Two separate defects, affecting true naïve (TNa) or virtual memory (VM) T cell precursors, combine to reduce naïve T cell responses with aging

Mice

Mice were bred and maintained in the animal facility at the University of Arizona and experiments conducted under guidelines and approval of the Institutional Animal Care and Use Committee of the University of Arizona. (B6.OT-I.Rag-KO x B6.Ly5-1) F1, (B6.P14.Rag-KOxB6.Thy1.1)F1, and (B6.gBT-1.Rag-KOxB6.Thy1.1)F1 mice were bred from the stocks of B6.OT-I.Rag-KO(11), B6.OT-II(12), B6.Ly5-1, and B6.PL mice, purchased from Taconic, NCI, and Jackson Labs respectively and from P14(13) and gBT-I(14) stocks generously provided to us by Dr H.P. Pircher via Dr J.A. Frelinger and by Dr F.R. Carbone, respectively. Old (18–23 mo.) C57Bl/6 (B6) mice were purchased from NIA and adult (12 week) B6 mice were purchased from Jackson. Mice with large spleens or other obvious abnormalities (tumors, etc.) were excluded from the study. Survival bleeds were performed retro-orbitally.

Flow cytometry

Samples were prepared as previously described ((15)). We used fluorochrome-conjugated Ab clones: CD8α (53-6.7), CD4 (RM4-5), CD44 (IM7), CD62L (MEL-14), Vβ5.1/5.2 (MR9-4), Vα2 (B20.1), IFN-γ (XMG1.2), CD3 (17A2), TCRβ (H57-597), CD5 (53-7.3), PD-1 (29F.1A12), LAG3 (C9B7W), CD122 (TM-β1), IL-4R (mIL4R-M1), phosho ser139 γH2AX (2F3) and CD49d (R1-2), purchased from eBioscience, Invitrogen, BD Biosciences, or Biolegend. Samples were analyzed on a 4-laser custom Fortessa cytometer (BDIS, Sunnyvale, CA), using the DiVA acquisition (BDIS) and Flow-Jo (Treestar, Ashland, OR) analysis software. For intracellular cytokine staining, samples were stimulated and prepared exactly as previously described (15).

Cell Sorting and Culture

Prior to sorting, CD8 T cells were enriched by magnetic separation (Miltenyi) using an AutoMACS pro. CD44^hi VM and CD44^lo TNa CD8 T cells were sorted based on CD44 and CD62L expression using a BD Aria. The sorted cells were stained with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) as described (15). The cells were cultured at 8×10⁵ cells/ml in RPMI-complete plus 100 U/ml rmIL-2 (eBioscience), with either 10⁻⁹ M SIINFEKL peptide or 10 ng/ml rmIL-12 (R&D Biosystems) and 10 ng/ml rmIL-18 (eBioscience) for 5 days. Alternatively cells were cultured with 10 ng/ml IL-7 (eBioscience) and 100 ng/ml IL-15 (eBioscience). For polyclonal stimulation, cells were cultured at 8×10⁵ cells/ml in RPMI-complete 1:1 with α-CD3/α-CD28 immobilized on beads (Miltenyi) and 100 U/ml rmIL-2 (eBioscience), On each day of analysis, cells were LIVE/DEAD stained (Invitrogen) according to manufacturer’s instructions, stained with surface antibodies, fixed/permeabilized with the FoxP3 Fix/Perm and stained with intracellular Ab. For cell cycle analysis, cells were stained with Vybrant DyeCycle Orange (Life Technologies) at 1.25×10⁻³ mM for 60 min. at room temperature and analyzed immediately. Cells were enumerated using CountBright Absolute Counting Beads according to the manufacturer’s instructions (Life Technologies).

TCRα PCR

5,000 CD44hi and CD44lo cells were sorted from OT-I mice as described above. RNA isolation was performed by standard chloroform/isoproponal purification. For cDNA synthesis, 15 μl containing 10 μl RNA (500 ng-5 μg), 1.5 μl Oligo(dT)20, 1.5 μl 10mM dNTP, and 2 μl H2O was incubated at 65° C for 5 minutes and then at 4° C for 2 minutes. 3 μl 10× PCR buffer, 6 μl 25 mM MgCl2, 3 ul 0.1 M DTT, 1.5 μl RNase Out (Life Sciences), and 1.5 μl Superscript III reverse transcriptase (Life Sciences) were added and incubated at 50° C for 50 minutes and then 85° C for 5 minutes. 1.5 μl RNaseH (Life Sciences) was added and mixture was incubated for 37° C for 20 minutes. TRAV primers and PCR reaction were as in (16) using Platinum Taq (Life Sciences). Each primer was used separately at 5 μM final concentration. PCR conditions were 95° C for 5 minutes followed by 40 cycles of 95° C for 20 seconds, 56° C for 20 seconds, and 72° C for 45 seconds, followed by a final extension at 72° C for 5 minutes. PCR products were visualized on a 1.8% agarose gel and band intensities calculated using Quantity One Software (Bio-Rad). Bands were enumerated by blind scoring by three independent examiners, who scored the presence or absence of bands.

Enrichment of tetramer positive CD8 T cells from wild type B6 mice

The tetramer-enrichment protocol was performed exactly as in (9) using the spleen, inguinal, cervical and axillary lymph nodes harvested from individual mice.

Data Analysis

Following the analysis of FCM data using Flow-Jo software, Graph Pad Prism was used for statistical analysis (unpaired Student’s t test, paired Student’s t test, linear regression and best fit line, and one-way ANOVA with Bonferroni post test). The following notations have been used to denote p values in all figures: *p < 0.05; **p < 0.01; ***p < 0.001.

Profound age-related conversion into VM cells in TCRTg CD8 T cells from unimmunized mice

We examined phenotypic changes with aging in a single, large clone of unimmunized CD8 T cells using OT-I Rag+ mice, specific for OVA_257–264:H-2K^b (11). Blood from 10, 12, 18 and 22 month (mo) unimmunized OT-I mice contained progressively increased CD44^hi T cell fraction, compared to 2–4 mo. mice (Fig. 1A), and this was paralleled by an increase in IFN-γ-producing cells responding to the cognate peptide (Fig. 1B). Large variability was observed in VM conversion in individual 10–12 mo. old mice (blood CD8CD44^hi =10–100%). However, with advanced age (in the rare OT-I mice surviving to 18–22 mo.), we found uniform and high representation of CD44^hi cells (>75%; Fig. 1A). In the experiments below we often used mice younger than the NIA-defined age cutoff of 18 months, because: (i) OT-I mice, in our colony, have a median lifespan of 15–16 mo. (not shown; colony B6 mice = 26–30 mo), which renders OT-I mice >18mo extremely scarce; (ii) 10–14 mo old OT-I mice showed a range of VM conversion from <20% to >70%, allowing us to analyze the results of functional and other assays in individual animals and test whether the VM conversion correlated with functional responsiveness (Fig. 1C). Finally, (iii) interventions to improve immunity would be implemented not only in the old age, but likely also in middle-aged individuals that still exhibit reasonable immune function. Importantly, all of our key observations were reproduced in bona fide old TCRTg and/or wt mice.

VM cells were reported to be of central memory (CM) CD44^hi62L^hi phenotype (9). Likewise, CD44^hi cells in TCRTg mice were overwhelmingly CD62L^hi (Fig. 1D), and CD44^hi62^lo effector memory (EM) cells did not significantly accumulate with age (Fig. 1E). Indeed, frequency of VM, but not EM, cells tightly correlated with %IFN-γ⁺ production by CD8 T cells in response to the OVA peptide (Fig. 1C and not shown; p<0.0001 and =0.19, respectively). We also determined the frequency of TCRTg cells that express CD49d, also known as α4-integrin, which associates with β7 (α4β7 or LPAM) and binds to vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, allowing T cells to enter inflamed tissue (16). Recently, it was shown that the CD49d^hi phenotype marked “true” memory T cells arising from antigen stimulation, while memory cells formed by homeostatic proliferation (VM) were CD49d^lo (10).We found that the majority of aged OT-I Rag+ CD44^hi62L^hi VM CD8 T cells are CD49d^lo (Fig. 1E), suggesting that they are not “true”, foreign Ag-driven memory, arising instead from alternate mechanisms. By contrast, about 30% of EM CD8 T cells (CD44^hi 62L^lo) were CD49d^hi, consistent with the explanation that at least a portion of this population had reacted to an antigen.

We did not observe reproducible and significant lymphopenia of CD8 T cells with aging (12–14 mo. mice = 2/4 expt. showed mild lymphopenia in blood and 1/3 experiments also in the spleen; 18–22 mo. = 0/1 experiments showed lymphopenia in blood or spleen). Collectively, these data show that TCRTg VM cells exhibiting the CM phenotype increase with age in absolute terms.

VM cells in both adult (10) and old (9) mice exhibit strong immediate effector function, unlike their TN CD44^lo counterparts. We found that the age of OT-I CD8 T cells and the CD44^hi phenotype correlated tightly with the acquisition of immediate cytokine production in these cognate Ag-inexperienced cells upon peptide stimulation (Fig. 1C). These results show that with aging TCRTg T cells exhibit generalized increased phenotypic and functional conversion into VM cells, similar to T cell precursors in aging wt mice (9).

Cytokine receptor expression in VM cells of TCRTg mice with aging

Even in unimmunized wild type (WT) or germ-free adult mice, 10–20% CD8 cells convert into the CD44^hi phenotype, likely due to stimulation by IL-7 during the neonatal period in the periphery (10, 17), and stimulation by NK-T cell-derived IL-4 of single-positive T cells in the thymus (18). To assess whether TCRTg VM cells show signs of differential cytokine maintenance, we examined expression of CD122, CD127 and IL-4R on TCRTg VM cells. We found variable expression of the IL-4R on VM cells in TCR TG mice, and the expression of CD127 (IL-7Rα chain) did not reproducibly correlate to VM phenotype (not shown). On the other hand, 14 mo. OT-I mice showed a significantly increased frequency of splenic CD8 CD122⁺ (IL-2/15Rβ chain) T cells compared to 5 mo. old animals (40–50% vs. 10–20%, Fig. S1A), and the mean fluorescence intensity (MFI) of CD122 expression was also higher (on the average by 2.5-fold; Fig. S1B). When compared between the VM and TN subsets, expression of CD122 on aged CD44^hi VM cells was the highest (Fig. S1C,D). However, even the CD44^lo cells of a 14mo old animal begun to express low levels of CD122 (Fig. 2D; 8–10× lower than in 14 mo old VM cells, but still higher than in 5-mo old counterparts, which were CD122⁻ by flow cytometry). This paralleled our findings in wt cells (ref. 11), indicating that CD122 MFI correlated tightly to the extent of VM conversion. That finding is consistent with the possibilities that: (i) homeostatic cytokines are playing a key role in VM conversion in TCRTg CD8 cells; or (ii) that VM conversion was mandated by other signals (TCR), and that activation of CD122 expression was part of a memory cell differentiation program.

Loss of the original TCRTg specificity in aged TCRTg T cells is critical for age-related VM conversion

Naïve precursors in young mice rely upon trophic signals from both TCR (recognition of self pMHC ligands) and homeostatic cytokines for maintenance and survival (19). These parameters may change with time: the self or environmental pMHC universe (e.g. food Ag, normal flora-derived Ag) could evolve with aging due to differential colonization, aberrant gene expression, etc.; likewise, homeostatic cytokine availability, T cell repertoire, abundance (particularly loss of naïve T cells – (4, 5) and cellular responsiveness to key homeostatic cytokines (9) could all be altered by aging.

We evaluated the expression of Tg TCR chains relative to the acquisition of the VM phenotype. Transgenic TCRVβ5 was stably expressed between 4–18 mo (not shown). Young (4 mo, Fig. S2A) OT-I CD8 T cells were almost exclusively (>99.8%) Va2^hi and exhibited a relatively low (<20%) and stable fraction of CD44^hi cells (Fig. 1). However, older OT-I mice contained a significant fraction of Vα2^int and Vα2^lo populations (Fig. S2A). Loss of Vα2 was accompanied by retention of overall TCR expression (pan-TCRβ staining, Fig. S2A), suggesting that these CD8 T cells expressed another TCRα chain and another potential TCR specificity, a scenario that physiologically occurs on up to ~30% of human and 15% murine T cells (20–22). Importantly, the Va2^lo/int phenotype correlated significantly with the CD44^hi phenotype in 12 mo OT-I mice (Fig. S2C).

To examine directly the role of TCRα replacement, we sorted CD44^lo (true naïve, TNa) and VM cells from mice of different ages in the presence or the absence of the Rag recombinase and performed PCR amplification of different T cell Receptor Alpha Variable (TRAV) gene rearrangements. As expected, 14 mo. OT-I Rag-KO mice exhibited a dominant TRAV14 band (encoding the TCRVα2 protein) and very few other bands (Fig. 2A); importantly, there was no difference in the number of bands in Rag-KO mice regardless of the age or the CD44 phenotype (Fig. 2D), indicating that these mice, as expected, cannot rearrange endogenous TRAV genes (bands on the gels that are appearing in a few TRAV families are artifacts of the PCR). Moreover, we found comparable low levels of endogenous TCRα rearrangements in 3 mo old VM samples, suggesting that young adult VM cells also do not exhibit pronounced endogenous rearrangements. (Fig. 2B), in accordance with the FCM analysis of protein expression (Fig. S2). TNa cells from 14mo old OT-I mice also did not carry an increased number of rearrangements compared to their younger counterparts (Fig. 2C, top). By contrast, VM cells at 14 mo of age contained rearrangements in many different TRAV families (see example in Fig. 4C, bottom). When quantified across groups of animals (by blind examination of three independent examiners) VM cells from old Rag-sufficient OT-I mice exhibited significantly increased expression of alternative TCRα families when compared to all other populations analyzed (Fig. 2D).

To examine whether the presence of secondary TRAV rearrangements impacted the frequency of VM cells across the lifespan, we aged different TCRTg strains on Rag-KO background. Up to 19 mo of age, the three different TCRTg Rag-KO mouse strains examined (OT-I, P14 (23) and gBT-I (14) did not downregulate the Vα2 chain, and also maintained a small (<5%) VM population that did not increase with age (Fig. 2E, filled bars), consistent with (10). This sharply contrasted with progressive, age-related VM accumulation in Rag+ OT-I mice, reaching >50% of all cells (Fig. 2E, open bars). Therefore, we conclude that secondary TCR rearrangements, and, therefore, the TCR-mediated signals, are essential for the age-related dominance of VM CD8 cells with, but are not necessary for the generation of neonatal VM cells in TCRTg mice.

Proliferative potential of VM cells declines with age in response to TCR but not to cytokine stimulation

We next examined functional responses of VM CD8 T cells with aging. Since VM cells exhibit increased expression of homeostatic cytokine receptors (Fig. S1), we tested whether these cells were more sensitive to IL-7/IL-15. Sorted, >98% pure VM (CD44^hi) and CD44^lo CD8 T cells from adult and old OT-I Rag+ mice were labeled with CFSE and cultured with IL-7/IL-15 for up to 7 days. At both 5 days (not shown) and 7 days of culture (Fig. 3) VM (CD44^hi) CD8 T cells from both 3 mo and 14 mo old mice proliferated more than their TNa (CD44^lo) counterparts based on percentages (Fig. 3A,B) and counts (not shown) of cells reaching 4^th division and above. Moreover, compared to their 3mo old counterparts, both VM (Fig. 3C) and TNa (Fig. 3D) cells from 14mo old mice exhibited significantly higher propensity to divide 4 or more times in response to IL-7+IL-15. Therefore, VM OT-I cells exhibit increased sensitivity to homeostatic cytokines, which increased with age and could contribute to their age-related accumulation.

Our results (Fig. 2) indicated that VM cell phenotype and accumulation in old mice must be driven/maintained by TCR signals. Over a lifetime, even low-level proliferation/ turnover could exert cumulative effects on old T cells. To examine functional impact of this interaction upon TCR-driven functions, sorted >98% pure VM and TNa cells from 3 and 14 mo OT-I mice were stimulated in vitro with the cognate SIINFEKL peptide, or with IL-12 + IL-18, to elicit IFN-γ secretion and proliferation. An increased proportion of VM OT-I T cells rapidly produced IFN-γ in response to both types of stimulation, compared to TNa counterparts (Fig. 4A,B), as was seen in WT VM cells (9, 10). Moreover, aging again accentuated this phenotype, because 14mo old VM cells produced IFN-γ at a much higher frequency (~30%) compared to their 3mo old counterparts (5–7%; Fig. 4B).

As seen in young adult VM cells from wt mice (10), young adult TCRTg VM CD8 T cells exhibited significantly enhanced proliferation when compared to TNa cells at both day 3 (not shown) and day 5 of culture (Fig. 4C). Importantly, 14mo (Fig. 4D) or older (not shown) VM cells exhibited delayed and reduced entry into advanced cell divisions, and these cells were clearly outperformed by old TNa cells in proliferation on both days 3 (not shown) and 5 post stimulation (Fig. 4D). Collectively, these results suggest that aging leads to selective proliferative impairment; old OT-I VM cells proliferate more than their TNa counterparts in response to homeostatic cytokines (likely explaining their gradual dominance with time) but are less capable to proliferate in response to cognate peptide (TCR) stimulation. To assess whether the proliferation difference between bulk adult and old naïve T cells may be due to differential behavior of TNa and VM cells, we directly compared adult and old VM (Fig. 4E) and TNa cells and found no significant differences between adult and old TNa (Fig. 4F). We were surprised that old TNa cells proliferated similarly to their adult counterparts. The literature: never separates between VM and TNa cells when comparing proliferation between adult and old (Refs. (24–27).

Old VM cells exhibit decreased viability when responding to peptide, but increased viability when responding to homeostatic cytokines

We next sought to address why old VM cells did not proliferate as extensively as old TNa cells, by examining cell cycle progression and survival in the course of proliferation. In response to peptide stimulation, we saw no difference in the ability of VM cells to enter into cell cycle or to cross to different phases of the cycle, when compared to TNa (Fig. 5A). However, when we measured viability, we found increased cell death, in old VM cells when compared to TNa (Fig. 5B, C), which corresponded to very few live VM cells by day 3 (Fig. 5D). By contrast, we found that VM cells stimulated with IL-7 and IL-15 exhibited superior survival and reduced cell death when compared to TNa (Fig. 5E), which resulted in increased cell numbers (Fig. 5F). These results strongly support our findings in Fig. 3, suggesting that survival advantage in response to cytokines and proliferative capacity in response to antigenic peptide may be dissociated in old VM cells.

Old VM cells proliferate extensively to polyclonal stimulation

We further investigated proliferate characteristics of the old VM cells in response cognate TCR stimulation, in relationship to their in vivo accumulation. These cells are more sensitive to homeostatic cytokines IL-7 and iL-15 (Fig. 3) and have increased expression of CD122 (Fig S1), but . Since alternative TCR’s may also play a role (Fig. 2), we were interested in whether old VM cells could proliferate to polyclonal stimulation. We stimulated sorted splenic old OT-I Rag+ VM and TNa CD8 T cells with αCD3 and αCD28 and found old VM cells proliferated similarly to TNa (Fig. 6A), and in fact had significantly increased frequency of cells in later cell divisions. This is likely due to increased viability - we found that old VM cells stimulated with αCD3 and αCD28 exhibit increased viability compared to VM cells stimulated with peptide (Fig. 6B).

Old precursors from unimmunized wild type mice exhibit the same VM conversion and CD122 upregulation seen in TCRTg precursors

One could argue that many of the above observations could be an artifact of the OT-I TCRTg model. To assess whether the above findings from TCRTg mice also extended to wt mice, we isolated different antigen-specific CD8 T cells from unimmunized adult (2–3 mo) and old (18–23 mo) mice using the tetramer enrichment method (28) as in our prior work (9). As expected (9), the frequency of CD44^hi VM CD8 T cell precursors specific for B8R (poxvirus immunodominant epitope, (29)), OVA and the West Nile virus NS4b (30) increased with age (9) (Fig. 7A). Importantly, with age, all three types of precursors also exhibited significantly higher expression of the IL-2/IL-15Rβ chain (CD122; Fig. 7B), consistent with findings from aged OT-I mice (Fig. S1). We have also found that old wt VM cells proliferate worse than TNa counterparts both in vitro and in vivo (Renkema, K.R., G. Li et al., in preparation). Collectively, these results demonstrate the essential similarities found between the TCRTg and wt old VM T cell precursors.

Expression of inhibitory receptors on TCRTg CD8 VM cells with aging

Several additional mechanisms could contribute to cellular proliferative senescence, including exhaustion/anergy; an age-dependent increase in inhibitory and exhaustion receptors on CD8 T cells was reported (rev. in (31)), even on naïve precursors in unimmunized mice (32). While we observed a significant increase in the frequency of both PD-1⁺(Fig. S3A,C) and LAG3⁺ (Fig. S3B) OT-I T cells at 14mo compared to adults (Fig. S3A,B), the representation of cells expressing these markers remained modest (>20% in OT-I 14mo mice, with the exception of a single outlier, Fig. S3A; and also in Tet+ 20mo old wt CD8 T cells, Fig. S3C). There were no significant changes in the expression of 2B4 on these cells from 2–14 months or on wt T cells up to 24mo of age (not shown). We conclude that these inhibitory markers are unlikely to be the sole (or perhaps even the main) explanation for the proliferation defects in aging VM precursors, particularly because we have seen them expressed exclusively on the EM (CD44^hi62L^lo), and not CM/VM (CD44^hi62L^hi) fraction of CD44^hi CD8 T cell precursors (not shown). They could, however, contribute to the overall reduction in proliferative capacity in old VM precursors.