Resident memory CD8 T cells occupy frontline niches in secondary lymphoid organs

Abstract

Resident memory CD8 T cells (T_RM) comprise a non-recirculating subset positioned in non-lymphoid tissues (NLT) to provide early responses to re-infection. While T_RM are associated with NLT, we asked whether they populated secondary lymphoid organs (SLO). We show that a subset of virus specific memory CD8 T cells in SLO exhibit phenotypic signatures associated with T_RM, including CD69 expression. Parabiosis revealed that SLO CD69⁺ memory CD8 T cells do not circulate, defining them as T_RM. SLO T_RM were overrepresented in IL-15 deficient mice, suggesting independent regulation compared to T_CM and T_EM. These cells were positioned at SLO entry points for peripheral antigens: the splenic marginal zone, red pulp, and lymph node sinuses. Consistent with a potential role in guarding SLO pathogen entry points, SLO T_RM did not vacate their position in response to peripheral alarm signals. These data extend the range of tissue resident memory to SLO.

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 SLO. Effector memory CD8 T cells (T_EM) recirculate through NLT. Resident memory CD8 T cells (CD8 T_RM) comprise a newly recognized subset that patrols non-lymphoid 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 antigen 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 re-infection and accelerate pathogen clearance (4, 5, 10, 11). Thus, their anatomic localization at initial sites of antigen exposure is critical for the conceptualization of T_RM function. Although these early responders are tightly associated with non-lymphoid tissue distribution, SLOs are compartmentalized and also contain “frontline” sites of initial antigen encounter (12). For instance, the subcapsular and medullary sinuses of lymph nodes 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 may also exist in the “frontline” portions of lymphoid tissue.

Materials and Methods

Mice and infections

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 p.f.u. lymphocytic choriomeningitis virus (LCMV, Armstrong strain) i.p. the next day. Memory OT-I cells were generated by transferring 5×10⁴ naïve transgenic CD45.1⁺ OT-I T cells into memory P14 chimeras. The next day, recipients were infected with either 1×10⁶ p.f.u. recombinant vesicular stomatitis-OVA (VSV-OVA) i.v., as described (16). Naïve CD45.2⁺ OT-I T cells were injected into CD45.1⁺ RAG^−/− mice as indicated. For local re-challenge experiments, 50μg of gp33 peptide was delivered transcervically (t.c.) as described (10) in a volume of 35μl delivered by modified gel loading pipette.

Parabiosis surgery

Parabiosis surgeries were performed as described (10). Briefly, mice were shaved along opposite lateral flanks. Skin was then 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.

Immunofluorescence microscopy and flow cytometry

For immunofluorescence microscopy, tissues were frozen in 2-methylbutane surrounded by dry ice. Frozen blocks were cut into 7μm sections, fixed in acetone, blocked in a 5% bovine serum albumin PBS solution for 1 h, and stained with DAPI (Invitrogen) and antibodies specific for Thy1.1 (OX-7, eBioscience), CD45.1 (A20, eBioscience), CD169 (AbD Serotec), CD69 (goat polyclonal, R&D), Collagen IV (rabbit polyclonal, Acris). Jackson Immunoresearch secondary antibodies conjugated to various fluorochromes were used to stain unconjugated antibodies. 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 were performed as previously described (10).

Statistical analysis

P values were determined by a two-tailed, unpaired Student’s t-test. Differences between groups were considered significant if P≤0.05.

Results & Discussion

A subset of memory CD8 T cells in SLO phenotypically resembles T_RM

To determine if a population of memory CD8 T cells within SLO expressed canonical markers of T_RM, we analyzed Thy1.1⁺ memory P14 CD8 T cells eight weeks after LCMV Armstrong infection (see methods). P14 small intestine intraepithelial lymphocytes (SI IEL) were used as a positive control because they have been 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 lymph node (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 similar to non-lymphoid tissue 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 antigen was not driving expression (Fig. 1C). To support this interpretation, we transferred naïve Rag^−/− OT-I CD8 T cells to lymphopenic RAG^−/− mice, which results in homeostatically expanded memory-like T cell differentiation without pathogen-derived antigen (6). Under these conditions, we also observed a CD8 T cell subset within SLO that exhibited a T_RM-like phenotype (Fig. 1D).

Figure 1. A subset of memory CD8 T cells in SLO phenotypically resembles T_RM.

A) Eight weeks after LCMV infection, P14 memory CD8 T cells in spleen were analyzed for the indicated markers by flow cytometry. All plots gated on Thy1.1⁺ P14 memory CD8 T cells. B) As in A, however P14 splenocytes were split into CD69⁺ CD62L⁻ (black), CD69⁻ CD62L⁻ (blue), and CD69⁻ CD62L⁺ (red) subsets. P14 isolated from small intestinal epithelium (green) were included as a positive control for previously defined T_RM. C) P14 splenocyte phenotype was compared on days seven and 40 after LCMV infection. D) 5×10⁴ naïve CD45.2⁺ OT-I were transferred to CD45.1⁺ RAG^−/− C57BL/6 mice. Sixty days later, OT-I splenocytes were analyzed for the indicated markers by flow cytometry. Data are representative of 6 mice from 2 independent experiments.

CD69⁺ SLO memory CD8 T cells are T_RM positioned at common antigen entry sites

We wished to test whether CD69⁺ memory CD8 T cells within SLO were T_RM. The vascular system of LCMV immune mice (generated as in figure 1) were surgically conjoined to naïve mice via parabiosis, which equilibrates T_CM and T_EM between hosts without equilibrating T_RM (5, 18). Figure 2A shows that the CD69⁺ SLO memory CD8 T cell population was specifically absent in the naïve 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 lymph node (Fig. 2B–E), which represent the primary initial site of antigen entry into these SLO.

Figure 2. CD69+ SLO memory CD8 T cells are T_RM positioned at common antigen entry sites.

A) P14 LCMV immune chimeric (black bars) and naïve (white bars) mice were surgically conjoined. 15 days later, the fraction of P14 that expressed CD69 was histologically enumerated within the SLO of both parabionts. B) The proportion of CD69⁺ P14 (black) and also CD69⁻ P14 (white) memory CD8 T cells present in each SLO subcompartment of the immune parabiont was determined histologically. C) Representative staining showing CD69+ memory CD8 T cells in the LN sinus. D) Representative CD69 staining in the red pulp, white pulp and marginal zone of the spleen. E) The fraction of P14 memory CD8 T cells within each SLO compartment of the immune parabiont that expressed CD69. *P<.01 and **P<.001. Data are from 6 mice from 2 independent experiments.

Reduced IL-15 dependence of CD8 T_RM in SLO

Previous reports show 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, figure 3A), similar to what has been 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 methods). 60 days after LCMV infection, we found that CD69⁺ SLO T_RM were dramatically overrepresented in the absence of IL-15 (Fig. 3B&C). These data suggest that SLO T_RM are less dependent upon IL-15 for their survival when compared to recirculating primary T_CM and T_EM, which further defines them as a unique subset.

Figure 3. Reduced IL-15 dependence of CD8 T_RM in SLO.

A) CD122 (IL-15Rβ) expression was analyzed by flow cytometry on CD69⁺ (black) and CD69⁻ (gray) memory P14 CD8 T cells isolated from spleen 60 days after LCMV infection. B&C) P14 immune chimeras were generated in WT (black) and IL-15^−/− (white) mice. 60 days later, CD69⁺ and CD69⁻ P14 were enumerated in LN and spleen. *P<.05, **P<.01 and ***P<.001. Data are from 5 mice per group from 2 independent experiments representative of 4 different experiments.

SLO T_RM do not redistribute in response to peripheral alarm signals

Upon antigen 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 would also mobilize to the female reproductive tract in this context. To this end, we transferred naïve OT-I CD8 T cells into P14 LCMV immune chimeras. The next day we infected these mice with vesicular stomatitis virus expressing ovalbumim (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 (two days after gp33 peptide rechallenge). Unlike recirculating memory OT-I, many of which vacated SLOs 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 non-lymphoid tissues 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 that distinguish them from recirculating T_CM and T_EM, particularly CD69 expression (4, 8, 17). T_RM differentiation is induced by local environmental cues, and thus T_RM ontogeny is intrinsically coupled with location (6, 8). It is therefore surprising that we defined a population of bona fide T_RM via parabiosis experiments, which could also be identified by CD69 expression, present within lymphoid tissues. It should be noted that CD69⁺ CD8 T cells have also 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 lymph nodes 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 antigen entry into lymphoid organs (12). In this light, there may be operational similarities between SLO T_RM and those in non-lymphoid 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 non-lymphoid tissue T_RM, accelerates alarm signals and pathogen control in response to re-infection.

Figure 4. SLO T_RM do not redistribute in response to peripheral inflammation.

Both P14 and OT-I memory CD8 T cells were established in mice following LCMV and VSV-OVA infection. Mice were then rechallenged transcervically with 50μg gp33 peptide. Two days later, CD69⁻ (left graph) and CD69⁺ (right graph) OT-I memory CD8 T cells were enumerated within spleen. *P<.01. Data are from 3 mice per group representative of 2 independent experiments.