Cutting Edge: Enhanced Clonal Burst Size Corrects an Otherwise Defective Memory Response by CD8⁺ Recent Thymic Emigrants Free

Recent thymic emigrants (RTEs) are T cells that have most recently left the thymus to enter the lymphoid periphery. RTEs are an important, yet understudied, population that helps to seed the population of naive peripheral T cells throughout life, as well as makes up the bulk of T cells in neonates and individuals recovering from accidental or targeted lymphoablation. RTEs can be identified unambiguously as GFP⁺ peripheral T cells in mice carrying a GFP transgene driven by the RAG2 promoter (1). Analyzing RTEs from unmanipulated RAG2p-GFP transgenic (Tg) reporter mice demonstrated that these youngest peripheral T cells are functionally distinct from their GFP⁻ mature naive (MN) counterparts (reviewed in Refs. 2, 3). CD4⁺ RTEs are Th2 skewed (4, 5), and in the context of infection, CD8⁺ RTEs exhibit increased responses to low-affinity Ags, proliferate more robustly, and are skewed toward a short-lived effector phenotype (6–8). In addition, RTEs express elevated levels of homing molecules that allow increased entry into peripheral tissues and enhanced resistance to tissue infection (6). Although RTEs have a surface phenotype that is consistent with increased effector function, they produce lower levels of the effector cytokines IFN-γ and TNF-α (6, 7, 9). These data suggest that, although RTEs respond more readily than mature T cells to a broader range of Ags in the context of a primary infection, their reduced cytokine production may help to prevent excessive tissue damage. Understanding how this distinct Ag-response profile translates into the generation of a memory T cell response was the goal of the current studies.

After infection is cleared, most responding CD8⁺ T cells die by apoptosis, but a small population converts to a central memory population that is characterized by increased expression of transcription factors, such as Eomesodermin (Eomes), Id3, and Bcl-6, efficient localization to secondary lymphoid organs, and improved re-expansion during secondary infection (10). The failure of T cells to properly differentiate into central memory cells can lead to decreased T cell longevity and an inability to control secondary infection (10). The current study addresses the capacity of RTEs to differentiate into central memory T cells and demonstrates that central memory conversion of RTEs is slowed relative to that of mature T cells postinfection. This slower rate of conversion results in altered tissue localization, with more Ag-specific RTEs than mature T cells residing in the spleen and blood rather than in the lymph nodes (LNs). Contrary to expectation, RTE-derived memory cells do not mount a poor proliferative response to secondary infection, but instead re-expand as well as their mature counterparts. Our data indicate that, although RTEs differentiate poorly into central memory T cells (Tcms) and show reduced effector cytokine production, these deficiencies are overcome by the enhanced proliferation of RTEs, a trait that persists through primary and secondary infection, well after RTEs have transitioned to the mature T cell compartment.

RAG2p-GFP Tg OT-I TCR Tg mice were backcrossed in our laboratory for 12 generations onto the C57BL/6 (B6) background with both CD45.1 and CD45.2 congenic markers. B6×B6.SJL-Ptprc^aPepc^b/BoyJ (CD45.2×CD45.1) adoptive recipients were bred in-house. All mice were housed under specific pathogen–free conditions and used in accordance with the University of Washington Institutional Animal Care and Use Committee.

Fluorochrome-conjugated Abs against the following surface and intracellular molecules were purchased from eBioscience, BD Biosciences, or BioLegend: CD45.1 (A20), CD45.2 (104), NK1.1 (PK136), CD4 (RM4-5), CD8α (53-6.7), Ter119 (Ter119), CD11b (M1/70), B220 (RA3-6B2), CD44 (IM7), CD62L (MEL-14), CXCR4 (L276F12), IL-2 (JES6-5H4), IFN-γ (XMG1.2), granzyme B (GB11), and Eomes (Dan11mag).

RBCs were water lysed from single-cell suspensions of splenocytes, blood, and bone marrow (BM) harvested from femurs and tibias. These populations and those from LNs were surface stained for 20 min at 4°C. For CXCR4 staining, cells were stained first with anti-CXCR4 at 37°C for 30 min, washed, and stained for other surface molecules as above. For intracellular cytokine staining, 2–4 × 10⁶ cells were incubated for 5 h at 37°C with brefeldin A (GolgiPlug; BD Biosciences) and 10 nM of the OVA-derived SIINFEKL peptide (Genemed) recognized by OT-I Tg T cells, surface stained, and then fixed and permeabilized with BD Cytofix/Cytoperm, according to the manufacturer’s instructions. For intranuclear staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer’s protocol. Single-color compensation controls and experimental samples were identically fixed. All samples were run on an LSR II or FACSCanto (BD Biosciences); live-gated, singlet populations were analyzed using FlowJo software (TreeStar).

Splenocytes and LN cells from RAG2p-GFP Tg OT-I TCR Tg mice were first enriched with a no-touch method using a CD8 T Cell Isolation Kit (STEMCELL Technologies), and FcRs were blocked with anti-CD16/32 (clone 2.4G2) before cells were sorted to >98% purity as GFP⁺ (RTEs) or GFP⁻ (MN) NK1.1⁻CD4⁻Ter119⁻CD11b⁻B220⁻ (dump gate) CD44^loCD62L^hi cells. Sorted RTEs and MN T cells were counted and mixed 1:1; unless otherwise noted, 10⁴ total T cells were injected i.v. into sex-matched congenic hosts. Where indicated, cells were labeled in the presence of 2.5–5 μM CFSE for 10 min at 37°C to enable quantification of cell proliferation. The dim GFP signal from RTEs does not interfere with this analysis. One day after T cell transfer, adoptive hosts were infected i.v. with 2000 CFU mid-log phase (0.3–0.7 OD₆₀₀) Listeria monocytogenes engineered to express chicken OVA (Lm.OVA), originally a gift from the Shen laboratory (11). For rechallenge experiments, 1–5 × 10⁶ PFU vesicular stomatitis virus engineered to express chicken OVA (VSV.OVA) (12) were injected i.v. For secondary transfers, spleens were processed on day 73 postinfection and analyzed for donor cell composition and 9 × 10⁶ donor cells were injected into hosts that were infected 1 d later with VSV.OVA. Donor cells were analyzed from secondary hosts 6 and 60 d after reinfection. In vivo killing assays were performed as described (13). Briefly, 3 × 10³ RTEs or MN T cells were transferred into separate mice, and 7 d days later, Lm.OVA-infected or uninfected hosts received 10⁶ SIINFEKL-pulsed splenocytes labeled with 2.5 μM CFSE and 10⁶ unpulsed targets labeled with 0.05 μM CFSE. The percentage killing at the 1-h time point was calculated as: 100 − [(% peptide pulsed in infected/% unpulsed in infected)/(% peptide pulsed in uninfected/% unpulsed in uninfected)] × 100.

As indicated, a paired or unpaired two-tailed Student t test was used for comparisons, and p < 0.05 was considered statistically significant.

Expansion and memory formation by RTE- and MN-derived T cells were examined in recipient mice that were infected with Lm.OVA 1 d after cotransfer of equal numbers of CD8⁺ RTEs and MN T cells sorted from RAG2p-GFP Tg OT-I TCR Tg mice. At 7 d postinfection, RTE-derived cells were present in 6-fold greater numbers than MN-derived donor T cells in the blood (Fig. 1A), consistent with previous data indicating that RTE-derived CD8⁺ T cells have increased clonal expansion compared with their mature counterparts (6). MN-derived T cells represented ∼1% of the total peripheral blood CD8⁺ T cells from 21 d postinfection onward (Fig. 1A). RTE-derived cells remained in circulation for a longer period, reaching stable levels at ∼60 d postinfection; they gradually increased, outnumbering MN-derived T cells by 2–3-fold for >250 d after Lm.OVA infection (Fig. 1A). This increase in RTE-derived memory cells was also observed in the spleen, where RTEs outnumbered MN T cells by ∼3-fold at 60 d postinfection (Fig. 1B). These data indicate that postinfection, RTEs have a greater burst size than mature T cells, and RTE-derived memory cells preferentially remain in the circulating and splenic memory pool.

We next determined whether the increased numbers of RTEs generated during a primary infection were sufficient to reverse the effects of their diminished effector cytokine production (6, 7). To address this, CFSE-labeled OT-I Tg RTEs and MN T cells were transferred into mice that were later infected with Lm.OVA. Five days later, a lower proportion of RTEs was granzyme B⁺ than mature T cells at each division (Fig. 1C, left panel). Representative flow plots (Supplemental Fig. 1) reveal minimal differences in the mean fluorescence intensity (MFI) of granzyme B staining in RTEs and mature T cells, suggesting that any influence on staining levels caused by degranulation are minimal at this time point. On a per-responder cell basis, RTE-derived effector cells lysed fewer OVA peptide–pulsed splenocytes (Fig. 1C, middle panel). However, the per-host complement of RTEs killed the bolus of transferred target cells even more efficiently than did their mature counterparts (Fig. 1C, right panel). Thus, RTEs have reduced levels of granzyme B and are poor at lysing target cells, but their abundance allows them to maintain a population level effector function that is equal to or better than that of their mature counterparts.

Postinfection, a subset of CD8⁺ T cells develops a Tcm phenotype, which is characterized by increased expression of the LN-homing molecules CD62L and CCR7, increased production of IL-2, and enhanced re-expansion potential (10). The increased number of RTEs in the circulation suggests that these cells may be slower to acquire a Tcm phenotype and localize to LNs. This hypothesis is supported by previous data demonstrating that fewer RTEs acquire a memory precursor phenotype during acute infection (6, 7). To measure conversion to a Tcm phenotype, expression of CD62L was analyzed on RTE- and MN-derived splenocytes 60 d postinfection. As T cells entered the memory phase, RTE-derived cells were slower to upregulate CD62L than were MN-derived cells (Fig. 2A, left panel; see Supplemental Fig. 2A for representative flow plots). CD62L expression was diminished in RTEs both in the proportion of positive cells (Fig. 2A) and in the per cell expression level (Supplemental Fig. 2B). Further phenotypic comparison of RTE- and MN-derived memory CD8⁺ T cells indicated that the former were reduced in their ability to produce IL-2 after in vitro stimulation (Fig. 2A, middle panel). Expression of Eomes, a transcription factor required for Tcm development (14), was also reduced in RTEs both by proportion and percent of positive cells (Fig. 2A, right panel) and by MFI (Supplemental Fig. 2B). Analysis of T cells in the blood indicated that it took 85 d after infection for 40% of RTE-derived memory cells to convert to a CD62L^hi phenotype, whereas mature T cells reached this point at 50 d (Fig. 2C). The failure of RTE-derived memory cells to efficiently re-express CD62L persisted for >120 d, even after infection with Lm.OVA expressing low-affinity altered peptide ligands (data not shown), to which RTEs respond relatively better than do mature T cells (6). These data indicate RTEs are slower than mature T cells to acquire a Tcm phenotype. This slow Tcm conversion parallels our earlier findings that naive CD8⁺ RTEs are IL-7Rα^lo (1) and that, in Listeria-infected hosts, responding RTEs are more prone than mature T cells to differentiate into KLRG1^hiIL-7Rα^lo short-lived effectors (6, 7). It would be informative to determine whether forced expression of IL-7R through transgenesis alters the kinetics of Tcm conversion in RTE-derived effector cells.

One feature of Tcms is their ability to preferentially home to the BM, where they access homeostatic cytokines, such as IL-7 and IL-15, and undergo increased proliferation compared with cells in other lymphoid organs (15, 16). Loss of Eomes is associated with reduced CXCR4 expression and reduced BM localization (14). We assessed the BM-homing capabilities of RTE- and MN-derived memory CD8⁺ T cells 60 d postinfection and found that they were present at similar frequencies, in contrast to the spleen, in which RTE-derived T cells outnumbered their MN counterparts by ∼2.8-fold (Fig. 3A). We next measured the expression of the chemokine receptor CXCR4 on RTE- and MN-derived memory T cells; it is upregulated on memory T cells and plays an important role in recruiting CD8⁺ T cells into the BM (17). The percentage of RTEs expressing CXCR4 in the spleen and even the BM was significantly reduced compared with their mature counterparts (Fig. 3B). Furthermore, the level of CXCR4 expression/cell, as measured by MFI, was also significantly reduced in splenic RTEs but not in those in the BM, perhaps a result of that chemokine receptor’s role in cellular recruitment to this site. These data demonstrate that RTE-derived memory cells express lower levels of CXCR4 and, accordingly, show reduced recruitment to, or retention within, the BM. Therefore, it became important to test whether reduced BM residency impacted the maintenance of long-term memory among RTE-derived T cells.

CD8⁺ Tcms are thought to be superior in their ability to re-expand upon secondary challenge, whereas effector memory T cells are capable of exhibiting more immediate effector function (10). The developing RTE-derived memory pool is defective in its ability to generate Tcms compared with MN-derived memory T cells, suggesting that, upon rechallenge, RTE-derived memory cells might be hindered in their re-expansion. To examine the recall potential of RTE- and MN-derived memory T cells, mice that received OT-I T cells and were primed with Lm.OVA 66 d earlier (and in which RTEs outnumber their mature counterparts but express less CD62L, Fig. 4A) were infected with VSV.OVA. Surprisingly, the re-expansion of RTE-derived memory T cells was similar to that of MN-derived T cells 5 d after VSV.OVA challenge (Fig. 4B, left panels). Functionally, RTE-derived memory T cells were imprinted with the same deficiencies observed upon initial Ag encounter, including reduced production of IL-2 and IFN-γ (Fig. 4B, right panels). The MFI for cytokine (IL-2, IFN-γ, and TNF-α) production was also lower for RTEs, indicating that the defect was measurable in terms of the proportion of cytokine-producing cells and in the amount of cytokine produced per cell (data not shown). Other mice were rechallenged with VSV.OVA at 252 d after primary infection, when RTE-derived memory T cells still outnumber their MN-derived counterparts but have similar expression of CD62L in the blood (Fig. 4C). Again, RTE-derived memory cells expanded at least as well as MN-derived T cells (Fig. 4D, left panels) but continued to display defects in cytokine production, with a lower percentage of RTEs producing IFN-γ and IL-2 (and TNF-α, data not shown) compared with their mature counterparts (Fig. 4D, right panels). These data indicate that the impaired transition of RTEs to Tcms and their diminished BM localization do not trigger a corresponding defect in the magnitude of their recall response, but that reduced cytokine production by RTE-derived T cells persists during secondary expansion. The fact that RTE-derived memory cells show reduced cytokine production >250 d after initial priming is striking, given that RTEs transition into the mature T cell pool after ∼3 wk of residence in the lymphoid periphery. Clearly, the stage of maturation at which peripheral T cells first meet Ag has a lasting impact on their immune function.

To definitively determine whether the skewed Tcm phenotype of RTEs alters expansion and cytokine production after secondary infection, we isolated RTE- and MN-derived memory CD8⁺ T cells from mice at 73 d postinfection and transferred them into naive animals. At this time point, RTE-derived memory cells displayed reduced expression of CD62L (Fig. 5A) compared with mature T cells. After T cell transfer, mice were infected with VSV.OVA, and T cells were analyzed at 6 and 60 d postsecondary infection. At day 6, RTEs outnumbered mature donor cells in the blood, spleen, LN, and BM (Fig. 5B, left panel), and RTE- and MN-derived memory cells expanded ∼2000-fold (Fig. 5C). At 60 d postinfection, RTEs continued to outnumber MN-derived secondary memory cells in the blood and spleen, but they were present in equal proportions in the LN and BM (Fig. 5B, right panels), as seen during primary memory.

We demonstrated that, compared with mature T cells, CD8⁺ RTEs proliferate more readily, remain in the circulation for an extended period of time, and exhibit a short-lived effector phenotype. However, this is at the expense of their conversion into Tcms, because RTEs fail to efficiently express Eomes, produce less IL-2, and home to the BM poorly compared with mature T cells. These data predict that, upon secondary challenge, RTEs would be outcompeted by MN-derived memory T cells; however, RTEs proliferated at least as well as mature T cells during secondary challenge. The ability of RTEs to proliferate more robustly than MN T cells has been attributed to increased surface expression of the TCR and CD3 and enhanced TCR signal transduction (6, 18); this could persist into the memory phase, allowing RTE-derived memory cells that are skewed toward an effector memory T cell phenotype to proliferate as well as MN-derived memory cells that are Tcm skewed. Hypermethylation of the Il2 promoter is observed in CD4⁺ RTEs and may limit IL-2 production (19), an epigenetic alteration that likely persists through multiple rounds of stimulation and may also regulate cytokine production by CD8⁺ RTEs. Overall, these data suggest that RTEs maintain an effector phenotype and patrol the body for a prolonged period, which would provide a substantial benefit in neonatal and other lymphopenic settings to ensure pathogen clearance and a more immediate response to reinfection. The unique biology of RTEs reflects a program that unfolds when the cells initially encounter Ag and extends for >8 mo thereafter, allowing this distinct cellular compartment to maintain enhanced readiness without a concomitant loss in proliferative capacity.

The authors have no financial conflicts of interest.