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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:568-571 (2006)
© 2006 The Gerontological Society of America

Age-Related Accumulation of a Novel CD44 + CD25low{gamma}{delta} T-Cell Population in Hematopoietic Organs of the Mouse

Jessica C. Langer, Ritu Kumar and Hans-Willem Snoeck

Department for Gene and Cell Medicine, Mount Sinai School of Medicine, New York.

Address correspondence to Hans-Willem Snoeck, MD, PhD, Department for Gene and Cell Medicine, Mount Sinai School of Medicine, Box 1496, Gustave L. Levy Place, New York, NY 10029. E-mail: hans.snoeck{at}mssm.edu


   Abstract
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 Abstract
Materials and Methods
 Results and Discussion
 References
 
We discovered a novel population of {gamma}{delta} T cells in the mouse that accumulates with age in hematopoietic organs, but not in epithelia. These cells are CD25low (an unusual phenotype for {gamma}{delta} T cells in the mouse); express higher levels of TCR{gamma}{delta} and CD44 than do CD25– {gamma}{delta} T cells; mainly express V{gamma}2, V{gamma}3, and V{gamma}4 chains; and are largely quiescent. A very similar cell population appears in the late stages of fetal thymus organ cultures, suggesting that the accumulation of CD44 + CD25lowTCR{gamma}{delta} + cells is a response to stress induced by aging in vivo or by culture in vitro. The precursors of CD44 + CD25lowTCR{gamma}{delta} + cells are generated during fetal or very young adult life, as this population was undetectable in aged recipients of bone marrow from old or young donors. CD44 + CD25lowTCR{gamma}{delta} + cells may be a biomarker of aging, but could also play a role in the inflammatory changes that accompany aging.

AGING in mice and humans is accompanied by inflammation and increased autoimmunity, together with decreased cellular and humoral immune responses to infectious agents and vaccines (1,2). These observations suggest that immune dysregulation is a contributing factor to or a secondary consequence of the aging process. Age-related changes in the immune system are therefore of interest to gain insight into organismal aging. These changes include a loss of lymphoid potential in hematopoietic stem cells, thymic involution and a concomitant depletion of naïve T cells and expansion of memory cells, a sharp decrease in the earliest B-cell precursors, and changes in the composition of the peripheral B-cell pool (3–6). Little is known about age-related changes in {gamma}{delta} T cells, a T-cell lineage whose role is still unclear. {gamma}{delta} T cells have been implicated, depending on their V{gamma} and V{delta} usage, in both the initiation of a specific, TCR{alpha}ß-mediated immune response against pathogens and in the resolution of inflammation after eradication of the infectious agent. Similarly, in various models of autoimmune disease, {gamma}{delta} T cells appear to play an immunoregulatory role although, in some models, they appear to play a role in pathogenesis. {gamma}{delta} T cells localize mainly to epithelia, and within each epithelium they express specific oligoclonal {gamma}– and {delta} – chain rearrangements (7–9). In humans, peripheral blood V{gamma}9V{delta}2 cells decrease in frequency but appear to acquire a lower activation threshold with age (10,11). Here we report the identification of a novel population of CD44 + CD25low {gamma}{delta} T cells of unknown function that accumulates with age in hematopoietic organs, but not in epithelia in mice.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results and Discussion
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Mice
C57BL/6J (Jackson Laboratories, Bar Harbor, ME) and C57BL/6.SJL-PtprcaPep3b/BoyJ (National Cancer Institute, Frederick, MD) mice were kept in a specific pathogen-free facility. Experiments and animal care were performed in accordance with the Mount Sinai Institutional Animal Care and Use Committee (IACUC).

Antibodies and Flow Cytometry
FITC-conjugated CD45.1, CD2, CD3, CD8{alpha}, CD4, B220, Ly6G/Gr1, Mac1, Ter119, TCRß, phycoerythrin-conjugated Sca1, and CD45.2 were purchased from Southern Biotechnologies (Birmingham, AL). FITC-conjugated anti-TCR{gamma}{delta}, phycoerythrin-conjugated CD25, and allophycocyanin-conjugated CD44 were purchased from Pharmingen (San Diego, CA). Cell cycle analysis was performed after propidium iodide staining of 80% cold ethanol-fixed samples in the presence of RNAse, and pulse shape analysis to exclude doublets.

Semiquantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA from purified thymic CD44 + CD25lowTCR{gamma}{delta} + or CD25–TCR{gamma}{delta} + cells was isolated using Trizol Reagent (Gibco BRL, Grand Island, NY) according to the manufacturer's instructions and treated with DNaseI, followed by reverse transcription using Superscript II (Gibco) for 1 hour at 42°C using oligo(dT) primers. For the {gamma} chain of the {gamma}{delta} T-cell receptor, primers spanning each of the variable regions (the same primer amplified V{gamma}1.1-1.3) and a primer common to the constant regions were used. The primers were: 5'-TATCGGTCACCAGAGCAACA-3' for V{gamma}1, 5'-GAAGAACCCTGGCTCACAAG-3' for V{gamma}2, 5'-TCAGCTCTCCTTTACCCGAA-3' for V{gamma}3, 5'-AGGGGAATCGAGGATACAGG-3' for V{gamma}4, 5'-CAATCACCAAGCTAGAGGGG-3' for V{gamma}5, and 5'-TTGCAATCCTTTTCTTTCCA-3' for the constant region (Garman nomenclature; 12).

Fetal Thymus Organ Cultures
D16 thymi were cultured as described in (13) for up to 25 days.

Bone Marrow Transplantation
Bone marrow cells (2 x 106) from 8-week- or 18-month-old C57BL/6.SJL-PtprcaPep3b/BoyJ (CD45.1+) mice were injected into lethally (950 cG) irradiated 8-week-old C57BL/6 mice (CD45.2+).

Statistical Analysis
A two tailed t test for unpaired samples was used. Results are given a mean ± standard error of the mean.


   RESULTS AND DISCUSSION
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We discovered a population of CD4–CD8–CD44 +&!hairsp;CD25low cells in the thymi of aged (16–20 months) C57BL/6 mice (Figure 1a) that was not detectable in young mice (2 months). By adding combinations of individual lineage antibodies to the CD4/CD8 stain used to define CD4–CD8– cells, it was found that the CD4–CD8–CD44 + CD25low subpopulation expressed TCR{gamma}{delta} (Figure 1a). Similar data were obtained in the thymi of 16-month-old DBA/2 mice (not shown). Direct staining of the thymus, bone marrow, spleen, and peripheral blood from old mice for CD44, CD25, and TCR{gamma}{delta} resolved the same cell population at similar frequencies in each tissue, although there was a tendency toward a lower frequency in the bone marrow, where variability also appeared the highest (thymus: 0.2 ± 0.07%, n = 6; spleen 0.219 ± 0.08%, n = 4; bone marrow 0.091 ± 0.04%, n = 5; peripheral blood 0.3 ± 0.1%, n = 3; mean + standard error of the mean) (Figure 1b). In young mice, however, CD44 + CD25lowTCR{gamma}{delta} + cells were 10-fold less frequent to undetectable in the same tissues (Figure 1b), and these cells accumulated exponentially with age (Figure 1c). As {gamma}{delta} T cells are mainly associated with epithelia (7–9), we analyzed intestine, skin, and tongue for the presence of CD44 + CD25lowTCR{gamma}{delta} + cells. In neither young nor old mice were CD44 +CD25lowTCR{gamma}{delta}+ cells observed in these tissues (shown for intestine in Figure 1b). The increase in the fraction of CD44 + CD25lowTCR{gamma}{delta} + cells in hematopoietic organs represents an increase in the absolute number of these cells and is not a reflection of changes in total cellularity. Bone marrow mononuclear cellularity was in fact higher in old mice than in young mice (2.9 x 107/femur in old vs 1.8 x 107/femur in young mice, p =.03, n = 3), whereas a small, statistically nonsignificant decrease in cellularity was observed in the spleen (not shown). Only in the thymus could the increase in the fraction of CD44 + CD25lowTCR{gamma}{delta} + cells be explained by a decrease in overall cellularity with age (from 6.1 x 107 at 8 weeks to 0.47 x 107 at 24 months, p =.0029). However, if the accumulation of CD44 + CD25lowTCR{gamma}{delta} + cells in the thymus were passive, then it would have to be assumed that the thymic niche for these cells is specifically maintained during thymic involution. Taking into account the Boggs estimate of the total number of bone marrow cells [20 x 107 in young mice (14), i.e., 20 x 2.9/1.8 = 32 x 107 in aged mice given our data on bone marrow cellularity in aged mice], together with our estimates of spleen cellularity (3 x 107) and thymic cellularity (0.5 x 107), and of the frequencies of CD44 + CD25lowTCR{gamma}{delta} + in these organs, we estimate that there are approximately 3.6 x 105 CD44 + CD25lowTCR{gamma}{delta} + cell in aged mice, mainly in the bone marrow.


Figure 01
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  		Figure 1. CD44 + CD25low {gamma}{delta} T cells in aged mice. a, Presence of population of CD4–CD8–CD44 + CD25low cells in the thymus of 18-month-old C57BL/6 mice (left, gated on CD4–CD8– cells). A CD44 + CD25low population was undetectable among lineage – (CD4–CD8–CD–TCRß–TCR{gamma}{delta}–NK1.1–Ter119–B220–Gr1–Mac1–) cells (middle, gated on lin– cells) in the thymus of 18-month-old C57BL/6 mice. The absence of a CD44 + CD25low population after gating on CD4–CD8–TCR{gamma}{delta}– cells shows that these cells express TCR{gamma}{delta} (right). Representative of five experiments. b, Representative examples of the frequency of CD44 + CD25lowTCR{gamma}{delta} + cells in the thymus, spleen, bone marrow, peripheral blood, and intestine of C57BL/6 mice at the ages of 8 weeks and 18 months. c, Age-associated accumulation of CD44 + CD25lowTCR{gamma}{delta} + cells in the thymus. d, Expression levels CD44 (left) and TCR{gamma}{delta} (right) on CD44 + CD25lowTCR{gamma}{delta} + and CD25–TCR{gamma}{delta} + cells in the thymus. e, Expression of messenger RNA for V{gamma}1-5 in CD44 + CD25lowTCR{gamma}{delta} + cells and in CD25–TCR{gamma}{delta} + cells isolated from the thymus of 18-month-old CD57BL/6 mice

 
The phenotype of this population is peculiar, as CD25 + {gamma}{delta} T cells have not been described yet in mice, although CD25 is an activation marker of {gamma}{delta} T cells in cattle (15), sheep (16), rats (17), and human peripheral blood V{gamma}9V{delta}2 cells (18–20). CD44 + CD25lowTCR{gamma}{delta} + cells were uniformly CD44 +, whereas CD25– {gamma}{delta} T cells expressed varying amounts of CD44 (Figure 1d). In addition, CD44 + CD25lowTCR{gamma}{delta} + cells expressed higher levels of TCR{gamma}{delta} than did CD25– {gamma}{delta} T cells (Figure 1d). These expression patterns suggest that CD44 + CD25lowTCR{gamma}{delta} + cells are highly activated. Despite their apparently activated status, the cell cycle activity of CD44 + CD25lowTCR{gamma}{delta} + cells was significantly lower than that of CD25– {gamma}{delta} T cells (fraction of S/G2/M cells 26 ± 9.4% in CD25– {gamma}{delta} T cells vs 2 ± 0.7% in CD44 + CD25lowTCR{gamma}{delta} + cells, n = 4; p =.03), indicating that CD44 + CD25lowTCR{gamma}{delta} + cells are not an expanding cell population, but probably arise from a CD25– precursor. The CD44 + CD25lowTCR{gamma}{delta} + cell population expressed V{gamma}2, V{gamma}3, and V{gamma}4, but low to undetectable levels of V{gamma}1 and V{gamma}5 [nomenclature from Garman and colleagues (12), Figure 1e], indicating that, although there is some selectivity in V{gamma} usage, this population does not express a canonical TCR{gamma}, in contrast to {gamma}{delta} T cells in many other tissues. One function of {gamma}{delta} T cells in epithelial tissues (such as intestine and skin) is the maintenance of epithelial integrity, mediated by the production of keratinocyte growth factor (KGF) (21–23). However, no KGF messenger RNA could be detected in purified CD44 + CD25low {gamma}{delta} T cells, whereas CD25– {gamma}{delta} T cells expressed KGF messenger RNA (not shown).

We next investigated whether CD44 + CD25lowTCR{gamma}{delta} cells could be generated in fetal thymus organ cultures (FTOC; 13). In these cultures, CD44 + CD25lowTCR{gamma}{delta} cells with a similar level of expression of TCR {gamma}{delta} (Figure 2a) and a similar pattern of expression of V{gamma} chains (Figure 2b) as CD44 + CD25lowTCR{gamma}{delta} + cells in aged mice progressively accumulated. The generation of CD44 +&!hairsp;CD25lowTCR{gamma}{delta} + cells after prolonged culture in FTOC indicates that age per se does not induce the development of CD44 + CD25lowTCR{gamma}{delta} + cells. As in vitro culture represents a severe form of oxidative stress to cells and tissues (24,25), our data suggest that stress, either during culture in vitro or during the aging process in vivo, is critical for the generation of CD44 + CD25lowTCR{gamma}{delta} + cells. It is interesting to note in this context that many {gamma}{delta} TCR can recognize nonclassical major histocompatibility (MHC) molecules expressed by stressed cells (7–9). To investigate whether the age of the environment or of the hematopoietic system determines the generation of CD44 + CD25lowTCR{gamma}{delta} + cells, we transplanted 2 x 106 bone marrow cells from old (18 months) and young (8 weeks) mice into lethally irradiated young recipient mice, which were aged to the age of 14 months. Using this procedure, 97% of the hematopoietic cells in the recipients were donor-derived (not shown). CD44 + CD25lowTCR{gamma}{delta} + cells were undetectable in any of the aged reconstituted animals (Figure 2c). Thus, neither young nor aged hematopoietic stem cells (HSC) can generate CD44 + CD25lowTCR{gamma}{delta} + cells de novo. Therefore, their precursors must have been generated during fetal or young adult life.


Figure 02
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  		Figure 2. Generation of CD44 + CD25lowTCR{gamma}{delta} + cells. a, Accumulation of CD44 + CD25lowTCR{gamma}{delta} + cells in fetal thymus organ cultures. Note that, as in CD44 + CD25lowTCR{gamma}{delta} + in hematopoietic organs of aged mice (see
Figure 1a), the expression of TCR{gamma}{delta} is higher than in CD25–TCR{gamma}{delta} + cells. b, Expression of messenger RNA for V{gamma}1-5 in CD44 + CD25lowTCR{gamma}{delta} + cells and CD25–TCR{gamma}{delta} + isolated from fetal thymus organs cultures. c, Frequency of CD44 + CD25lowTCR{gamma}{delta} + cells 12 months after reconstitution of lethally irradiated recipients with bone marrow from young (left) or aged (right) mice

 
Summary
We describe the accumulation of a novel population of CD44 + CD25lowTCR{gamma}{delta} + cells in the hematopoietic tissues of aged mice and in the late phase of FTOC. The accumulation of CD44 + CD25lowTCR{gamma}{delta} + cells with age together with their activated phenotype and the known role of {gamma}{delta} T cells in immune regulation (7–9) suggests a role in the aging process, which is characterized by enhanced inflammation and autoimmunity (1,2). It is also possible, however, that the frequency of CD44 + CD25lowTCR{gamma}{delta} + cells in hematopoietic organs is a biomarker of aging and a response to the aging of hematopoietic tissues such as the thymus.


   Acknowledgments
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 Results and Discussion
 References
 
This work was supported by National Institutes of Health grants RO1 AG16327 and R01 HL073760 to Dr. H.-W Snoeck.


   Footnotes
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 Results and Discussion
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Decision Editor: James R. Smith, PhD

Received September 16, 2005

Accepted November 17, 2005


   References
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  1. Boren E, Gershwin ME. Inflamm-aging: autoimmunity, and the immune-risk phenotype. Autoimmun Rev. 2004;3:401-406.[Medline]
  2. Caruso C, Lio D, Cavallone L, Franceschi C. Aging, longevity, inflammation, and cancer. Ann N Y Acad Sci. 2004;1028:1-13.[Medline]
  3. Geiger H, Van Zant G. The aging of lympho-hematopoietic stem cells. Nat Immunol. 2002;3:329-332.[Medline]
  4. Allman D, Miller JP. The aging of early B-cell precursors. Immunol Rev. 2005;205:18-29.[Medline]
  5. Miller JP, Allman D. The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors. J Immunol. 2003;171:2326-2330.[Abstract/Free Full Text]
  6. Linton PJ, Dorshkind K. Age-related changes in lymphocyte development and function. Nat Immunol. 2004;5:133-139.[Medline]
  7. Hayday AC. {gamma}{delta} T cells: a right time and a right place for a conserved third pathway of protection. Annu Rev Immunol. 2000;18:975-1026.[Medline]
  8. Hayday A, Tigelaar R. Immunoregulation in the tissues by gammadelta T cells. Nat Rev Immunol. 2003;3:233-242.[Medline]
  9. Carding SR, Egan PJ. {gamma}{delta} T cells: functional plasticity and heterogeneity. Nat Rev Immunol. 2002;2:336-345.[Medline]
  10. Re F, Poccia F, Donnini A, Bartozzi B, Bernardini G, Provinciali M. Skewed representation of functionally distinct populations of Vgamma9Vdelta2 T lymphocytes in aging. Exp Gerontol. 2005;40:59-66.[Medline]
  11. Romano GC, Potestio M, Scialabba G, et al. Early activation of gammadelta T lymphocytes in the elderly. Mech Ageing Dev. 2000;121:231-238.[Medline]
  12. Garman RD, Doherty PJ, Raulet DH. Diversity, rearrangement, and expression of murine T cell gamma genes. Cell. 1986;45:733-742.[Medline]
  13. Ueno T, Liu C, Nitta T, Takahama Y. Development of T-lymphocytes in mouse fetal thymus organ culture. Methods Mol Biol. 2005;290:117-133.[Medline]
  14. Boggs DR. The total marrow mass of the mouse: a simplified method of measurement. Am J Hematol. 1984;16:277-286.[Medline]
  15. Koo HC, Park YH, Hamilton MJ, et al. Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves. Infect Immun. 2004;72:6870-6883.[Abstract/Free Full Text]
  16. Whist SK, Storset AK, Johansen GM, Larsen HJ. Modulation of leukocyte populations and immune responses in sheep experimentally infected with Anaplasma (formerly Ehrlichia) phagocytophilum. Vet Immunol Immunopathol. 2003;94:163-175.[Medline]
  17. Fujioka T, Purev E, Kremlev SG, Ventura ES, Rostami A. Flow cytometric analysis of infiltrating cells in the peripheral nerves in experimental allergic neuritis. J Neuroimmunol. 2000;108:181-191.[Medline]
  18. Conti L, Casetti R, Cardone M, et al. Reciprocal activating interaction between dendritic cells and pamidronate-stimulated gammadelta T cells: role of CD86 and inflammatory cytokines. J Immunol. 2005;174:252-260.[Abstract/Free Full Text]
  19. Chen L, Cencioni MT, Angelini DF, Borsellino G, Battistini L, Brosnan CF. Transcriptional profiling of gamma delta T cells identifies a role for vitamin D in the immunoregulation of the Vgamma 9V delta2 response to phosphate-containing ligands. J Immunol. 2005;174:6144-6152.[Abstract/Free Full Text]
  20. Jouen-Beades F, Paris E, Dieulois C, et al. In vivo and in vitro activation and expansion of gammadelta T cells during Listeria monocytogenes infection in humans. Infect Immun. 1997;65:4267-4272.[Abstract]
  21. Jameson J, Ugarte K, Chen N, et al. A role for skin gammadelta T cells in wound repair. Science. 2002;296:747-749.[Abstract/Free Full Text]
  22. Chen Y, Chou K, Fuchs E, Havran WL, Boismenu R. Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proc Natl Acad Sci U S A. 2002;99:14338-14343.[Abstract/Free Full Text]
  23. Erickson M, Morkowski S, Lehar S, et al. Regulation of thymic epithelium by keratinocyte growth factor. Blood. 2002;100:3269-3278.[Abstract/Free Full Text]
  24. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol. 2003;5:741-747.[Medline]
  25. Busuttil RA, Rubio M, Dolle ME, Campisi J, Vijg J. Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture. Aging Cell. 2003;2:287-294.[Medline]




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