Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs

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IMMUNOLOGY

135, 118- 131 ( 1991)

Influence of Aging on intracellular Free Calcium and Proliferation of Mouse T-Cell Subsets from Various Lymphoid Organs ANCELIKA GROSSMANN,*,~,'LILLIAN MAGGIO-PRICE,?JOHN C.JINNEMAN,* ANDPETERS.RABINOVITCH* Departments of Pathology,,* and Comparative Medicine,? University of Washington, Seattle, Washington 98195 Received November 2, 1990; acceptedJanuary 15, 1991 The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6Fl (C57Bl/6J X WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatestage-related changeswere seenin CD4+ cells derived from spleensof 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2’li signaling and decreasedproliferation in the presenceof exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca’+& but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca’+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2’li or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentageof CD4+ cells was decreasedin all lymphoid organsfrom old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sourcesand subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans. Q 1991 Academic press. hc.

INTRODUCTION Aging in mice is accompanied by a reduction in splenocyte proliferative response to mitogens (l-3). It has been suggestedthat deficiencies in transmembrane signaling may be responsible for this age-related decreasein cell proliferation (4, 5). An early event in mitogenic signaling is a transient increasein intracellular free calcium ([Ca2+]J2 (6). When stimulated with concanavalin A (Con A), splenocytes from old CBA mice (7) and old C57B1/6 mice (6) showed reduced peak calcium concentration. Our recent studies on T-lymphocytes of CBA mice have shown that both the CD4+ and CD8+ lymphocytes exhibit reduced peak [Ca*+]i responsesto anti-CD3 monoclonal antibody (mAb); in addition, the percentage of cells which can respond to the stimuli is also ’ To whom correspondence and reprint requests should be addressed. ’ Abbreviations used: BrdU, S-bromodeoxyuridine; BSA, bovine serum albumin; Con A, concanavalin A; [Ca*+]r, intracellular free calcium; EB, ethidium bromide; FITC, fluorescein isothiocyanate; IL-2, interleukin-2; mAb, monoclonal antibody; ME, &mercaptoethanol; PE, R-phycoerythrin; SPF,specific pathogen free. 118 0008~8749/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved

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reduced in old mice (8). In the above cited murine studies, lymphocytes were derived from spleen. Unlike studies performed in mice, immunosenescence in man has been studied using lymphocytes derived from peripheral blood. Reductions in [Ca2’]i responses were noted in CD4+ cells from aged human donors when stimulated with anti-CD3 mAb; there was not an associated reduction in proliferation of this subset in culture (9). We were, therefore, interested in whether the reported differencesbetween mice and humans might be related to differences between age-related changes in Tlymphocytes from different lymphoid organs. In the present study we measured the proliferative response of T-cell subsetsfrom peripheral blood, spleen, and lymph nodes from old and young mice after stimulation with anti-CD3 mAb. We also examined whether observed differences were related to altered [Ca2+]i responses or receptor density of CD3 on CD4+ and CD8+ cells. In addition, we analyzed whether altered responsescould be due to age-related changes in relative T-cell subset proportions or increased numbers of memory cells within these subsetsin aged mice. Our results indicate that aging changesin peripheral blood lymphocytes are similar in mice and humans in CD4+ cells. CD4+ lymphocytes from blood of old mice have decreased calcium signaling without alteration in the proliferative response. These observations are similar to those made previously in cells from elderly human donors (9). In contrast, CD4+ and CD8+ splenocytes from old mice have reduced calcium signaling and proliferation and T lymphocytes from lymph nodes have no age-related changes in signal transduction or proliferation. MATERIALS AND METHODS Mice. C57B1/6J were crossedwith WB/Re mice to produce WBB6Fl mice. Twelve WBB6Fl mice were 6 months of age (young) and twelve were 27-30 months of age (old). WBB6Fl mice were reared at the University of Washington, School of Medicine in an AAALAC accredited facility and housed under specific pathogen-free (SPF) barrier conditions. A quality assuranceprogram at the University of Washington has ascertained that the colony is serologically negative for major rodent viral pathogens. culture negative for common bacterial pathogens, and free of rodent ecto- and endoparasites. Only mice showing no macroscopic abnormalities at necropsy were used in our study. mAb and reagents. R-phycoerythrin (PE), fluorescein isothiocyanate (FITC)-conjugated, and unconjugated anti-CD3 mAb (145-2Cll) were a gift from Dr. J. Ledbetter (Oncogen, Seattle, WA). PE-conjugated anti-CD4 mAb (anti-L3T4) and FITC-conjugated anti-CD8 mAb (anti-Lyt2) were obtained from Becton-Dickinson (Mountain View, CA). Anti-Pgpl was a kind gift from Dr. Ian Trowbridge (Salk Institute, San Diego, CA). Human recombinant interleukin-2 (IL-2) was a gift from Cetus Corp. (Emeryville, CA) and indo-l was obtained from Molecular Probes (Portland, OR). @ mercaptoethanol (ME) was obtained from Aldrich Chemical Co. (Milwaukee, WI), 5’-bromodeoxyuridine (BrdU) from Sigma (St. Louis, MO) and penicillin-streptomycin and RPM1 1640 were from GIBCO (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone Laboratories Inc. (Logan, UT). Preparation of PBL, lymph node cells, and splenocytes. For studies which required PBL, animals were first anesthetized with ketamine/xylazine (0.22 ml of xylazine, 20 mg/ml: 0.65 ml ketamine, 100 mg/ml; 9.13 ml saline: dosage of 0.3 to 0.7 ml intra-

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peritoneal injection) and blood (0.5- 1.O ml) was collected with a Pasteur pipet from the retroorbital sinus. Mice were then sacrificed by cervical dislocation. Axillary and inguinal lymph nodes and spleens were retrieved using aseptic technique. Cell suspension was prepared by mechanical dissociation of spleens. Splenocytes (2 X lo7 cells) and blood were each layered over 3 ml of Lympholyte M (Cedarlane Laboratories, Hornby, Ontario) and centrifuged at 400g for 30 min. The buffy coat was removed and cells were washed three times in RPM1 1640 supplemented with 5% FBS. Lymph node cell suspensionswere prepared by passing the cells over a sterilized stainless steel mesh and then through a 23-gauge needle. Cells were counted in a hemocytometer. To obtain enough cells for the analyses,splenocytes, PBL and lymph node cells from two age-matched WBB6Fl mice were pooled for each experiment. Cell surface staining and indo-l loading. For quantitation of T-cell subsetsand CD3 receptors, 0.5 X lo6 cells were suspended in 50 &RPM1 medium containing 10% FBS. PE- or FITC-conjugated mAb was added in saturating amounts. For cell sorting, 1 X IO7 cells were stained with PE-conjugated anti-CD4 and FITC-conjugated antiCD8 mAb for 20 min, washed, and stored in the dark until use. For [Ca2+]i analysis, cells were loaded with 3 pg/ml indo-l acetoxymethyl ester for 45 min at 37°C at a concentration of 10’ cells/ml. Cells were washed and stained with PE- and FITCconjugated mAb as described above. Measurement of[Ca”]i. Measurement of [Ca2+]i was performed on an Ortho Cytofluorograph 50-H with a Model 2 150 computer (Ortho Diagnostic Systems, Westwood, MA), as previously described (10). In brief, uv excitation from an argon ion laser (100 mW at 35 1 to 364 nm) was used to analyze indo-l fluorescence. FITC and PE fluorescence were excited by a second argon laser (488 nm). Violet indo- 1 emission was detected at 383 to 407 nm and green indo- 1 fluorescence was measured at 5 15 to 535 nm. The spatially and temporally separatedgreen FITC emission was detected through the same filter but a second fiberoptic cable and photomultiplier was used. PE emission was collected at 563 to 589 nm. Forward angle uv scatter was used to gate cells from debris. For each cell falling into the PE or FITC region, the logarithm of the ratio of violet/green fluorescence was calculated by subtraction of log green from violet and displayed on a two-parameter cytogram as a function of time. For each experiment, anti-CD3 mAb was added to 0.5 ml cell suspension (1 X lo6 cells) 2 min after the start of the analysis. Mean indo-l ratio and [Ca2+]i vs time and percentage of responding cells vs time were performed as previously described ( 10) on an IBM PC/AT using the software MULTITIME (Phoenix Flow Systems,San Diego, CA). In order to minimize possible effectsof day to day variations, data were analyzed using a paired, two-tailed t test to compare results from young and old mice studied on the same day. Cell sorting. PE-labeled CD4+ and FITC-labeled CD8+ T cells were sorted using the same machine and configuration (but visible laser only) as described above, using a flow rate of 1500 cells/set as previously described (9). Culture conditions. Round-bottom, 96-well microtiter plates (Nunc) were precoated with anti-CD3 mAb (0.5 pg/ml/well) for 30 min and washed once with RPM1 1640 medium. Sorted cells were cultured at 5 X lo4 cells/well for 72 hr in growth medium consisting of RPM1 1640, supplemented with 10% FBS, 1.5 X 1Oe4M BrdU, 100 U/ ml penicillin, 100 pg/ml streptomycin, and 5 X 10d5ME. Selectedsamples were also supplemented with 50 U/ml recombinant IL-2.

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Cell cycle analysis. Three days after incubation at 37°C in the presence of BrdU, medium was removed from the culture wells and replaced with 0.2 ml Hoechst staining solution which consists of: 0.1 M Tris, pH 7.4, 0.9% NaCl, 1.O mM MgCl*, 0.2% bovine serum albumin (BSA), 0.1% Nonidet P-40, and 1 pg/ml Hoechst 33258 (Sigma, St. Louis, MO) after 30 min. An additional 0.2 ml staining solution was added which contained 5 pg/ml ethidium bromide (EB) (Calbiochem, La Jolla, CA) in addition to the above and cells were incubated for a further 30 min before flow cytometric analysis. Analyses were performed on an ICP flow cytometer (Ortho Diagnostic Systems) interfaced to an IBM PC/AT, using the software ACQ-CYTE (Phoenix Flow Systems, San Diego, CA). Ultraviolet excitation (UGl filter) was used and Hoechst 33258 emission detected at 425 to 500 nm, and EB fluorescence detected above 600 nm. The histogram of Hoechst vs EB fluorescence was stored for 3-4 X lo3 cells and cell cycles were analyzed as previously described (11).

RESULTS Comparison of T-cell subsets in diflerent lymphoid organs from young and old mice.

There was a significant decreasein the percentage of CD4’ cells in all lymphoid organ examined in old mice (Fig. 1A). The percentage of CD8+ cells was similar in each lymphoid organ, comparing old vs young mice (Fig. 1B). To determine whether there were alterations in cell populations within these subsets,the percentage of cells with high Pgp1 was evaluated in CD4+ or CD8+ cells. There was increased Pgp1 expression in CD4+ and CD8+ subsetsin all lymphoid organs from old mice (Figs. 1C and ID), with the exception of lymph node, in which differences in CD4+ cells did not reach statistical significance. Comparison of percentage of prolqerating cells in CD4+ and CD8+ subsets of splenocytes, lymph nodes, and PBL from young and old mice. Figures 2A-2F summarize

the proliferative responseof CD4+ and CD8+ cells from spleen, peripheral blood. and lymph node with and without the addition of IL-2 to the cultures. CD4+ splenocytes from old mice had significantly decreasedproliferation and this was not corrected by optimization of cultured conditions by the addition of IL-2 (Fig. 2A). While lymph node CD4+ cells (Fig. 2B) from young and old animals grew similarly whether or not IL-2 was added, CD4+ cells from PBL (Fig. 2C) from young and old mice both showed enhanced proliferation after IL-2 supplementation; however, there were no age-related growth differences. It was of note that CD8+ cells from lymph nodes (Fig. 2E) and PBL (Fig. 2F) from old mice grew significantly better than CD8+ cells from young donors. With the addition of IL-2 to lymph node and PBL cultures, there was increased proliferation of young CD8+ cells but the old CD8+ did not increase proliferation suggestingthat they were already growing maximally. In contrast, CD8+ cells from spleens of young and old mice grew similarly without IL-2, but while the addition of IL-2 increasedproliferation of young cells, the CDS’ cell growth in old animals was unaffected by IL-2 (Fig. 2D). Comparison of cell cycle progression of cells exiting G, in young and old mice. It

has been suggestedthat the proportion of cells which can respond to a stimulus is reduced in old animals, and that those which do respond are functionally similar to cells from younger animals (12). This hypothesis can be directly tested by evaluating the cell cycle progression of responding subsetsof cells in young and old animals since the BrdU/Hoechst technique allows analysis of the percentage of cells in the first,

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FIG. 1. The percentage of CD4+ and CD8+ cells (A and B) and the percentage of Pgpl high cells within these subsets(B and D) were measuredin cells derived from splenocytes,peripheral blood, and lymph nodes from young and old animals. Differences are significant at (**), P < 0.0 1.

second, or third cell cycle after stimulation. Cell cycle progression into the second and third cell cycles was similar in CD4+ and CDV lymphocytes from all three tissues, spleen, peripheral blood, and lymph nodes, from young and old mice and was not affected by the addition of IL-2 to the cultures (Fig. 3). Comparison of antigen density of CD3 on young and old CD4+ or CD8+ cells. To assesswhether differences in the response to anti-CD3 mAb in different lymphoid organs of young and old mice were related to CD3 receptor density, CD4+ or CDV cells were simultaneously stained with anti-CD3 mAb and CD4 mAb or CD8 mAb. Identical levels of CD3 binding were seenin cells derived from splenocytes and lymph nodes; however, T cells derived from peripheral blood of old mice showed 67% (CD4) and 33% (CD8) higher CD3 binding (Table 1). [Ca”‘]i and maximal percentage of responding cells of CD4+ and CD8+ cells derived from splenocytes, lymph nodes, and PBL. To determine whether alterations in cell proliferation might be related to differences in efficiency of transmembrane signaling,

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FIG. 2. The mean percentages of proliferating cells +_ SEM are shown for CD4’ and CD8’ lymphocytes from six 3-month-old (solid bars) and six 27- to 30-month-old (hatched bars) WBB6F 1 mice for cells derived from splenocytes, lymph nodes, and peripheral blood. The percentage of proliferating cells, defined as cells that have exited G,/G, and entered S-phase or further, was determined in sorted CD4+ and CD8’ cells cultured for 72 hr with and without supplementation of IL-2. Differences are significant at (*), P < 0.05.

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FIG. 3. Comparison of cell cycle progression of CD4+ and CD8+ cells which have exited G, for young (solid) and old (hatched) WBB6FI mice. Cells grown with and without IL-2 were analyzed as the proportion of proliferating cells which had progressedto the second or the third cell cycle by 72 hr after anti-CD3 stimulation. Differences are significant at (*), P < 0.05.

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TABLE I CD3 Receptor Density on CD4’ and CD8+ Cells of Young and Old Animals Cells examined

Organ

Differences between young and old (%)’

CD4’ CD8’ CD4’ CD8’ CD4+ CD8’

Spleen Spleen Lymph node Lymph node PBL PBL

3.8 ~2.6 -6.7 -8.3 67.2 33.8

” Values are expressedas the percentage of the difference between positively vs negatively stained cells

we measured mitogen-induced [Ca2’]i in CD4+ and CD8+ cells derived from splenocytes (Fig. 4A), lymph nodes (Fig. 4B), and peripheral blood (Fig. 4C) from young and old mice. Cells were stimulated with either 1 or 10 pg/ml anti-CD3 mAb. CD4’ and CD8+ cells derived from splenocytes from young mice (Fig. 4A) showed significantly higher [Ca’+]i levels after treatment with both concentrations of anti-CD3 mAb than were seen in corresponding cells from old mice. The pattern of lower levels of intracellular calcium responsesin older animals was similar in PBL, except that no significant age-relateddifference was seenin CD8+ cells from PBL with the lower dose of anti-CD3 mAb stimulus (Fig. 4C). In contrast, no significant age-related changes in [Ca2’], were seen in lymph nodes (Fig. 4B). To ascertain if the above differences were due to lower numbers of cells in old mice which could respond to the stimulus, we measured the percentage of cells in which [Ca’+]i rose above baseline levels. In general, PBL and splenocytes from old mice which had a reduced [Ca2’]i responsealso showed a decreasedpercentageof responding cells (Fig. 5) and this was statistically significant at the higher concentration of antiCD3 mAb. This would suggestthat the reduced mean [Ca’+]i responseswere due in part to a decreasedpercentage of responding cells. DISCUSSION The purpose of this study was to evaluate age-relatedchangesin lymphocyte subsets derived from different lymphoid organs in the mouse and determine whether the ageassociated decreasedproliferative capacity of lymphocytes in culture was related to alterations in signal transduction, changes in T-cell subsets, percentage of cells responsive to activation, or to lymphocyte CD3 receptor density. We also wished to determine if lymphocytes from old mice were a good model for the understanding of age-related changes reported in lymphocytes from elderly humans (9). Our results showed that the proliferative response was only decreased in T cells from spleens of old WBB6Fl mice, but not PBL or lymph nodes (Fig. 2A). This is in contrast to studies by Kay et al. (13) in which a significant reduction in proliferation of T cells from lymph nodes of old mice was noted. This may have been due in part to the decreasedpercentageof CD4+ cells in nodes of old mice (Fig. 1A) in unseparated lymph node preparations. Gahring and Weigle (14) analyzed antigen-specific T-cell proliferative responsein CBA CD4+ cells derived from lymph nodes of old mice which

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FIG. 4. Comparison of peak [Ca*+], + SEM of CD4+ and CD8+ lymphocytes derived from splenocytes, lymph nodes, and peripheral blood of young (solid) and old (hatched) WBB6Fl mice. For measurements of [Ca’+], cells were stimulated with 1 and 10 &ml anti-CD3 mAb. Differences are significant at (*), P < 0.05.

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showed significantly decreasedproliferation, whereas cells from C57BL/6 mice were not affected. It has been suggestedby Miller ( 12) that the decreasedproliferative responseof cells in old mice may be a result of a decreasein the proportion of cells responding to the

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stimulus, but that among the responders there may be no age-related change. Our data strongly support this hypothesis; we found that the proliferative defect in old cells is in the transition from Go to Gr of the first cell cycle, but that for the subset of cells from old animals which do enter the cycle, progression through the second and third cell cycles is similar to that of cells from young animals (Fig. 3). This differs somewhat from our previous results obtained with human peripheral blood T cells (9) in which a reduction in cell cycle progression of responding cells was seen in CD8+ cells from old donors, as well as a reduced proportion of responding cells. A reduction in proportion of responding cells has also been reported previously in human PBL by StaianoCoico et al. (15) and Kubbies et al. (16). Differences noted in T-cell proliferation in old animals could be related to differences in percentagesof “naive” vs “memory” cells in T-cell subsetsfrom various lymphoid organs. Naive cells derive from recent thymus emigration and convert upon antigen stimulation into memory cells that have increased expression of Pgp1 ( 17). Such memory cells are thought to increase with age and exhibit lower [Ca2+]i and proliferative responsesupon stimulation with Con A when compared with naive T cells (5, 18). Lemer et al. (18) reported that although the percentage of CD4+ and CD8’ Pgp1 high cells increased with age, this increase was similar in lymph node, spleen, and blood T cells of B6DFl mice. Our studies showed that CD4+ cells from spleensof old WBB6Fl mice had a higher percentage of Pgpl high cells (Fig. 1) than cells derived from lymph nodes or peripheral blood. This may partly explain the decreasedcalcium and proliferative response we noted in splenocytes from old animals. To further examine the basis of proliferative defects in cells from old animals, we examined CD3 receptor density on CD4+ and CD8 cells from various lymphoid organs. CD3 receptor density was not significantly different in both T-cell subsets derived from spleen and lymph nodes of young and old mice (Table 1). Interestingly, both CD4 and CD8 cells from PBL of old animals had a higher CD3 receptor density. These results differ from human studies in which CD3 receptor density is not altered with age in PBL (9). Increased receptor density in cells from old mice did not compensate for defects in transmembrane signaling, since [Ca’+]i was nevertheless decreased. We further investigated whether the reduced proliferative response in old mice could be related to decreased transmembrane signaling after mitogen stimulation. Although [Ca2+]i is thought to play an important role in T-cell activation, the relationship between the magnitude of transient changesin [Ca2+]iand T-cell proliferation has been unclear. Using Con A stimulation, Philosophe and Miller (5) showed that lymphocytes exhibiting a high [Ca2+]i had a better proliferative capacity than that of cells exhibiting a low [Ca2+]i, but the T-cell subsetswere not examined in these studies. Other groups have also linked increased [Ca2+]i with increased cell proliferation ( 19), but this relationship could not be confirmed with T-cell lines (20-22). In this report, we found multiple cell subsets in which an age-related difference in [Ca2+li was not correlated with a changein proliferation, or vice versa.We further attempted to correlate peak [Ca2+]i and proliferative capacity in CD4+ and CD8+ cells from different tissues of an individual mouse and we found little correlation between the two parameters (r = .08 for CD4+ cells, r = .21 for CD8+ cells, data not shown). Additionally the percentage of cells which could respond to the stimulus by increasing their [Ca2+]i 2 SD over baseline did not correlate to the percentage of cells which could exit Gi (data not shown). Differences between Philosophe and Miller (5) and the studies described here

Spleen BDFl CBA C57BL/6 WBB6Fl Peripheral blood WBB6F 1 Lymph node WBB6Fl

Mouse

Human Peripheral blood

t

J-

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4

4

i

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t

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% Responding cells

CD4+

zz

=

:

i

% Responding cells

Proliferation

=

4

=

Cell cycle progression

(+IL-2)

=

J

4

i

=

Magnitude

W’l,

4

4

i =

=

CD8 +

i

4 =

=

4

% Responding cells

Proliferation

mAb) and Cell Proliferation

% Responding cells

and Percentage of Cells Responding to the [Ca”] Stimulus (10 pg/ml Anti-CD3 in the Presence of IL-2 between Young and Old Human and Mouse

[Ca2+li

of [Ca”]

Magnitude

Comparison

TABLE 2

=

=

4 = =

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4

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could be related to differences in the activation stimulus or different assaysused to evaluate lymphocyte proliferation. Con A preferentially stimulates naive cells, whereas anti-CD3 mAb mainly stimulates memory cells (23). The role of murine viral infections should also be considered. Mice used in Philosophe’s studies were not described as being specific pathogen free, and ubiquitous viruses such as mouse hepatitis virus present in conventional animal colonies can alter immunologic function (24, 25). One of the goals of this work was to evaluate the relationship of age-related changes in T lymphocytes from various lymphoid organs in the mouse to those previously seenin human PBL; if the mouse is to be a good animal model for human age-related defects,there should be a concordance with human proliferation and signaling-related changes.Table 2 summarizes the results of murine and human studies which we have performed regarding T-cell subset analysis in this and previous reports. In elderly humans, [Ca2+]iis reduced in CD4+ cells derived from peripheral blood but this transmembrane signaling is not accompanied by a reduced percentage of responding cells or a decreasedproliferative response. Similarly, CD4 cells from peripheral blood of WBB6Fl mice also exhibit a reduced [Ca2+]i, which like human cells, is not accompanied by a decreased proliferative response. One difference is that, unlike human cells, the percentage of responding cells was decreasedin murine WBB6Fl CD4+ cells. In general, however, age-related defects in CD4+ cells derived from peripheral blood appear to be similar between mice and humans. In contrast, CD4+ splenocytes from four mice strains exhibited a reduced [Ca2+]i, a decreasedpercentage of responding cells and a reduced proliferative response. CD4+ lymphocytes derived from lymph nodes of WBB6Fl mice did not show age-related defects in [Ca2+]i or proliferation as previously discussed. CD8+ cells from peripheral blood of elderly humans exhibited a reduced proliferative response but [Ca2+]i and the percentage of responding cells were not decreased.The opposite result was seen in CD8+ cells from peripheral blood of old WBB6Fl mice; [Ca2’]i and the percentage of responding cells were decreased but the proliferative response was similar to that of young mice. The response of CD8+ splenocytes from different mouse strains was variable. CD8+ cells from old CBA and WBB6Fl mice showed reduced [Ca2+]i responses,decreasedpercentage of responding cells, and decreased cell growth. In BDFl mice, [Ca2+]i responsesand percentagesof responding cells were decreasedwith age,but proliferative responsewas the sameas that for young BDFl mice. C57B1/6J mice showed no age-related defects in any of the parameters measured. Once again, CD8+ cells from lymph nodes showed no age-related changes, Our results would suggestthat CD4+ cells from peripheral blood of older mice may be a satisfactory model to study the age-related defects noted in human peripheral blood CD4+ cells. However, it must be noted that although blood lymphocytes are the main source of cells used to study human immunosenescence, these cells might permit only a restricted view of the immune system (26) since cells derived from other lymphoid organs (e.g., spleen) may be more altered by the aging process. ACKNOWLEDGMENTS We thank Ms. Faith Shiota for lymphocyte preparation, Mr. Chong Kim and Robert Lee for their assistance with flow cytometric analyses,and Janice Garr for preparing the manuscript. This work was supported by Grant AGO1751 from the NIH.

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