Cutting Edge: Resident Memory CD8 T Cells Occupy Frontline Niches in Secondary Lymphoid Organs

Memory CD8 T cells are widely distributed throughout the body and are partitioned into subsets based on trafficking properties (1, 2). Central memory CD8 T cells (T_CM) recirculate through secondary lymphoid organs (SLO). Effector memory CD8 T cells (T_EM) recirculate through nonlymphoid tissues (NLT). Resident memory CD8 T cells (T_RM) are a newly recognized subset that patrols nonlymphoid frontline sites of pathogen exposure without recirculating (3–5). CD69 is expressed upon TCR stimulation by early effectors and T cells responding to chronic infections. CD69 is also constitutively expressed by T_RM after Ag clearance, suggesting a separate pathway for regulation (6, 7). Tissue microenvironments induce constitutive CD69 expression by T_RM, tying this phenotype to location (4, 6–9). Moreover, CD69 expression is associated with retention in tissues, suggesting that this marker plays a defining functional role in T_RM differentiation (8).

T_RM play a specialized sentinel role and are positioned to rapidly alert the host in the event of reinfection and accelerate pathogen clearance (4, 5, 10, 11). Thus, their anatomic localization at initial sites of Ag exposure is critical for the conceptualization of T_RM function. Although these early responders are tightly associated with NLT distribution, SLO are compartmentalized and also contain “frontline” sites of initial Ag encounter (12). For instance, the subcapsular and medullary sinuses of lymph nodes (LN) capture lymph-borne pathogens derived from tissues (13, 14). In spleen, hematogenous pathogens are first encountered in the red pulp or trapped in the marginal zone (15). Thus, we hypothesized that T_RM also may exist in the “frontline” portions of lymphoid tissue.

All mice were used in accordance with the guidelines of the Institutional Animal Care and Use Committees at the University of Minnesota. C57BL/6J mice were purchased from The Jackson Laboratory. Thy1.1⁺ P14, CD45.1⁺ OT-I, and CD45.2⁺ OT-I mice were fully backcrossed to C57BL/6J mice and maintained in our animal colony. P14 immune chimeras were generated by transferring 5 × 10⁴ naive transgenic Thy1.1⁺ P14 T cells into naive wild-type or IL-15^−/− C57BL/6J mice and infecting with 2 × 10⁵ PFU lymphocytic choriomeningitis virus (LCMV, Armstrong strain) i.p. the next day. Memory OT-I cells were generated by transferring 5 × 10⁴ naive transgenic CD45.1⁺ OT-I T cells into memory P14 chimeras. The next day, recipients were infected with 1 × 10⁶ PFU recombinant vesicular stomatitis virus expressing OVA (VSV-OVA) i.v., as described (16). Naive CD45.2⁺ OT-I T cells were injected into CD45.1⁺ RAG^−/− mice, as indicated. For local rechallenge experiments, 50 μg gp33 peptide was delivered transcervically, as described (10), in a volume of 35 μl delivered by modified gel loading pipette.

Parabiosis surgeries were performed as described (10). Briefly, mice were shaved along opposite lateral flanks. Skin was wiped clean of fur with alcohol prep pads and further cleaned with Betadine solution and 70% isopropyl alcohol. Mirrored incisions were made on lateral aspects of both mice, and 5-0 VICRYL thread was used to suture skin to conjoin the mice. Additional sutures were placed through the olecranon and knee joints to secure the legs.

For immunofluorescence microscopy, tissues were frozen in 2-methylbutane surrounded by dry ice. Frozen blocks were cut into 7-μm sections, fixed in acetone, blocked in a 5% BSA PBS solution for 1 h, and stained with DAPI (Invitrogen) and Abs specific for Thy1.1 (OX-7; eBioscience), CD45.1 (A20; eBioscience), CD169 (AbD Serotec), CD69 (goat polyclonal; R&D Systems), and Collagen IV (rabbit polyclonal; Acris). Jackson ImmunoResearch secondary Abs conjugated to various fluorochromes were used to stain unconjugated Abs. Tiled images were acquired with an automated Leica DM5500B microscope, and analysis was performed with ImageJ and Adobe Photoshop. Cell isolations and flow cytometry were performed as previously described (17). Quantification of microscopy images was performed as previously described (10).

The p values were determined by a two-tailed, unpaired Student t test. Differences between groups were considered significant if p ≤ 0.05.

To determine whether a population of memory CD8 T cells within SLO expressed canonical markers of T_RM, we analyzed Thy1.1⁺ memory P14 CD8 T cells 8 wk after LCMV Armstrong infection (see 2Materials and Methods). P14 small intestine intraepithelial lymphocytes were used as a positive control because they were shown to be T_RM in this infection model (3). Flow cytometric analysis revealed a subset of CD69⁺ memory CD8 T cells within both the LN (data not shown) and the spleen (Fig. 1A). CD69⁺ memory CD8 T cells within the SLO lacked CD62L, a cardinal feature of recirculating T_CM. This unusual SLO population was distinct from canonical T_EM (CD69⁻ CD62L⁻) and T_CM (CD69⁻ CD62L⁺) but was similar to NLT-derived T_RM, by expression of Ly6C, KLRG1, and Eomes (Fig. 1B). Unlike T_RM that have been characterized in or near epithelial surfaces, CD69⁺ memory CD8 T cells within the SLO did not express CD103 (i.e., αeβ7 integrin; data not shown). It should be noted that CD69 expression was upregulated after clearance of LCMV, suggesting that persistent foreign Ag was not driving expression (Fig. 1C). To support this interpretation, we transferred naive Rag^−/− OT-I CD8 T cells to lymphopenic RAG^−/− mice, which results in homeostatically expanded memory-like T cell differentiation without pathogen-derived Ag (6). Under these conditions, we also observed a CD8 T cell subset within SLO that exhibited a T_RM-like phenotype (Fig. 1D).

We wished to test whether CD69⁺ memory CD8 T cells within SLO were T_RM. The vascular systems of LCMV-immune mice (generated as in Fig. 1) were surgically conjoined to naive mice via parabiosis, which equilibrates T_CM and T_EM between hosts without equilibrating T_RM (5, 18). Fig. 2A shows that the CD69⁺ SLO memory CD8 T cell population was specifically absent in the naive parabiont, demonstrating that this population was indeed resident within SLO. Immunofluorescence microscopy localized these T_RM preferentially to the marginal zone and red pulp of the spleen and the subcapsular and medullary sinuses of the LN (Fig. 2B–E), which represent the primary initial sites of Ag entry into these SLO.

Previous reports showed that maintenance of T_CM and T_EM is critically dependent on IL-15 (19). We found that CD69⁺ T_RM within SLO express reduced levels of CD122 (IL-15Rβ, Fig. 3A), similar to what was reported for T_RM located within the epithelium of the small intestine (17). We hypothesized that a distinguishing feature between SLO T_RM and recirculating memory CD8 T cells could be dependence upon IL-15. To test this hypothesis, we compared memory P14 differentiation and maintenance in wild-type and IL-15–deficient LCMV-immune chimeras (see 2Materials and Methods). Sixty days after LCMV infection, we found that CD69⁺ SLO T_RM were overrepresented in the absence of IL-15 (Fig. 3B, 3C). These data suggest that SLO T_RM are less dependent upon IL-15 for their survival compared with recirculating primary T_CM and T_EM, which further defines them as a unique subset.

Upon Ag recognition within the female reproductive mucosa, reactivated local memory CD8 T_RM precipitate alarm signals (10). These signals recruit bystander resting memory CD8 T cells, which redistribute from spleen and blood to the site of reactivation in the mucosa (10). We asked whether this redistribution function was restricted to recirculating memory CD8 T cells or whether CD8 T_RM in the SLO also would mobilize to the female reproductive tract in this context. To this end, we transferred naive OT-I CD8 T cells into P14 LCMV-immune chimeras. The next day, we infected these mice with VSV-OVA. Thirty days later, these mice contained populations of both OT-I and P14 memory CD8 T cells. They were then rechallenged transcervically with gp33 peptide, as described (10), to reactivate P14 within the female reproductive tract and precipitate local alarm signals that induce redistribution of memory CD8 T cells. CD69⁺ and CD69⁻ OT-I CD8 T cells were enumerated within the spleen (2 d after gp33 peptide rechallenge) (Fig. 4). Unlike recirculating memory OT-I CD8 T cells, many of which vacated SLO to respond to the site of peripheral P14 CD8 T cell reactivation, the number of CD69⁺ memory OT-I CD8 T cells was unaffected. This indicates that, unlike recirculating memory CD8 T cell subsets, T_RM maintain their presence in the SLO even in the context of peripheral alarm signals.

T_RM have been identified in many NLT, as well as the thymus, where they are positioned to rapidly provide protection against pathogens re-encountered at anatomic barriers (4, 5, 7, 11, 20). T_RM express unique phenotypic signatures, including CD69 expression, that distinguish them from recirculating T_CM and T_EM (4, 8, 17). T_RM differentiation is induced by local environmental cues; thus, T_RM ontogeny is intrinsically coupled with location (6, 8). Therefore, it is surprising that we defined a population of bona fide T_RM via parabiosis experiments, which also could be identified by CD69 expression, present within lymphoid tissues. It should be noted that CD69⁺ CD8 T cells also have been detected in human SLO (21, 22).

It remains possible that local inductive cues also regulate T_RM ontogeny within SLO microenvironments. Indeed, SLO T_RM occupied specific niches within lymphoid organs, particularly the subcapsular and medullary sinuses of LN and the marginal zones of spleen, which showed minimal overlap with T_CM and T_EM. These compartments also represent major portals for pathogen and Ag entry into lymphoid organs (12). In light of these findings, there may be operational similarities between SLO T_RM and those in NLT tissues: as guardians of frontline sites of pathogen exposure. It is telling that, unlike CD69⁻ recirculating memory CD8 T cell populations, SLO T_RM did not abandon the lymphoid tissue in response to distant inflammatory cues. Perhaps they fulfill a specialized immunosurveillance role that, like NLT T_RM, accelerates alarm signals and pathogen control in response to reinfection.

The authors have no financial conflicts of interest.