Abstract]
43. De Rie, M. A., I. Cairo, R. A. Van Lier, J. D. Bos. 1996. Expression of the T-cell activation antigens CD27 and CD28 in normal and psoriatic skin. Clin. Exp. Dermatol. 21:104.[Medline]
44. Roos, M. T., R. A. van Lier, D. Hamann, G. J. Knol, I. Verhoofstad, D. van Baarle, F. Miedema, P. T. Schellekens. 2000. Changes in the composition of circulating CD8⁺ T cell subsets during acute Epstein-Barr and human immunodeficiency virus infections in humans. J. Infect. Dis. 182:451.[Medline]
45. Lempicki, R. A., J. A. Kovacs, M. W. Baseler, J. W. Adelsberger, R. L. Dewar, V. Natarajan, M. C. Bosche, J. A. Metcalf, R. A. Stevens, L. A. Lambert, et al 2000. Impact of HIV-1 infection and highly active antiretroviral therapy on the kinetics of CD4⁺ and CD8⁺ T cell turnover in HIV-infected patients. Proc. Natl. Acad. Sci. USA 97:13778.[Abstract/Free Full Text]
46. Reome, J. B., D. S. Johnston, B. K. Helmich, T. M. Morgan, N. Dutton-Swain, R. W. Dutton. 2000. The effects of prolonged administration of 5-bromodeoxyuridine on cells of the immune system. J. Immunol. 165:4226.[Abstract/Free Full Text]
47. Gerdes, J., H. Lemke, H. Baisch, H. H. Wacker, U. Schwab, H. Stein. 1984. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J. Immunol. 133:1710.[Abstract]
48. Mohri, H., S. Bonhoeffer, S. Monard, A. S. Perelson, D. D. Ho. 1998. Rapid turnover of T lymphocytes in SIV-infected rhesus macaques. Science 279:1223.[Abstract/Free Full Text]
49. DeMaria, M. A., M. Casto, M. O’Connell, R. P. Johnson, M. Rosenzweig. 2000. Characterization of lymphocyte subsets in rhesus macaques during the first year of life. Eur. J. Haematol. 65:245.[Medline]
50. Rosenzweig, M., M. A. DeMaria, D. M. Harper, S. Friedrich, R. K. Jain, R. P. Johnson. 1998. Increased rates of CD4⁺ and CD8⁺ T lymphocyte turnover in simian immunodeficiency virus-infected macaques. Proc. Natl. Acad. Sci. USA 95:6388.[Abstract/Free Full Text]
51. Veazey, R. S., K. G. Mansfield, I. C. Tham, A. C. Carville, D. E. Shvetz, A. E. Forand, A. A. Lackner. 2000. Dynamics of CCR5 expression by CD4⁺ T cells in lymphoid tissues during simian immunodeficiency virus infection. J. Virol. 74:11001.[Abstract/Free Full Text]
52. Kaur, A., C. L. Hale, S. Ramanujan, R. K. Jain, R. P. Johnson. 2000. Differential dynamics of CD4⁺ and CD8⁺ T-lymphocyte proliferation and activation in acute simian immunodeficiency virus infection. J. Virol. 74:8413.[Abstract/Free Full Text]
53. Kuroda, M. J., J. E. Schmitz, W. A. Charini, C. E. Nickerson, C. I. Lord, M. A. Forman, N. L. Letvin. 1999. Comparative analysis of cytotoxic T lymphocytes in lymph nodes and peripheral blood of simian immunodeficiency virus-infected rhesus monkeys. J. Virol. 73:1573.[Abstract/Free Full Text]
54. Arlettaz, L., C. Barbey, F. Dumont-Girard, C. Helg, B. Chapuis, E. Roux, E. Roosnek. 1999. CD45 isoform phenotypes of human T cells: CD4⁺CD45RA^-RO⁺ memory T cells re-acquire CD45RA without losing CD45RO. Eur. J. Immunol. 29:3987.[Medline]
55. Faint, J. M., N. E. Annels, S. J. Curnow, P. Shields, D. Pilling, A. D. Hislop, L. Wu, A. N. Akbar, C. D. Buckley, P. A. Moss, et al 2001. Memory T cells constitute a subset of the human CD8⁺CD45RA⁺ pool with distinct phenotypic and migratory characteristics. J. Immunol. 167:212.[Abstract/Free Full Text]
56. Wills, M. R., A. J. Carmichael, M. P. Weekes, K. Mynard, G. Okecha, R. Hicks, J. G. Sissons. 1999. Human virus-specific CD8⁺ CTL clones revert from CD45RO^high to CD45RA^high in vivo: CD45RA^highCD8⁺ T cells comprise both naive and memory cells. J. Immunol. 162:7080.[Abstract/Free Full Text]
57. Borthwick, N. J., M. Lowdell, M. Salmon, A. N. Akbar. 2000. Loss of CD28 expression on CD8⁺ T cells is induced by IL-2 receptor chain signalling cytokines and type I IFN, and increases susceptibility to activation-induced apoptosis. Int. Immunol. 12:1005.[Abstract/Free Full Text]
58. Labalette, M., E. Leteurtre, C. Thumerelle, C. Grutzmacher, B. Tourvieille, J. P. Dessaint. 1999. Peripheral human CD8⁺CD28⁺ T lymphocytes give rise to CD28^- progeny, but IL-4 prevents loss of CD28 expression. Int. Immunol. 11:1327.[Abstract/Free Full Text]
59. Freitas, A. A., B. Rocha. 2000. Population biology of lymphocytes: the flight for survival. Annu. Rev. Immunol. 18:83.[Medline]

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				M.-C. Gauduin, Y. Yu, A. Barabasz, A. Carville, M. Piatak, J. D. Lifson, R. C. Desrosiers, and R. P. Johnson Induction of a virus-specific effector-memory CD4+ T cell response by attenuated SIV infection J. Exp. Med., November 27, 2006; 203(12): 2661 - 2672. [Abstract] [Full Text] [PDF]

				Y. Sun, J. E. Schmitz, A. P. Buzby, B. R. Barker, S. S. Rao, L. Xu, Z.-y. Yang, J. R. Mascola, G. J. Nabel, and N. L. Letvin Virus-Specific Cellular Immune Correlates of Survival in Vaccinated Monkeys after Simian Immunodeficiency Virus Challenge J. Virol., November 15, 2006; 80(22): 10950 - 10956. [Abstract] [Full Text] [PDF]

				J. J. Mattapallil, D. C. Douek, A. Buckler-White, D. Montefiori, N. L. Letvin, G. J. Nabel, and M. Roederer Vaccination preserves CD4 memory T cells during acute simian immunodeficiency virus challenge J. Exp. Med., June 12, 2006; 203(6): 1533 - 1541. [Abstract] [Full Text] [PDF]

				N. A. Wilson, J. Reed, G. S. Napoe, S. Piaskowski, A. Szymanski, J. Furlott, E. J. Gonzalez, L. J. Yant, N. J. Maness, G. E. May, et al. Vaccine-Induced Cellular Immune Responses Reduce Plasma Viral Concentrations after Repeated Low-Dose Challenge with Pathogenic Simian Immunodeficiency Virus SIVmac239. J. Virol., June 1, 2006; 80(12): 5875 - 5885. [Abstract] [Full Text] [PDF]

				U. Wille-Reece, B. J. Flynn, K. Lore, R. A. Koup, A. P. Miles, A. Saul, R. M. Kedl, J. J. Mattapallil, W. R. Weiss, M. Roederer, et al. Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates J. Exp. Med., May 15, 2006; 203(5): 1249 - 1258. [Abstract] [Full Text] [PDF]

				P. M. Acierno, J. E. Schmitz, D. A. Gorgone, Y. Sun, S. Santra, M. S. Seaman, M. H. Newberg, J. R. Mascola, G. J. Nabel, D. Panicali, et al. Preservation of Functional Virus-Specific Memory CD8+ T Lymphocytes in Vaccinated, Simian Human Immunodeficiency Virus-Infected Rhesus Monkeys J. Immunol., May 1, 2006; 176(9): 5338 - 5345. [Abstract] [Full Text] [PDF]

				Z. Wang, B. Metcalf, R. M. Ribeiro, H. McClure, and A. Kaur Th-1-Type Cytotoxic CD8+ T-Lymphocyte Responses to Simian Immunodeficiency Virus (SIV) Are a Consistent Feature of Natural SIV Infection in Sooty Mangabeys J. Virol., March 15, 2006; 80(6): 2771 - 2783. [Abstract] [Full Text] [PDF]

				T. Kuwata, H. Dehghani, C. R. Brown, R. Plishka, A. Buckler-White, T. Igarashi, J. Mattapallil, M. Roederer, and V. M. Hirsch Infectious Molecular Clones from a Simian Immunodeficiency Virus-Infected Rapid-Progressor (RP) Macaque: Evidence of Differential Selection of RP-Specific Envelope Mutations In Vitro and In Vivo J. Virol., February 1, 2006; 80(3): 1463 - 1475. [Abstract] [Full Text] [PDF]

				S. M. Murray, L. J. Picker, M. K. Axthelm, and M. L. Linial Expanded Tissue Targets for Foamy Virus Replication with Simian Immunodeficiency Virus-Induced Immunosuppression J. Virol., January 15, 2006; 80(2): 663 - 670. [Abstract] [Full Text] [PDF]

				C. K. Asiedu, K. J. Goodwin, G. Balgansuren, S. M. Jenkins, S. Le Bas-Bernardet, U. Jargal, D. M. Neville Jr, and J. M. Thomas Elevated T Regulatory Cells in Long-Term Stable Transplant Tolerance in Rhesus Macaques Induced by Anti-CD3 Immunotoxin and Deoxyspergualin J. Immunol., December 15, 2005; 175(12): 8060 - 8068. [Abstract] [Full Text] [PDF]

				U. Wille-Reece, B. J. Flynn, K. Lore, R. A. Koup, R. M. Kedl, J. J. Mattapallil, W. R. Weiss, M. Roederer, and R. A. Seder HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates PNAS, October 18, 2005; 102(42): 15190 - 15194. [Abstract] [Full Text] [PDF]

				S.-h. Ho, L. Shek, A. Gettie, J. Blanchard, and C. Cheng-Mayer V3 Loop-Determined Coreceptor Preference Dictates the Dynamics of CD4+-T-Cell Loss in Simian-Human Immunodeficiency Virus-Infected Macaques J. Virol., October 1, 2005; 79(19): 12296 - 12303. [Abstract] [Full Text] [PDF]

				M. Vaccari, C. J. Trindade, D. Venzon, M. Zanetti, and G. Franchini Vaccine-Induced CD8+ Central Memory T Cells in Protection from Simian AIDS J. Immunol., September 15, 2005; 175(6): 3502 - 3507. [Abstract] [Full Text] [PDF]

				Y. Nishimura, C. R. Brown, J. J. Mattapallil, T. Igarashi, A. Buckler-White, B. A. P. Lafont, V. M. Hirsch, M. Roederer, and M. A. Martin Resting naive CD4+ T cells are massively infected and eliminated by X4-tropic simian-human immunodeficiency viruses in macaques PNAS, May 31, 2005; 102(22): 8000 - 8005. [Abstract] [Full Text] [PDF]

				N. T. Pande, C. Powers, K. Ahn, and K. Fruh Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11 J. Virol., May 1, 2005; 79(9): 5786 - 5798. [Abstract] [Full Text] [PDF]

				Y. Sun, J. E. Schmitz, P. M. Acierno, S. Santra, R. A. Subbramanian, D. H. Barouch, D. A. Gorgone, M. A. Lifton, K. R. Beaudry, K. Manson, et al. Dysfunction of Simian Immunodeficiency Virus/Simian Human Immunodeficiency Virus-Induced IL-2 Expression by Central Memory CD4+ T Lymphocytes J. Immunol., April 15, 2005; 174(8): 4753 - 4760. [Abstract] [Full Text] [PDF]

				G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. McClure, M. B. Feinberg, and S. I. Staprans Divergent Host Responses during Primary Simian Immunodeficiency Virus SIVsm Infection of Natural Sooty Mangabey and Nonnatural Rhesus Macaque Hosts J. Virol., April 1, 2005; 79(7): 4043 - 4054. [Abstract] [Full Text] [PDF]

				G. Ramesh, X. Alvarez, J. T. Borda, P. P. Aye, A. A. Lackner, and K. Sestak Visualizing Cytokine-Secreting Cells In Situ in the Rhesus Macaque Model of Chronic Gut Inflammation Clin. Vaccine Immunol., January 1, 2005; 12(1): 192 - 197. [Abstract] [Full Text] [PDF]

				M. Conklyn, C. Andresen, P. Changelian, and E. Kudlacz The JAK3 inhibitor CP-690550 selectively reduces NK and CD8+ cell numbers in cynomolgus monkey blood following chronic oral dosing J. Leukoc. Biol., December 1, 2004; 76(6): 1248 - 1255. [Abstract] [Full Text] [PDF]

				L. J. Picker, S. I. Hagen, R. Lum, E. F. Reed-Inderbitzin, L. M. Daly, A. W. Sylwester, J. M. Walker, D. C. Siess, M. Piatak Jr., C. Wang, et al. Insufficient Production and Tissue Delivery of CD4+ Memory T Cells in Rapidly Progressive Simian Immunodeficiency Virus Infection J. Exp. Med., November 15, 2004; 200(10): 1299 - 1314. [Abstract] [Full Text] [PDF]

				K. Sestak, M. M. McNeal, A. Choi, M. J. Cole, G. Ramesh, X. Alvarez, P. P. Aye, R. P. Bohm, M. Mohamadzadeh, and R. L. Ward Defining T-Cell-Mediated Immune Responses in Rotavirus-Infected Juvenile Rhesus Macaques J. Virol., October 1, 2004; 78(19): 10258 - 10264. [Abstract] [Full Text] [PDF]

				M. Moniuszko, T. Fry, W.-P. Tsai, M. Morre, B. Assouline, P. Cortez, M. G. Lewis, S. Cairns, C. Mackall, and G. Franchini Recombinant Interleukin-7 Induces Proliferation of Naive Macaque CD4+ and CD8+ T Cells In Vivo J. Virol., September 15, 2004; 78(18): 9740 - 9749. [Abstract] [Full Text] [PDF]

				Y. Nishimura, T. Igarashi, O. K. Donau, A. Buckler-White, C. Buckler, B. A. P. Lafont, R. M. Goeken, S. Goldstein, V. M. Hirsch, and M. A. Martin Highly pathogenic SHIVs and SIVs target different CD4+ T cell subsets in rhesus monkeys, explaining their divergent clinical courses PNAS, August 17, 2004; 101(33): 12324 - 12329. [Abstract] [Full Text] [PDF]

				K. Abel, L. La Franco-Scheuch, T. Rourke, Z.-M. Ma, V. de Silva, B. Fallert, L. Beckett, T. A. Reinhart, and C. J. Miller Gamma Interferon-Mediated Inflammation Is Associated with Lack of Protection from Intravaginal Simian Immunodeficiency Virus SIVmac239 Challenge in Simian-Human Immunodeficiency Virus 89.6-Immunized Rhesus Macaques J. Virol., January 15, 2004; 78(2): 841 - 854. [Abstract] [Full Text] [PDF]

				Y. Yue, S. S. Zhou, and P. A. Barry Antibody responses to rhesus cytomegalovirus glycoprotein B in naturally infected rhesus macaques J. Gen. Virol., December 1, 2003; 84(12): 3371 - 3379. [Abstract] [Full Text] [PDF]

				B. Gansuvd, W. J. Hubbard, A. Hutchings, F. T. Thomas, J. Goodwin, S. B. Wilson, M. A. Exley, and J. M. Thomas Phenotypic and Functional Characterization of Long-Term Cultured Rhesus Macaque Spleen-Derived NKT Cells J. Immunol., September 15, 2003; 171(6): 2904 - 2911. [Abstract] [Full Text] [PDF]

				M. C. Strain, H. F. Gunthard, D. V. Havlir, C. C. Ignacio, D. M. Smith, A. J. Leigh-Brown, T. R. Macaranas, R. Y. Lam, O. A. Daly, M. Fischer, et al. Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: Intrinsic stability predicts lifelong persistence PNAS, April 15, 2003; 100(8): 4819 - 4824. [Abstract] [Full Text] [PDF]

				C. Sugimoto, K. Tadakuma, I. Otani, T. Moritoyo, H. Akari, F. Ono, Y. Yoshikawa, T. Sata, S. Izumo, and K. Mori nef Gene Is Required for Robust Productive Infection by Simian Immunodeficiency Virus of T-Cell-Rich Paracortex in Lymph Nodes J. Virol., April 1, 2003; 77(7): 4169 - 4180. [Abstract] [Full Text] [PDF]

				T. J. Fry, M. Moniuszko, S. Creekmore, S. J. Donohue, D. C. Douek, S. Giardina, T. T. Hecht, B. J. Hill, K. Komschlies, J. Tomaszewski, et al. IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates Blood, March 15, 2003; 101(6): 2294 - 2299. [Abstract] [Full Text] [PDF]

				E. V. Ravkov, C. M. Myrick, and J. D. Altman Immediate Early Effector Functions of Virus-Specific CD8+CCR7+ Memory Cells in Humans Defined by HLA and CC Chemokine Ligand 19 Tetramers J. Immunol., March 1, 2003; 170(5): 2461 - 2468. [Abstract] [Full Text] [PDF]

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