Altered aging-related thymic involution in T cell receptor transgenic, MHC-deficient, and CD4-deficient mice

Mechanisms of Ageing and Development 114 (2000) 101 – 121 www.elsevier.com/locate/mechagedev

Altered aging-related thymic involution in T cell receptor transgenic, MHC-deficient, and CD4-deficient mice Lisa L. Lau, Lisa M. Spain * Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA Received 24 November 1999; received in revised form 28 December 1999; accepted 29 December 1999

Abstract During aging in mice and humans, a gradual decline in thymus integrity and function occurs (thymic involution). To determine whether T cell reactivity or development affects thymic involution, we compared the thymic phenotype in old (12 months) and young (2 months) mice transgenic for rearranged ab or b 2B4 T cell receptor (TCR) genes, mice made deficient for CD4 by gene targetting (CD4 − / − ), mice made deficient for major histocompatability complex (MHC) class I (b2M− / −) or class II genes (Abb − / − on C57Bl/6 background) or both. The expected aging-related reductions in thymic weights were observed for all strains except those bearing disruption of both class I and class II MHC genes. Therefore, disruption of MHC class I and class II appeared to reverse or delay aging-related thymic atrophy at 12 months. Immunohistochemical analysis of aging-associated alterations in thymic morphology revealed that TCR ab transgenes, CD4 disruption, and MHC class II disruption all reduced or eliminated these changes. All strains examined at 12 months showed alterations in the distribution of immature thymocyte populations relative to young controls. These results show that aging-associated thymic structural alterations, size reductions, and thymocyte developmental delays can be separated and are therefore causally unrelated. Futhermore, these results suggest that the T cell repertoire and/or its development play a role in aging-related thymic involution. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

* Corresponding author. Present address: Department of Immunology, Holland Laboratory for the Biomedical Sciences, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. Tel.: + 1-301-7380732; fax: +1-301-5170344. E-mail address: [email protected] (L.M. Spain) 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 0 9 1 - 9

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Keywords: Aging; Thymus; Transgenic; Knock-out; T cell receptors

1. Introduction Aging-associated involution of the mammalian thymus is a well-documented process in which the thymus gradually regresses in size, weight, and cellularity (reviewed in Bodey et al., 1997). In humans, deposits of adipose tissue displace and disrupt the thymic stroma, particularly the cortical epithelial network which supports early T cell differentiation and selection (George and Ritter, 1996). The cortical and medullary regions, and corticomedullary junction, become less well defined. In addition, expression of various cortical and medullary epithelial cell markers, including the MHC class II restricting molecule, is reduced in the aged thymus (Farr and Sidman, 1984; Farr and Anderson, 1985). These changes have been linked to reduced generation and export of naive T cells in aged animals, with the peripheral repertoire eventually shifted toward a less diverse, predominantly memory phenotype. Reduced thymic function with aging is a consequence of changes in both thymocyte precursors and thymic stromal cells. For example, studies showed diminished development of hematopoietic precursors from old compared to young donors in fetal thymic organ culture or in lethally irradiated recipients (Hirokawa et al., 1986; Eren et al., 1988; Sharp et al., 1990). However, the transplantation of young bone marrow to old recipients was unable to reverse decreased T cell regeneration in old thymus (Mackall et al., 1998). The thymic microenvironment in aged hosts supported repopulation in the periphery of about 50% of the number of T cells recovered from young hosts. Thus, the aged thymic microenvironment has a diminished capacity to support thymocyte differentiation. The underlying mechanism of age-related thymic involution has not been determined, although experimental support for hormonal (Simpson et al., 1975; Bellamy et al., 1976) or genetic bases (Takeda et al., 1981; Aspinall, 1997; Kuro-o et al., 1997; Nabarra et al., 1997) exists. Most relevant to the present work is the hypothesis that aging-related alterations in thymocyte development contribute to thymic involution. For example, in the adult thymus, T lineage development of bone marrow-derived precursors can be monitored by expression of the TCR component CD3, and the coreceptors CD4 and CD8. The earliest T cell progenitors are CD4−CD8− or double negative (DN). Within DNs, transient surface expression of CD44 and CD25 further defines thymocyte maturation (Godfrey and Zlotnik, 1993). The most immature are CD44+CD25−. As they differentiate, they become CD44+CD25+ and then CD44−CD25+. TCR chain gene rearrangement, the first step in T cell committment, has been shown to occur at the CD44−CD25+ transition in the DN subset (Godfrey et al., 1994). Further progression to the CD44−CD25− phenotype and to CD4+CD8+ double positive (DP) thymocytes hinges on successful rearrangement of a functional TCR chain (b-selection). Studies have shown that aged thymus contains altered ratios of these early thymocyte

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subsets, showing increases in CD44+25− subsets and decreases in CD44-CD25+ subsets (Thoman, 1995; Aspinall, 1997; Thoman, 1997a,b). Although the precise stage of the aging-related disruption is more consistent with changes in the efficiency of T cell commitment, others have proposed that aging-related failure of early development is due to a disruption in TCR rearrangement and b-selection efficiency in older animals. This possibility was supported by a recent study by Aspinall (1997), showing that aging-related thymic involution was relieved by TCR transgenes. Although suggestive, this earlier study cannot rule out other potential mechanisms by which TCR transgenes rescue thymic involution. For example, TCR transgenes restrict T cells’ antigenic specificity and skew T cell subset development, as well as bypass TCR trarrangement. In the present study, we have investigated these alternative possibilities by examining thymic involution in several additional mouse models. To address the specificity issue, we have examined another transgenic strain which bears a TCR of different specificity (2B4, I− Ek/pigeon cytochrome C restricted). In addition, we have examined mice transgenic for the 2B4 TCR b chain gene alone which bypasses b rearrangement but does not restrict TCR specificity or T cell development. Finally, we have examined mice deficient for the CD4 coreceptor, b2M (MHC class I) and Abb (MHC class II) genes, which have deficiencies in T cell subset development but in which TCR rearrangements are not altered.

2. Materials and methods

2.1. Mice 2B4 TCRab and TCRb Tg mice, and non-Tg littermates (H− 2k), were maintained at the Wistar animal facility under specific pathogen free conditions in sterile microisolater cages with unlimited autoclaved food and water. Young mice were 1− 2 months old, and old mice were 12 months old. Male and female mice were used, as similar results were obtained with both sexes. Young (1 month) and old (20 months) DBA/2 and C57BL/6 mice from the National Institute on Aging (Bethesda, MD) were used within 2 weeks of delivery. MHC-deficient mice on a C57Bl/6 background were retired breeders from Jackson Laboratories and Taconic Laboratories and maintained in our facility until 12 months of age. Aged CD4-deficient mice on a C57Bl/6 background were a gift from R. Ahmed, Emory University, Atlanta, GA.

2.2. Antibodies and reagents FITC anti-CD4 (clone CT-CD4), PE anti-CD8a (CT-CD8a), APC anti-CD8a, PE anti-CD3 (500A2), biotin anti-Vb8.1, 8.2 (KJ16) were from Caltag Laboratories (Burlingame, CA). Biotin anti-V3 (KJ25), biotin anti-CD44 (IM7), and PE antiCD25 (3C7) were from Pharmingen (San Diego, CA). Streptavidin Red670 was from Life Technologies (Gaithersburg, MD). Biotinylated UEA I, biotin anti-rat

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IgG, Vectastain HRP APC kit, DAB substrate kit, and methyl green were from Vector Laboratories (Burlingame, CA). 6C3 hybridoma (Adkins et al., 1988) supernatant was prepared in the lab.

2.3. Preparation and staining of cells Thymuses were removed from euthanized mice. One lobe was processed for immunohistochemistry. The second lobe was weighed in an eppendorf tube and then disrupted with a cone homogenizer into a single cell suspension in staining medium (HBSS with 2.5% FCS and 0.1% NaN3). Viable cell counts (by eosin Y exclusion) were based on an average of four fields counted per sample on a hemacytometer. Thymocytes were incubated with antibodies in staining medium, and stored in 1% formalin in PBS/BSA. Samples were analyzed within 1 week on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software. Diseased mice (mostly lymphoid tumors) were excluded. Tumor-bearing old mice were observed in TCR transgenic and non-transgenic littermates at similar frequencies ( 50%). These high tumor incidences are characteristic of the B10 strain background (Smith et al., 1973).

2.4. Immunohistochemistry Thymus lobes were embedded in Tissue-tek OCT (Sakura Finetek, Torrance, CA), and snap frozen in isopentane. Six micrometer sections cut onto multiwell slides (Carlson Scientific, Peotone, IL) were fixed for 10 min in ice-cold acetone, air-dried, and rehydrated with PBS. Sections were quenched with staining medium and incubated with biotinylated UEA I, or with 6C3 supernatant followed by biotinylated anti-rat IgG. Staining with the Vectastain HRP ABC kit, color development, and counterstaining were done according to manufacturer’s instructions. Slides were then dehydrated, xylene-cleared, and coverslipped using Micromount (Surgipath, Richmond, IL). Several sections from each thymus sample were also stained with hematoxylin and eosin (H&E) for further analysis of tissue integrity and cellularity.

2.5. Statistics Statistical significance was determined by a Student’s t-test using Microsoft Excel software.

3. Results

3.1. TCR transgenes do not pre6ent aging-related reductions in thymus size and cellularity Total thymic weights and cell densities (number of thymocytes per mg of tissue)

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were determined from young (1 –2 months) and old (12 months) 2B4ab (ab) and 2B4b (b) Tg mice (Berg et al., 1989b), and non-Tg littermates. Mice at 12 months of age were used since pilot experiments showed that control animals were dramatically involuted at 12 months compared to young mice. Older mice were not used because they were unavailable, since B10.BR mice (whether transgenic or not) tended to rapidly succumb to lymphomas or become unanalyzable due to tumor tissue after one year of age. A high incidence of lymphoma is characteristic of the B10 strain background (Smith et al., 1973). Thymus weight and cell density were significantly (P B 0.03) reduced in old mice of B10.BR non-Tg strains and ab and b 2B4 TCR transgenics (Fig. 1A, Fig. 2A). The difference between old and young mice of the 2B4 ab strain was less striking mainly because the thymus of young ab TCR Tg mice was smaller and less dense than non-Tg littermates. Similar reductions in thymus cellularity in young mice has also been reported in other H− 2k TCR Tg systems, and may reflect either enhanced negative selection on self-ligands, reduced positive selection due to the avidity of TCR/MHC interactions, or reductions in thymocyte expansion due to more rapid developmental transitions (Brabb et al., 1997). Nevertheless, aging-related size and cell density changes were observed in ab TCR transgenic mice. Thus, the ability to bypass b rearrangement (in either the b alone or ab together TCR transgenic strains) was not sufficient to prevent reduced cellularity in the thymus. Reduced thymic size and cellularity is therefore unlikely to be a consequence of aging-related reductions in TCR rearrangement efficiency.

3.2. MHC deficiencies pre6ent aging-related reductions in thymic weight and cellularity Thymic weights and cell densities were determined from young (1–2 months) and old (12 months) CD4 knock-out mice (McCarrick et al., 1993), b2M − /− (MHC class I deficient), Abb −/ − (MHC class II deficient on the C57Bl/6 background) and b2M−/ − xAbb −/ − doubly-deficient mice, and C57Bl/6 controls. Old C57Bl/6 mice showed significantly reduced thymic weights and cell densities by 12 months of age (Fig. 1B, Fig. 2B). In contrast, mice bearing Abb disruptions did not show any reduction in thymocyte cell densities with age, while mice deficient for b2M showed no significant reduction in thymus weight with age (Fig. 1B, Fig. 2B). Although it is unclear why thymic weight and cell density measurements are not concordant as expected, in b2M −/ −x/Abb −/ − doubly-deficient mice both measures of thymic size are similar in young and old mice. CD4-deficient animals still showed significant reductions in both measurements with age. These data suggest that deficiencies in MHC class I or class II are capable of partially reversing or delaying aging-related thymic atrophy. Again, T cell rearrangements still occur, and b-selection processes are normal in these MHC-deficient animals. Therefore, failure of TCR rearrangement is unlikely to underly aging-related thymic involution. However, these data do suggest that either aging-related changes in T cell development, most likely during positive or negative selection stages when MHC expression is required, or changes in mature T cells, contribute to aging-related thymic involution.

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Fig. 1. Thymic weight changes during aging. One lobe of the dissected thymus from young and old mice of the indicated genotypes was weighed. Each sample is indicated by circles while the mean of each group is indicated by bars. Statistical significance of differences between old and young (Student’s t-test) is indicated by a P value B0.05; n.s. (not significant) indicates P \0.05.

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Fig. 2. Thymic cell density changes during aging. One lobe of the dissected thymus from old and young mice was dispersed into a cell suspension, cells counted, and cells per mg of thymic weight displayed. Each sample is indicated by a circle while the mean of each group is indicated by bars. Statistical significance of differences between old and young (Student’s t-test) is indicated by a P value B0.05; n.s. (not significant) indicates P\ 0.05.

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3.3. Aging-related changes in DN thymocyte subsets occur in all strains To determine whether early T cell development was also affected by aging in the genetically altered strains, we examined thymocyte distribution by staining with CD4, CD8, TCR, and CD44 and CD25. Of the four major subsets defined by CD4, CD8, and TCR expression, old mice in all B10. BR background groups (non-Tg or Tg) had a significantly (P B0.05) greater percentage of immature TCR−CD4−8− thymocytes compared to young mice (Table 1). Among the old mice on the C57Bl/6 background, only CD4−/ − old mice showed a statistically significant increase in the percentage of TCR−CD4−8− thymocytes (Table 1). Subset shifts among CD4 and CD8 expressing thymocytes appears to be strain-dependent, we see greater increases in TCR−CD4−8− thymocytes, and decreases in CD4+8+ thymocytes in the DBA background (data not shown). Thymocytes within the DN subset were analyzed for expression of the early differentiation markers CD44 and CD25 (Fig. 3). In ab Tg mice there is an expansion of TCR+ CD4−8− thymocytes. We therefore compared the ratio of CD25+44− to CD25−44+ thymocytes (25+/44−) in all groups, which factors out the excess CD25−44− cells in ab Tg mice. In every case, old thymus suffers a decrease in T cell progression from the earliest CD44+CD25− progenitors to the CD44−25+ stage (Fig. 3). 2B4 TCR transgenes, or MHC or CD4 deficiencies, do not appear to protect against this alteration of the early stages of T cell differentiation in aged animals.

Table 1 Aging-related changes in thymocyte distribution Strain

Age

% CD4-8-TCR-

Non-Tg

1 m. 12 m. 1 m. 12 m. 1 m. 12 m. 1 m. 12 m. 1 m. 12 m. 1 m. 12 m. 1 m. 12 m. 1 m. 12 m.

3.69 0.2 8.79 1.0* 3.0 90.6 7.6 91.1* 3.5 90.8 7.0 9 1.1* 2.5 9 0.3 4.8 9 0.7* 3.1 9 0.5 3.49 0.1 4.3 9 1.0 4.89 0.7 4.1 9 0.6 4.1 9 0.7 2.2 9 0.3 3.29 1.2

2B4b 2B4ab CD4−/− C57B1/6 b2M−/− Abb−/− b2M/Abb−/−

* PB0.05, Student’s t-test.

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Fig. 3. Aging-related changes in CD44 and CD25 expression on developing thymocytes. CD4−8−- gated thymocytes from young and old mice were analyzed for CD44 and CD25 expression by flow cytometry. The ratio of percentages of CD25+44− to CD25−44+ subsets is displayed. Statistical significance of differences between old and young (Student’s t-test) is indicated by a P value B0.05.

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Table 2 Thymic medullary rings are suppressed in CD4−/−, 2B4 ab TCR Tg and Abb−/− mice Number of medullary rings per section

B10.BR 2B4 b 2B4 ab B6 b2M−/− CD4−/− Abb−/−

Young

Old

0 0 0 0 0 0 0

1.4 3.2 0 0.6 2.5 0.16 0

3.4. Analysis of thymus architecture in aged mice Thymus sections from b and non-Tg mice showed evidence of aging-related stromal disruption. We examined the integrity of the cortex and the medulla, respectively, using the antibody 6C3, which detects an aminoendopeptidase expressed on thymic cortical epithelial cells and during B cell development (BP-1) (Lin et al., 1998), and the lectin UEA I, which stains a subset of thymic medullary epithelial and dendritic cells (Burkly et al., 1995). Between 5 and 22 sections from each strain were scored blind for the following features: visual estimation of cortex volume; definition of corticomedullary junction using 6C3 stain; intensity of UEA-1 stain; compaction or diffuseness of UEA-1 stain and the presence and number of discrete medullary rings (see below). Representative sections for each strain are shown (Fig. 4). The most distinctive morphological finding is the presence of UEA-1 positive ring structures within the medulla of aged mice (medullary rings). Examples of rings are shown in Fig. 4B (arrows). The presence and number of rings observed in sections from each strain is summarized in Table 2. No medullary rings were observed in young mice of any strain. Old control mice, both non-Tg B10.BR and C57Bl/6, contained numerous medullary rings, as did old b2M-deficient and 2B4 b TCR transgenic mice (Table 2). Only one ring was detected in six sections examined from CD4-deficient mice, while none were detected in 2B4 ab TCR Tg or Abb-deficient mice (\ 10 sections examined for each, Table 2). Data for b2M/Abb double-knock-out mice are not shown due to the suppression of medullary tissue in both young and old mice, but not surprisingly, no medullary rings were detected. Thus it appears that disruption of helper T cell development through TCR transgenes, CD4-deficiency or MHC class II deficiency suppresses the aging-related disturbance of medullary architecture characterized by medullary rings. We also scored sections for the presence or absence of particular morphological features more common in older mice (Table 3). Although subjective, these observations follow trends similar to the more quantitative scoring of medullary rings. For example, 6C3 staining was relatively disorganized, resulting in indistinct corticomedullary junctions, and cortical volume was reduced in sections from most

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Fig. 4. Representative examples of thymic morphology from old and young mice. Serial frozen sections from each sample were stained with antibody 6C3 (cortical thymic epithelium, left two columns) or the lectin UEA-1 (medullary stromal cells, right two columns). Old and young sections shown were stained in parallel on the same day. Methyl green counterstain for cell nuclei was used. Medullary rings are indicated by arrows. (A) typical sections from ab 2B4 TCR Tg, b 2B4 TCR Tg, and non-Tg are shown as indicated; (B) typical sections from CD4 −/ −, b2M −/ −, Abb −/ −, b2M/Abb −/ −, and C57Bl/6 controls are shown as indicated. Magnification, 100 ×.

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Fig. 4. (Continued)

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older control, b Tg, CD4-deficient, and b2M-deficient animals (Fig. 4, Table 3). 2B4 ab TCR Tg showed more distinct corticomedullary junctions; however, the medullary regions were scattered throughout the cortex, and cortical volume was reduced, even in young animals, as previously noted (Brabb et al., 1997). However, by the criteria of cortical volume and distinctness of the corticomedullary junction, older Abb-deficient and b2M/Abb double-knock-out animals were nearly indistinguishable from young controls. Thus, it appears that for these measures of aging-related disruptions of cortical thymic architecture, reduction of CD4 SP T cell development through MHC class-II deficiency is protective. UEA-1 lectin staining also revealed aging-related morphological changes. Two features were scored: the intensity of the UEA-1 lectin stain, and whether UEA-1 positive cells are diffusely scattered thoughout the section or remain together forming a compact medullary region. Intensity was judged relative to young controls stained in parallel to the blinded test sections. As shown in Fig. 4 and summarized in Table 3, all older mice showed some decrease in the intensity of UEA-1 staining and an increase in UEA-1 positive cell diffusion. However, as described above, young 2B4 ab TCR Tg animals showed an elevated diffuseness of UEA-1 staining primarily due to the scattering of medullary regions. Taking this into account, the aging-related changes were less apparent in this strain. In addition, Abb-deficient animals also showed relatively minor alterations in the diffuseness of UEA-1 staining patterns with age.

3.5. Receptor expression and allelic exclusion in aged 2B4ab and 2B4b transgenic mice Age-related changes in receptor expression and allelic exclusion at the TCRb locus were examined in Tg and non-Tg mice. Young 2B4 mice are efficient excluders of endogenous Vb rearrangement (Berg et al., 1989a). Nevertheless, to test our model that rearrangement deficiency does not precipitate thymic involution, we needed to ensure that the levels of TCRb expression, and subsequent allelic Table 3 Thymic structural changes during aging

B10.BR 2B4 b 2B4 ab B6 b2M−/− CD4−/− Abb−/−

Reduced cortex area (%)

Indistinct junction (%)

Faint UEA-1 (%)

Diffuse UEA-1 (%)

Young

Old

Young

Old

Young

Old

Young

Old

22 20 80 8 0 0 0

60 100 88 50 32 0 0

0 0 0 0 0 0 0

80 42 27 20 86 60 8

11 0 20 0 0 0 36

91 80 36 60 68 100 67

0 20 80 0 0 0 12.5

91 100 100 70 86 83 38

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Fig. 5. TCRb expression and allelic exclusion in aged 2B4b and 2B4ab transgenic mice. Thymocytes from young and old mice were stained for CD4 and CD8, and for Vb3 transgene and endogenous Vb8 expression. Representative flow cytometric profiles of Vb3 and Vb8 expression on combined CD4 and CD8 SP thymocytes from young (left panels) and old (right panels) mice are shown. Non-Tg (top), 2B4ab (middle), 2B4b (bottom).

exclusion, were maintained in older Tg animals. Therefore, anti-Vb8, which reacts against a relatively large fraction of endogenous TCRs (Staerz et al., 1984), was used to detect thymocytes expressing an endogenous TCR Vb (Fig. 5). Increasing age affected the percentage of thymocytes expressing transgenic and endogenous Vb chains in both and mice; a small but significant population of thymocytes from old mice in both groups were observed to be endogenous or dual Vb-expressing compared to thymocytes from young mice (PB 0.05). Although the increase in dual-expressors is low, only one Vb class was examined, suggesting that the actual number of dual expressors in aged animals is higher. Nevertheless, the 2B4 TCR transgenic mice maintained high levels of b transgene expression with age, supporting the validity of our conclusion that TCR ß transgene expression does not suppress thymic involution.

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3.6. Down-regulation of surface TCR expression on mature thymocytes from aged 2B4b and non-Tg, but not 2B4ab, mice In studies with DBA/2 and C57BL/6 mice, we found that thymocytes from old mice expressed 2.5-fold lower levels of individual TCR Vb and CD3o compared to young mice (data not shown). We also examined TCR modulation in ab, b, and non-Tg mice. Again, ab mice differed from b and non-Tg mice by displaying little or no reduction in Vb transgene or CD3o levels with increased age (Fig. 6). In addition, neither CD4-deficient, b2M-deficient, nor Abb-deficient aged mice showed any reduction in TCR expression on single-positive thymocytes (data not shown), suggesting that these genetic changes suppress this aging-related alteration. Interestingly, we observed a statistically significant decrease in the ratio of CD4 to CD8 SP thymocytes in aged TCR b Tg mice (young 7:1 versus old 4:1, PB 0.01). Mice transgenic for the 2B4 b chain have been previously shown to be significantly skewed to the CD4 SP lineage. This skewing is dependent on the level of MHC class II I− Ek expression, indicating that cells expressing Tg b with endogenous a TCRs are more effectively positively selected on the I− Ek molecule (51). The decreased CD4/CD8 ratio in aged mice therefore suggests that aging may reduce the efficiency of positive selection to the CD4 SP lineage. We also observed a statistically significant (P B 0.01), nearly two-fold reduction with age in the percentage of CD4 single-posiitve thymocytes in thymus of the b2M-deficient strain, possibly consistent with aging-related decreases in positive selection of CD4 T cells. No significant aging-related alterations in the percentage of CD8 single positive thymocytes was observed in any strain.

4. Discussion We show that TCR transgenes, CD4-deficiency, and MHC deficiency can alter some but not all aspects of aging-related thymic involution. Our findings demonstrate that the developmental and structural alterations of the aging thymus are distinct and separable, since all mice exhibited developmental alterations but only some structural abnormalities were observed. Significantly, we also show that any aging-protective effect of TCR transgenes requires both a and b TCR antigen-binding subunits, since the aging phenotype of a TCR b transgenic was indistinguishable from non-Tg controls. Therefore, our data demonstrate that an aging-related blockade of TCR b rearrangements does not cause involution. b2M −/ − xAbb −/ − doubly-deficient animals appeared to be the most protected against aging-related thymic involution at 12 months. These animals showed no significant aging-related reductions in thymic weight or cell densities, and retained all of the structural features of younger animals. The only aging-related defect shown by these animals was a decrease in the ratio of CD25/CD44 immature thymocyte subsets. Clearly therefore, the alterations in the immature subsets does not cause nor is caused by the structural, size, or cell density changes commonly seen during aging.

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Fig. 6. Reduced TCR expression on mature thymocytes from old 2B4b and non-Tg mice, but not on thymocytes from old 2B4ab mice. CD3+ cells were gated on total CD4 and CD8 SP thymocytes. The dark line on the histogram overlays shows CD3 fluorescence on young thymocytes, and the dashed line indicates CD3 fluorescence on old thymocytes. A, non-Tg; B, 2B4ab; C, 2B4b. Similar reduction in TCR expression was also observed with advanced age in DBA/2 and C57BL/6 strains of mice (data not shown).

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After the double MHC knock-out, MHC class II deficency alone was the next most protective. These animals again showed thymic cell densities and most structural features similar to younger animals. They were, however, deficient in thymic weight and in CD25/44 immature thymocyte ratios. CD4 deficiency was less protective than MHC deficiency, but still provided some suppression of aging-related medullary ring formation, and of cortex volume reduction. CD4-deficiency does not completely abrogate the formation and function of helper T cells (Locksley et al., 1993; Rahemtulla et al., 1994; Matechak et al., 1996), and is therefore less severe than the MHC class II deficiency, which might explain the differences in suppression of involution in the two strains. Nevertheless, CD4-deficiency does reduce helper T cell numbers (Law et al., 1994) and blocks TH2 differentiation (Brown et al., 1997; Fowell et al., 1997). In addition, CD4-deficient mice have reduced numbers of memory cytotoxic T cells (von Herrath et al., 1996). These features may provide some protection against aging-related structural changes. Finally, ab TCR Tg mice were protected against most structural abnormalities, but still showed size, cell density, and developmental abnormalities of aging mice. Our results therefore differ from those of Aspinall who showed that the F5 TCR transgenes suppressed size and some developmental features of thymic involution (morphological changes were not assessed). The F5 TCR transgenes encode a class I restricted TCR, and in addition, are expressed using a different expression cassette. Furthermore, the strain background is different, C57Bl/6 versus B10.BR. Any of these differences, as well as specificity differences alone, could contribute to the differences in aging phenotypes observed. However, our preliminary data using a limited population of HA-TCR transgenics (which are MHC class II restricted but also select some CD8 single-positive T cells) show aging-related involution concurrent with the 2B4 data reported here. Several transplantation and regeneration studies, including those involving TCR Tg mice, have shown that aged thymus cannot be fully restored by young marrow, suggesting that aging-related defects reside in the stroma (Mackall et al., 1998). However, it has also been shown that T cell progenitor activity declines in aging bone marrow populations (Hirokawa et al., 1986; Fridkis-Hareli et al., 1992). It seems likely that the aging-related shift to immaturity, which appears to occur in all strains regardless of involution status, observed here and by others (Aspinall, 1997; Thoman, 1997a) is a consequence of aged stem cells, since it is dissociated from observable stromal changes. How might we explain the ability of TCR transgenes, CD4 and MHC-deficiencies to suppress thymic involution? Based on our data, we speculate that certain T cell specificities, most likely MHC class II restricted, mount a destructive autoimmune attack of the thymic stroma (Hartwig, 1992; Hartwig and Steinmann, 1994). This model is based on the following observations. First, the observation that class-II restricted TCR transgenes, which limit the helper T cell repertoire, as well as CD4 deficiency, and MHC class II deficiency, all prevent thymic structural changes to varying degrees while at the same time suppressing the formation of helper T cells to varying degrees. MHC class I deficiency not only does not protect but may

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exacerbate stromal destruction, based on the higher incidence of medullary rings observed. Second, structural damage to the thymus during aging strongly resembles the destruction that occurs in mice with graft-versus-host disease (GVHD) (Seemayer et al., 1977; Seddik et al., 1980; Fukushi et al., 1990; Desbarats and Lapp, 1993; Hollander et al., 1994). For example, MHC class II expression is down-regulated during GVHD (Desbarats and Lapp, 1993), similar to the class II down-regulation observed in stromally involuted aged thymus (Farr and Sidman, 1984). Furthermore, TCR levels are down-regulated on thymocytes in GVHD (Desbarats and Lapp, 1993), as they are in our study of aged b-Tg and non-Tg mice, but not those strains showing protection from aging-related structural involution. Aging-related changes in thymic size and density are only fully suppressed by disruptions in both MHC class I and class II genes. It is possible that either the absence of T cell selection and mature thymocyte formation is somehow protective against aging-related thymic involution, or alternatively, that both helper and cytotoxic mature T cells are involved functionally in thymic involution. These studies, as well as those of others (Dubiski et al., 1989; Gonzalez-Quintial et al., 1995; Nabarra and Andrianarison, 1996), indicate that the phenotypes of aging in normal mice are highly dependent on background strain (note the differences between C57Bl/6 and B10.BR mice in our assays). This suggests that the complex phenotypes of aging have a genetic component. Experiments in progress are attempting to resolve and map some of the genes that influence aging phenotypes (Miller, 1997; Miller et al., 1997a,b; Jackson et al., 1999; Miller et al., 1999). If further tests confirm that thymic stromal involution is caused by T cell-mediated autoimmune destruction, it will be important to understand how such autoreactive T cells escape thymic negative selection during aging. Previous studies have indicated that thymic negative selection is relatively intact in aging animals (Gonzalez-Quintial and Theofilopoulos, 1992; Crisi et al., 1996). Of interest, we found allelic exclusion of the b locus to be less efficient in older Tg animals. Similar findings have been reported in aged AND TCR Tg mice (Balomenos et al., 1995; Linton et al., 1996). The analysis of transgenic mice on a RAG-deficient background will be necessary to determine whether endogenous receptors contribute to aging-related thymic involution. Dual receptor T cells may contribute to autoimmunity, since studies have shown that such dual-specificity T cells can be reactivated to cause autoimmune disease (Zal et al., 1996; Sarukhan et al., 1998). Our results indicate that deterioration of the thymic microenvironment does not fully explain the reduction in T cell differentiation with advanced age. On the other hand, despite dramatic stromal deterioration, aged thymus continues to support T cell generation and export, although to a lesser extent (Mackall et al., 1993), throughout adulthood. Recent studies indicate that the aging-related decline in thymic output is accelerated in HIV-infected patients (Doucek et al., 1998). However, after the initiation of highly active anti-retroviral therapy, renewed thymic function was observed. The ability to enhance T cell production and export from the aging thymus has important implications for improving immune responses in aged and immunocompromised individuals. Future experiments will focus on the

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causes of thymic stromal and developmental involution, and consequences of these aging-related thymic changes on the immune response.

Acknowledgements We thank D. Wilsker for excellent technical assistance. We also thank Drs. J. Erikson, D. Izon, and A. Eaton for critical reading of the manuscript, A. Farr and S. Carding for comments on immunohistochemistry, Elsa Aglow at the Wistar core histotechnology facility for tissue sectioning and H&E staining, A. Eaton, E. Pure and C. Buck for help and use of equipment for photographing tissue sections, and C. Healey for help with graphics. L.M.S. was supported by National Institutes of Health Grant AI364553 and NASA NAG9-832 and L.L.L. by NIH Postdoctoral Training Grant 5T32 CA 09140.

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