Aging induced decline in T-lymphopoiesis is primarily dependent on status of progenitor niches in the bone marrow and thymus

Age-related alterations in the myeloid vs. lymphoid differentiation of young BM progenitors may be due to the age of the host BM niches

It is well known that aged BM progenitors follow myeloid-biased differentiation [10, 12]. But it is largely unknown whether this is due to primary alteration in BM progenitors themselves or their niches (endogenous microenvironment). We asked what differentiation profile would be if young BM progenitors replace aged BM progenitors and stay in aged niches. This stimulated our efforts to obtain direct evidence whether the aged niches are responsible for the differentiation.

The OP9-DL1 stromal cell monolayer has been previously used for successful development of a singlesource of BM progenitors [33]. In this culture setting, LPCs from old BM differentiated fewer T-lineage cells than myeloid lineage cells compared to LPCs from young BM [12]. Based on this finding, we developed a BMT combining OP9-DL1 system.

By taking advantage of IL7R^−/− mouse WT-equivalent stromal cells and vacant niches [24, 25, 27], we first transplanted the CD45.1⁺ young BM cells from the same pool into either young (~2-month-old) or old (22-month-old) non-irradiated CD45.2⁺ IL7R^−/− recipient host mice. Five weeks after the BMT, we checked donor (CD45.1⁺) BM progenitor constituted BM cell numbers in the hosts (CD45.2⁺), and found that the reconstituted donor BM cells did not have significant quantitative difference in the grafted young and aged hosts (Supplemental Figs. S3A and C left panel, and data not shown). And then, we sorted donor (CD45.1⁺) LSK (lineage- and host-CD45.2 negative, Sca-1⁺, and c-Kit⁺) cells (Supplemental Fig. S1) from the grafted host BM, plated the same number of sorted LSK cells from the grafted young and old hosts on the OP9-DL1 stromal cell monolayers and co-culture them as previously described [12] and as shown in workflow diagram (Fig. 1A). After two weeks in culture, the young LPCs modulated by an old BM microenvironment differentiated fewer T-lineage cell than myeloid lineage cells, similar to old LPCs [12], when they were compared to young LPCs from a young microenvironment (Figs. 1B-D). Our results indicate that differentiation of the skew of T-lymphoid to myeloid lineage is associated with the stem cells modulated (maybe receiving different signals) in BM niches of different ages.

Aged LPCs can compete with their young counterparts in a cKCT setting, although they cannot do so in a conventional BMT setting

It is well known that co-transplantation of young and old BM progenitor cells into a young WT host in conventional BMT, old BM progenitor cells could not compete with their young counterparts in generating T-lineage cells, no matter whether the hosts are irradiated [10] or non-irradiated [12]. The conventional BMT has an in vitro manipulation process of BM progenitors. Evidence showed that when aged BM progenitors stay in vitro, they display decreased adhesion to stroma [31]. We asked whether aged BM progenitors recruited in vivo by a grafted fetal thymus are able to compete with their young counterparts, because when we examined in vivo repopulation in the fetal thymus with aged LPCs over 1 to 4 weeks in a KCT model and in a time-course manner [1, 34], we found that LPCs from old BM were able to generate the same number of thymocytes (Fig. 2A) with similar differentiation profiles as young BM progenitor did (Fig. 2B). The longer the progenitors remained in the fetal microenvironment, the less distinction was observed between the young and old groups as defined by CD4 vs. CD8 differentiation profiles (Fig. 2B). These results were independent of whether cells matured in dGUO-treated or intact fetal thymus at any of the sampled time points. This encourages us to develop a novel in vivo competitive method based on KCT.

We, therefore, developed a cross-KCT (cKCT) model (Supplemental Fig. S2) by transplanting dGUO-treated CD45.1⁺CD45.2⁺ RAG^−/− fetal thymic lobes, which are a good in vivo model to accumulate WT LPCs [1, 34], into young CD45.1⁺ or old CD45.2⁺ mice (first hosts). Five days after the first host KCT, cKCT was performed by transplanting the fetal thymic lobes carrying seeded LPCs from the first hosts into age-reversed and CD45-subtype-reverseed second hosts to recruit LPCs from the second hosts for additional 5 days. The lobes containing “young and old” or “old and young” LPCs were then subjected to FTOC for one week. The later seeded LPCs from the second hosts of either young or old mice became a dominant population in the lobes after FTOC (Fig. 3A, open triangles and open circles). The KCT young LPCs were then compared with KCT old LPCs (both from the first hosts), while the cKCT young LPCs were compared with the cKCT old LPCs (both from the second hosts). In this in vivo competitive experiment we found no significant difference in T-lineage cell numbers generated (Fig. 3A) and in the differentiation of CD4⁺ and CD8⁺ single positive T cells (Fig. 3B) derived from young and old LPCs in the fetal thymic lobes. Taken together, our results indicate that the old LPCs did not lose their competence for T-cell generation and were equal in this respect with young LPCs when allowed to naturally seed the thymus but were not subjected to in vitro manipulation.

Aged BM progenitors, recruited by grafted fetal thymic lobes in vivo, compete with young BM progenitors even subjected subsequently to in vitro culture

We were curious if natural thymus-seeding aged BM progenitors, accumulated in vivo during fetal thymus KCT, can compete in T-cell generation with their young counterparts in an in vitro culture model. To test this, we designed and performed competitive cultures on an OP9-DL1 stromal cell monolayer (workflow in Fig. 4A). After young CD45.1⁺ and old CD45.2⁺ natural thymus-seeding cells were recruited in vivo from dGUO-treated CD45.1⁺CD45.2⁺ RAG^−/− fetal thymic lobes in KCT of young CD45.1⁺ and old CD45.2⁺ host mice for 7 days, we selected CD4⁻8⁻ DN population from the lobes, and loaded the DN cells at equal numbers on the same monolyayer co-culture for 10 days. The results showed that LPCs from young- and old-sources had the capacity to generate equal numbers and proportions of T-lineage cells (Figs. 4B and 4C) and an equal % of CD4⁺CD8⁺ double positive (DP) thymocytes (Figs. 4D and 4E). It suggests two possible scenarios: 1) Aged BM contains mixed population of defective and functional progenitors, and only the functional progenitors can naturally seed the fetal thymus. Therefore they can compete with young LPCs; 2) The competence of T-cell generation from the natural thymus-seeding old BM progenitors can be recovered to the young progenitor levels after the exposure to the fetal thymic microenvironment for a certain period of time.

Aging of LPCs though unavoidable occurs more slowly than aging of their niche cells

Although we did not find a significant reduction in the capacity of aged LPCs to generate T-lineage cells either in the young thymic microenvironment (Figs. 2 and 3), or after “rejuvenation” by a young thymic microenvironment (Fig. 4), these aged LPCs are not immortal. Aging, after all, induces stress in BM progenitors, as is evident from conventional BMT [12]. To determine at which age and how quickly LPCs show a marked increase in transplantation stress, associated with the age-related disability of their BM niche cells (osteoblastic cells in the BM) and thymic niche cells (TECs in the thymus), we designed an experiment, in which competence of thymic T-lymphopoiesis associated with LPC age or niche-cell age was observed in the recipient thymus.

When WT mouse BM cells are transplanted into IL7R^−/− host mice, they can restore the small thymus size of IL7R^−/− mouse to normal size and develop normal number of mature T cells [27]. This is because IL7R^−/− host mice have WT-equivalent niche cells in the BM and thymus, and these niches can accept and support development of WT BM progenitors. The process is dependent upon lympho-stromal interaction between donor LPCs and host niches. Based on this, we designed a set of experiments (Experiments A and B, workflow is shown in Fig. 5A). In experiment-A, WT BM cells, isolated from 2-, 8-, 12-, 18-, and 22-month-old WT mice were transplanted into age-mismatched young (~2-month-old) IL7R^−/− host mice. This allows determination of age at which the LPCs show significant loss of transplant efficacy and undergo a subsequent reduction in their ability of thymic T-lymphopoiesis in young BM and thymic microenvironments. In experiment-B, we then transplanted a pool of BM cells, isolated from young (~2-month-old) WT mice, into age-mismatched IL7R^−/− host mice at 2-, 6-, 8-, 12~13-, 18-months of age to provide niches of different ages. This allows us to determine the age at which the niche cells in the BM and thymus lose their ability to support LPCs in T-cell generation. Thymocyte numbers of each group were compared to those in 2-month-old group respectively in order to determine the age at which the T-lymphopoisis was significantly reduced.

Our results showed that although the competence of LPCs to generate thymic T-lymphopoiesis dynamically declined with increasing age, only when the LPCs reached 22 months of age, was the thymic T-lymphopoisis significantly reduced when compared to that of young LPCs (Fig. 5B). However, the capacity of the niche cells to support young LPC's thymic T-lymphopoiesis was significantly reduced from 8-12 months of age, much earlier than in the case of old LPCs (Fig. 5C). Comparing thymocyte numbers from Exp-A and Exp-B in the same linear regression plot, a slope of decline based on niche cell age (Exp-B) showed more steeper than the one based on BM cell age (Exp-A), and the test for equal slopes for these two groups gave a (2-sided) p-value of 0.016 (significantly different) (Fig. 5D, left panel). The thymocyte numbers generated from young LPCs supported by 11~13-month-old niche cells were significantly lower than those generated from the same aged LPCs in young niches (Fig. 5D, right panel), clearly supporting the conclusion that LPCs have longer lifespan than their niche cells on the thymic T-lymphopoisis.

Unlike peripheral lymphoid organs, such as the spleen, the thymus is not the site of executing the immunological rejection. The decrease in CD45.1⁺ WT donor-derived thymocytes with increased recipient ages in the CD45.2⁺ IL7R^−/− thymus is unlikely due to immunorejection, although IL7R^−/− recipient mice are considered incompletely immunodeficient. In order to determine whether there is an age-related immunological difference in IL7R^−/−hosts, we checked the chimerism of the CD45.1-subtype progenitors in the CD45.2⁺ BM of different ages (Supplemental Figs. S3A and C left panel). No differences of CD45.1⁺ donor-derived BM cells were found among 2- and 8-month-old CD45.2⁺ IL7R^−/− hosts, whereas, 8-month-old CD45.2⁺ IL7R^−/− thymic niches have already exhibited a significantly reduced competence to support CD45.1⁺ donor thymocyte development (Fig. 5C, and Supplemental Figs. S3B and C right panel). By observing peripheral lymphocytes, while donor-derived numbers of splenic B cells were slightly increased (Supplemental Figs. S3B and D), donor-derived numbers (Supplemental Fig. S3D) and % (Supplemental Fig. S4) of splenic CD4⁺T cells were significantly decreased at the age of 8-12 months old, which is probably due to decreased thymic output, and it is one of reported immune system aging phenotypes [35].

Murine models and animal care

Mice from the C57BL/6 genetic background that expressed CD45.2⁺ or CD45.1⁺ congenic markers on the hematopoietic cell surface were used. Young (±2-month), early middle-aged (6~9-month), middle-aged (±12-month) and aged (over 18-month) (ages indicated in each figure) wild-type (WT), IL7R^−/− mice [26], and RAG^−/− fetal mice were originally purchased from the National Cancer Institute, National Institute for Aging /National Institutes of Health (NIH) (Bethesda, MD), and Jackson Laboratory (Bar Harbor, ME), respectively, and bred in our SPF animal facility. E15 indicates fetal mouse at embryonic day-15. All animal experiments were in compliance with the protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Tyler and the University of North Texas Health Science Center at Fort Worth, in accordance with guidelines of the NIH, USA.

Comparison of myeloid vs. lymphoid fates in age-associated BM microenvironments

Erythrocyte-depleted BM cells (RBC lysing buffer from Sigma), from a pool of young CD45.1⁺ WT donor mice, were retro-orbitally transplanted (BMT) into 2- and 18-month-old CD45.2⁺ IL7R^−/− host mice (2 × 10⁶ cells/mouse). The donor LPCs [donor CD45.1⁺, lineage-negative, host CD45.2-negative, ckit⁺, andSca1⁺ BM population, i.e. LSK cells [52]] were sorted (FACS-Aria, Supplemental Fig. S1) from the young or old CD45.2⁺ IL7R^−/− hosts, 5 weeks after the BMT. These cells were cultured on an 80% confluent OP9-DL1 (kindly provided by Dr. J.C. Zuniga-Pflucker)[33] stromal cell monolayer (~2000 sorted donor LSK cells per culture well), in presence of Flt3 ligand (5 ng/ml) and IL-7 (5 ng/ml) (R&D). On the culture day 7, non-adherent cells were transferred to a fresh OP9-DL1 monolayer, and after 14 days in culture the cells were harvested and analyzed via flow cytometry for the differentiation of Mac1⁺ and Thy1.2⁺ cells derived from these sorted LSK cells (workflow in Fig. 1A).

Kidney capsule transplantation (KCT)

Single-host KCT was performed as previously described [1] with the following modifications: (i) In group one the grafted fetal thymic lobes prior to KCT were treated with dGUO (2’-Deoxyguanosine, Sigma) for 5 days in fetal thymic organ culture (FTOC) for depleting hematopoietic cells in the lobes, while in group two the grafted fetal thymic lobes were intact prior to KCT; (ii) Thymocyte numbers and thymocyte differentiation profiles in the grafted fetal thymic lobes were monitored over time (1, 2, 3, and 4 weeks after the KCT). For cross KCT (cKCT), RAG^−/− mouse (CD45.1⁺CD45.2⁺ double congenic marker positive) E15 fetal thymic lobes from same littermate were subjected to KCT into first hosts of young (~2-month) CD45.1⁺ or aged (~22-month) CD45.2⁺ WT mice for 5 days. And then, cKCT was performed by transplanting the fetal thymic lobes carrying seeded LPCs from the first hosts into age-reversed and CD45-subtype-reversed second hosts to recruit natural thymus-seeding LPCs from the second hosts for additional 5 days. The thymic lobes contain young and aged (group-1) or aged and young (group-2) LPCs were harvested and cultured in vitro in 95% oxygen FTOC system [53] for ~7 days to expand the recruited LPCs before flow cytometric analysis (detailed workflow is shown in supplemental Fig. S2).

Competitive reconstitution of natural thymus-seeding young and aged LPCs through an in vivo recruitment

The dGUO-treated fetal thymic lobes (E15 CD45.1⁺CD45.2⁺RAG^−/−) were transplanted under the kidney capsules of young (~2-month) WT (CD45.1⁺) and aged (22-month) WT (CD45.2⁺) host mice, respectively, to recruit natural thymus-seeding LPCs over a 7-day period. The CD4 and CD8 double negative (DN, with CD3⁻) thymocytes from the grafted thymic lobe were then sorted by negative selection using magnetic beads (IMag^TM streptavidin particles, BD bioscience). The sorted cells (1:1 ratio of young and aged sources ~5000 cells each) were loaded on 80% confluent monolayers of OP9-DL1 stromal cells [33]in 12-well plates and culture in the presence of exogenously supplied IL-7 (5ng/ml) and Flt3 ligand (5ng/ml) (R&D). On the 5^th day of the culture non-adherent cells were transferred to a fresh OP9-DL1 monolayers, and ten days after the co-culture, the cells were isolated and analyzed by flow cytometry for CD45.1 vs. CD45.2, and CD4 vs. CD8 cell populations (workflow in Fig. 4A).

Competence of BM progenitors or their niche cells of different ages to generate T cells

Experiment-A: to test the competence for thymic T-lymphopoiesis by BM progenitor cells from various ages, BM cells were obtained from CD45.2⁺ WT mice of ~2-, 8-, 12-, 18-, and 22-months of ages. About 2 × 10⁶ erythrocyte-depleted BM cells were retro-orbitally injected into non-irradiated CD45.1⁺ young IL7R^−/− host mice to reconstitute the donor T-cell pool in the host thymus. Experiment-B: to test competence for thymic T-lymphopoiesis in LPC niches (both BM and thymic niches) of different ages, ~2 × 10⁶ erythrocyte-depleted BM cells from a pool of young CD45.1⁺ WT mice were retro-orbitally injected into ~2-, 6-, 8, 12~13-, and 18-month-old non-irradiated CD45.2⁺ IL7R^−/− host mice. Five weeks after the injection, the donor-derived thymic T-lymphopoiesis was analyzed by flow cytometry (workflow in Fig. 5A).

Flow cytometric assay

Cell counting was carried out using the BioRad TC10^TM automated cell counter or a hemacytometer. For flow cytometric analysis, thymocytes isolated with a cell-strainer were blocked with anti-FcR (clone 2.4G2) to diminish nonspecific binding, and then stained with combinations of fluorochrome-conjugated antibodies (as indicated in each figure). Data were acquired by a dual-laserFACS-Calibur system and analyzed with CellQuest and FlowJo software.

Statistics

Statistical significance was analyzed by unpaired Student's t-test with Welch's correction (if variances shown unequal) or Mann-Whitney test (if data shown non-parameter). Linear regression is used to fit straight lines for the two groups. By using a dummy variable to fit two regression lines in a single equation, the equality of the two slopes is tested based on a t-test. Differences were considered statistically significant at values of p < 0.05.