The Molecular Signature of Tissue Resident Memory CD8 T Cells Isolated from the Brain

Linda M. Wakim, Amanda Woodward-Davis, Ruijie Liu, Yifang Hu, Jose Villadangos, Gordon Smyth and Michael J. Bevan
J Immunol October 1, 2012, 189 (7) 3462-3471; DOI: https://doi.org/10.4049/jimmunol.1201305

Abstract

Tissue resident memory (Trm) CD8 T cells represent a newly described memory T cell population. We have previously characterized a population of Trm cells that persists within the brain after acute virus infection. Although capable of providing marked protection against a subsequent local challenge, brain Trm cells do not undergo recall expansion after dissociation from the tissue. Furthermore, these Trm cells do not depend on the same survival factors as the circulating memory T cell pool as assessed either in vivo or in vitro. To gain greater insight into this population of cells, we compared the gene expression profiles of Trm cells isolated from the brain with those of circulating memory T cells isolated from the spleen after an acute virus infection. Trm cells displayed altered expression of genes involved in chemotaxis, expressed a distinct set of transcription factors, and overexpressed several inhibitory receptors. Cumulatively, these data indicate that Trm cells are a distinct memory T cell population disconnected from the circulating memory T cell pool and display a unique molecular signature that likely results in optimal survival and function within their local environment.

Introduction

Classically, memory CD8 T cells are broadly divided into two subsets termed central memory (Tcm) and effector memory (Tem). Tcm CD8 T cells express the adhesion molecule L-selectin (CD62L) and the chemokine receptor CCR7, traffic through lymph nodes, spleen, and blood, and represent a long-lived population of memory cells with high proliferative capacity upon rechallenge. Tem CD8 T cells lack CD62L and CCR7, are present in spleen and blood, recirculate through the peripheral tissues, and maintain immediate cytotoxic potential (1, 2). Nonlymphoid tissues house a large proportion of the memory T cell pool (3). Previously, it was thought that these were simply Tem cells trafficking through the tissue as part of their immunological surveillance. Recent studies have shown that a proportion of these memory T cells reside in the tissue and represent a distinct memory T cell population (4). These peripherally deposited memory T cells, termed tissue resident memory (Trm) cells, are a self-sustaining population that persist long term within nonlymphoid tissue, commonly at sites of prior infection. Trm cells have been identified in a variety of peripheral tissues including the skin and sensory ganglia of mice latently infected with HSV (4–6) and in the gut (7), brain (8), lung (9, 10), and salivary glands (11). These cells rapidly acquire effector function upon secondary pathogenic encounter (6), and those in the skin are highly protective against subsequent local infection (4, 12).

We have recently characterized the memory CD8+ T cell population that persists within the brain after an acute systemic vesicular stomatitis virus (VSV) infection (8). We showed that memory T cells persisting within the brain survive without replenishment from the circulation. These cells selectively expressed the integrin CD103, the expression of which was dependent on Ag recognition within the tissue. Ag persistence was not required for memory T cell retention within the brain. The memory CD8 T cells isolated from the brain died rapidly upon isolation from the tissue and failed to undergo recall expansion after adoptive transfer into the bloodstream of Ag-challenged recipients. Cumulatively, these data showed that memory CD103+ CD8 T cells that persist within the brain after an acute virus infection are bona fide Trm cells.

Recent work by several groups in both mice and human studies provides compelling evidence to support the presence of a locally confined memory T cell population that is deposited at former sites of Ag encounter (13). However, to date there was no molecular genetic profile defining these peripherally deposited memory T cells. Using a previously characterized model of intranasal infection with VSV, we genetically profiled Trm CD8 T cells that develop within the brain and compared them to memory T cells that are part of the circulating memory T cell pool. Our data clearly indicate that Trm cells are a distinct memory T cell population with a unique molecular signature.

Materials and Methods

Mice

C57BL/6 and B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen-free conditions in the animal facilities at the University of Washington (Seattle, WA). OT-I TCR transgenic mice congenic for Ly5.1 or deficient in bim and MHC class II-deficient mice were bred and maintained in the same facilities. All experiments were done in accordance with the Institutional Animal Care and Use Committee guidelines of the University of Washington.

T cell adoptive transfer and infections

Mice received 104 naive OT-I.CD45.1 CD8+ T cells via i.v. injection prior to infection. Cells were sorted from the brain and spleens of mice at the indicated times postinfection using a FACSAria II, and 3 × 103 cells were transferred into mice either intracranially (i.c.) or i.v. prior to infection.

Mice were infected intranasally (i.n.) with 5 × 104 PFU or i.p. with 2 × 107 PFU of a recombinant VSV that expresses GFP and a secreted form of OVA (14). Mice were infected i.c. with 10 CFU of either wild-type or a recombinant Listeria monocytogenes that expresses a secreted form of OVA (LM-OVA). Growth and quantification of LM-OVA was performed as described previously (15).

For in vitro stimulation experiments, 20,000 memory OT-I T cells were cultured at a 1:1 ratio with SIINFEKL pulsed (1 μM) or unpulsed irradiated splenocytes. The absolute number of live cells was determined 60 h later.

Viral titer determination

The presence of infectious VSV in tissue samples was determined using standard PFU assays on confluent Vero cell monolayers as described previously (8).

Flow cytometry

Single-cell suspensions were prepared from spleens and lymph nodes (LN) by mechanical disruption. Brains were enzymatically digested for 1 h at 37°C in 3 ml collagenase type 3 (Worthington) (3 mg/ml in RPMI 1640 supplemented with 2% FCS), and lymphocytes were separated on a Percoll gradient. Cells were stained for 25 min on ice with the appropriate mixture of mAbs and washed with PBS with 1% BSA. The following conjugated mAbs were obtained from BD Biosciences or eBioscience: anti-CD8α, CD45.1, CD103. Cells were analyzed on a FACSCanto II using FlowJo software (Tree Star).

Survival assay

Cells were cultured for 18 h in RPMI 1640 supplemented with 2% FCS, 2 mM glutamine, 5 × 10−5 M 2-mercaptoethanol, and antibiotics and with 10 ng/ml of either recombinant IL-7 or IL-15. Annexin V staining was performed using an annexin V staining kit (BD Biosciences) following the manufacturer’s instructions.

RNA samples

Mice received 104 naive OT-I.CD45.1 CD8+ T cells via i.v. injection prior to i.n. infection with VSV-OVA. CD103+ and CD103− OT-I cells were sorted from the brain and spleens of mice on day 20 postinfection (p.i.) using a FACSAria II. Cells were subjected to two successive rounds of sorting. All samples were maintained at 4°C for the duration of the sort, and purity was 95–99% for all populations. The treatments and cell sorts were repeated for three independent pools of mice. One or two samples were extracted of each cell population from each mouse pool, making 13 samples in total. RNA was isolated and hybridized to Affymetrix Mouse Gene 1.0ST GeneChips by the Immunological Genome Project (www.immgen.org), following standard operating procedures.

Microarray analysis

The microarray intensities were background corrected, normalized, and summarized according to the robust multichip average algorithm (16) using the aroma.affymetrix software package (http://www.aroma-project.org). The chip definition file MoGene-1.0-st-v1,r3.cdf was used to define a core set of probe sets with reliable gene annotation.

Differential expression analysis was undertaken using the limma software package for R (17). Genes were filtered as nonexpressed if they failed to exceed the bottom 25% of expression values on at least three arrays. Differential expression was assessed using empirical Bayes moderated t-statistics and F-statistics (18). The analysis was adjusted for correlation between samples from the same mouse pool using the duplicate correlation function. The p values were adjusted to control the false discovery rate (FDR) using the method of Benjamini and Hochberg (19).

Results

Brain Trm cells do not undergo recall expansion after dissociation from the tissue in which they reside

Intranasal infection with VSV results in a transient systemic infection that is cleared from all organs including lung, spleen, and brain by day 8 postinfection. Using a recombinant virus that expresses the model Ag OVA (VSV-OVA) in combination with OT-I TCR transgenic cells, we have previously demonstrated that long after the resolution of the acute VSV-OVA infection, memory OT-I CD8 T cells persist within the brain (8). The memory CD8 T cells persisting within the brain can be subdivided into two populations based on CD103 expression although by day 20 p.i. the vast majority of memory OT-I cells persisting within the brain express this integrin (Fig. 1A). Further characterization of these brain-residing CD103+ memory T cells revealed that they are Trm cells (8).

FIGURE 1.
FIGURE 1.

Resident memory T cells do not undergo recall expansion after dissociation from the tissue in which they reside. Mice were seeded with OT-I.CD45.1 T cells before i.n. infection with VSV-OVA. On day 20 p.i., OT-I cells were recovered from the brain and spleen of mice, sorted into CD103+ and CD103−, and adoptively transferred into naive recipient mice that were challenged with VSV-OVA (i.n.). (A) Representative flow cytometry profile of brain on day 20 p.i. demonstrating the OT-I.CD103+ and OT-I.CD103− subsets. (B) Schematic diagram representing the experimental setup. (C) The proportion of OT-I of the total CD8+ T cell population in the brain and spleen on day 8 p.i. after i.v. transfer of memory T cells. Data represent the mean + SEM (n = 3 to 5), and data are pooled from two independent experiments. (D) Mice were seeded with either bim−/− OT-I.CD45.1 or wild-type (wt) OT-I T cells before i.n. infection with VSV-OVA. On day 20 p.i., OT-I cells were recovered from the brain and spleen of mice, sorted into CD103+ and CD103−, and adoptively transferred (i.v.) into naive recipient mice that were challenged with VSV-OVA (i.n.). Depicted is the proportion of OT-I of the total CD8+ T cell population in the spleen on day 8 p.i. Data represent the mean + SEM (n = 4–8), and data are pooled from two independent experiments. (E) As described for (C) except memory T cells were transferred intracranially prior to i.n. challenge with VSV-OVA.

We have previously shown that brain CD103+ Trm cells undergo poor recall expansion after dissociation from the brain tissue. Specifically, when we sorted CD103+ and CD103− OT-I CD8+ T cells from the brains and spleens of mice on day 20 after i.n. infection with VSV-OVA and adoptively transferred these T cells i.v. into naive recipient mice and subsequently challenged these animals with VSV-OVA, we observed that all memory populations expanded in response to secondary challenge except the brain resident CD103+ memory T cells (8) (Fig. 1B, 1C). Delaying isolation of the spleen and lung memory T cells until a later memory time point (day 50 p.i.) to allow further maturation of the memory cells failed to restore the proliferation potential of brain CD103+ Trm cells (Supplemental Fig. 1A). The lack of recall expansion by the CD103+ memory T cell population after isolation and adoptive transfer may reflect a defect in survival after tissue dissociation. It is noteworthy that we could not rescue the inability of brain CD103+ memory T cells to expand when we repeated the experiments described earlier using OT-I cells deficient in the proapoptotic Bcl-2 family member Bim (OT-I.bim−/−) (Fig. 1D), which is known to be critical for controlling normal homeostasis of memory T cells (20). This suggests that if the inability of brain CD103+ memory T cells to mount a secondary recall response is due to a defect in survival after dissociation from the tissue, then this is a bim-independent death.

We next sought to determine if we could enhance the recall response of brain CD103+ memory T cells by reintroducing them back into the microenvironment of the brain. To do this, we sorted CD103+ and CD103− OT-I CD8+ T cells from the brains and spleens of mice on day 20 p.i. with VSV-OVA and adoptively transferred low numbers of these memory T cells directly into the brain (i.c.) of naive recipient mice. We subsequently challenged these animals with VSV-OVA. Even when reseeded back into the brain tissue, brain CD103+ memory T cells failed to mount a recall response (Fig. 1E). This is in contrast to both CD103− brain and splenic memory T cells, which underwent expansion. Brain CD103+ Trm cells also displayed impaired recall expansion when stimulated ex vivo. We sorted CD103+ and CD103− OT-I CD8+ T cells from the brains and spleens of mice on day 20 after i.n. infection with VSV-OVA. These cells were cultured in vitro with SIINFEKL peptide-loaded splenocytes. We observed that all memory populations expanded in response to ex vivo stimulation except the brain resident CD103+ memory T cells (Supplemental Fig. 1B). Cumulatively, these data demonstrate that brain CD103+ memory T cells are functionally distinct from brain CD103− and splenic CD103− memory T cell populations.

Brain memory T cells do not depend on the same survival factors as circulating memory T cells both in vitro and in vivo

We previously reported that memory CD8 T cells isolated from the brain die rapidly in vitro (8). We wished to determine whether the addition of cytokines known to be important in memory T cell survival could improve ex vivo survival of these tissue-residing memory T cells. CD103+ and CD103− OT-I cells were sorted from the brain and spleen of mice on day 20 postinfection with VSV-OVA. The cells were cultured in vitro in the presence of IL-7 or IL-15, and survival was assessed 18 h later by annexin V and 7-aminoactinomycin D (7-AAD) staining. Although IL-7 and IL-15 greatly enhanced the survival of splenic memory T cells, neither cytokine rescued either brain memory T cell population (Fig. 2A).

FIGURE 2.
FIGURE 2.

Brain memory T cells do not depend on the same survival factors as circulating memory T cells in vitro or in vivo. (A) Mice were seeded with OT-I.CD45.1 T cells before i.n. infection with VSV-OVA. On day 20 p.i., memory OT-I.CD103+ and CD103− cells were sorted from the brain and spleen of mice and cultured in vitro overnight in the presence of IL-7 or IL-15. Cells were stained with annexin V and 7-AAD, and the percentage of dead cells (annexin V+/− and 7-AAD+) was determined by flow cytometry. (B) Mice (either B6 or MHC class II KO) were seeded with OT-I.CD45.1 T cells before i.n. infection with VSV-OVA. Graphs depict the percentage or number of OT-I cells of the total CD8 T cell population in the spleen, lymph node (LN), and brain on day 60 p.i. Bars represent the mean + SEM (n = 9–17). Data are pooled from four independent experiments. **p < 0.005 (Student t test).

Memory T cells within the brain also do not appear to rely on the same survival factors as splenic memory T cells in vivo. We demonstrated this by monitoring the persistence of memory CD8 T cells within the brain of MHC class II-deficient mice. It has been previously reported that memory CD8 T cells undergo a gradual decay in MHC class II-deficient mice (21). To assess whether brain resident memory T cells were vulnerable to a similar decay, we adoptively transferred naive OT-I cells into either B6 or MHC class II-deficient mice prior to i.n. infection with VSV-OVA. Analysis of the brain, LN, and spleen of these mice on day 60 p.i. revealed that although memory OT-I within the LN and spleen from MHC class II KO mice underwent significant decay (Fig. 2B, 2C), consistent with the original studies (21) we observed no difference in the number of memory T cells within the brain when comparing B6 to MHC class II-deficient mice (Fig. 2D). Thus, brain memory T cells and circulating memory T cells do not depend on the same survival factors for their long-term maintenance. Furthermore, these data further support the notion that the memory T cell population within the brain exists independently of the circulating memory T cell pool because if this population was continually supplemented by circulating memory T cells, then we would also expect a decay in brain memory T cell numbers mirroring the events within the circulation.

Brain Trm cells provide protection during a localized infection

Brain CD103+ Trm cells exhibit survival defects ex vivo although when left in situ, they do provide an overt advantage during a localized secondary infection. To demonstrate this, we took advantage of the fact that only local virus infection of the brain results in the generation of CD103+ Trm cells. Mice seeded with low numbers of OT-I.CD45.1 T cells were infected either i.n. or i.p. with VSV-OVA. Irrespective of the route of infection, we observed a similar-sized memory OT-I response in the LN and spleen (Fig. 3A, 3B). Unlike i.n. administration of VSV-OVA, i.p. infection does not result in virus infection of the brain (Supplemental Fig. 2). Analysis of the OT-I cell population within the brains of mice infected with VSV-OVA either i.p. or i.n. 20 d earlier shows that in both cases there are approximately equivalent numbers of CD103− OT-I cells. However, only after i.n. immunization, which results in a virus infection of the brain, did we observe the development and persistence of CD103+ memory OT-I cells (Fig. 3C). In summary, this model provides us with a system where we have equivalent circulating memory T cell populations in the LN and spleen and similar numbers of brain CD103− memory T cells. The only difference between the two cohorts of mice is the presence of CD103+ memory OT-I cells in the animals that had been primed intranasally.

FIGURE 3.
FIGURE 3.

Resident memory T cells within the brain can provide protection against local infection. Mice were seeded with OT-I.CD45.1 T cells before i.n. or i.p. infection with VSV-OVA. On day 20 p.i., mice were challenged intracranially with LM-OVA. The proportion of OT-I T cells of the total CD8+ T cell population in the (A) spleen and (B) lymph node and (C) the number of OT-I CD8 T cells in the brain on day 20 p.i. Bars represent the mean + SEM (n = 6). Data are representative of two independent experiments. (D) Survival (measured as a loss of >20% of starting weight) of animals primed either i.p. or i.n. with VSV-OVA and challenged i.c. with LM-OVA. Shown is the mean + SEM (n = 15). Data are pooled from three independent experiments.

Mice primed 20 d earlier with VSV-OVA via either the i.p. or i.n. route were challenged intracranially with a recombinant Listeria that expresses OVA (LM-OVA) and were monitored for survival (Fig. 3D). We observed 100% survival among animals primed with VSV-OVA i.n. and therefore containing brain CD103+ memory T cells. In contrast, by day 3 after challenge with LM-OVA, 40% of mice primed via the i.p. route, and therefore lacking brain CD103+ memory OT-I T cells, had succumbed to infection. This protection is not merely due to presence of innate immune mechanisms present in the brain of mice primed i.n. with VSV-OVA, as when we intracranially challenge these mice with a wild-type Listeria (not expressing OVA) we failed to observe any protection (Supplemental Fig. 3). Thus, brain CD103+ memory T cells are functional in situ and provide enhanced protection during a localized infection.

Gene expression profile analysis of brain CD8+ Trm cells

To characterize brain CD103+ Trm cells further, we performed gene array analysis. To define the molecular signature of CD103+ Trm cells, we compared the transcriptomes of CD103+ and CD103− OT-I T cells sorted from the brain to CD103− memory OT-T cells isolated from the spleen of mice 20 d after i.n. infection with VSV-OVA. Note that there were too few CD103+ memory cells in spleen to include in the analysis. An example of pre-sort and post-sort analysis of purified cell subsets is shown in Supplemental Fig. 4.

Multidimensional scaling (MDS) was used to display the leading fold-changes between the expression profiles, demonstrating that the three cell populations have distinct profiles and that brain CD103+ OT-I cells are most similar to brain CD103− cells (Fig. 4A). Furthermore, when comparing the brain populations to the splenic memory T cell pool, it is the brain CD103− OT-I cells that share more genetic similarity to the splenic memory T cell population.

FIGURE 4.
FIGURE 4.

Brain resident memory T cells have a different molecular signature compared with circulating memory T cells. (A) An MDS plot depicting the relationship between brain CD103+, brain CD103−, and spleen CD103− OT-I T cell populations. Distances between samples represent leading fold change, the average log2-fold change between the 500 genes with largest differences. (B) Number of genes differentially expressed between brain resident and splenic memory T cells (FDR < 0.05). (C) Heat map of expression profiles (normalized log2 intensities) of brain OT-I.CD103+, brain OT-I CD103−, and spleen OT-I.CD103− memory T cells. Heat map shows the 50 genes with the most significant difference between the cell populations.

Although the two memory OT-I populations isolated from the brain are closely related, their genetic profiles indicate that they do represent distinct cell populations. We find that there are a total of 490 genes that are significantly differentially expressed (FDR < 0.05) when comparing the brain CD103+ to brain CD103− memory OT-I cells (Fig. 4B). There were more differences in gene expression comparing either CD103− or CD103+ brain OT-I to the splenic memory T cell population with 3941 and 5411 genes being upregulated or downregulated, respectively (Fig. 4B).

Fig. 4C shows a heat-map analysis of the 50 most differentially expressed genes when comparing brain CD103+, brain CD103−, and spleen CD103− memory OT-I cells. These data further highlight that the gene expression profiles of brain CD103+ and CD103− memory T cells are closely related. However, these two brain-derived subsets were markedly different from splenic memory T cells in terms of their gene expression profile. A list of the top 10 most differentially expressed genes when comparing the three subsets is presented in Table I, and Table II presents a list of genes that are significantly differentially expressed between the three cell subsets and grouped into immune system-related categories of interest.

View this table:
Table I. Fold changes (log2) and p values for the top 10 differentially expressed genes for each subtype comparison
View this table:
Table II. Fold changes (log2) for differentially expressed genes for each subtype comparison

Brain CD103+ T cells express genes characteristic of activated effector cells, including granzyme B and IL-2Ra at higher levels than spleen memory cells. Notably, two inhibitory receptors in the CD28:CTLA-4 family (22) (CTLA-4 and PD-1) are upregulated in brain CD103+ Trm cells compared with brain CD103− and splenic CD103− memory T cells. Furthermore, transcripts encoding key regulators of T cell differentiation such as eomesodermin (eomes), T cell factor 1 (Tcf-1), lef1, and T-bet are downregulated in CD103+ Trm cells compared with the other cell subsets. Brain CD103+ Trm cells also express the highest levels among all the subsets of several IFN-stimulated genes including IFN-induced transmembrane protein 3 (IFITM3) in addition to Irf4 and Isg20.

Both CD103+ and CD103− memory T cell populations isolated from the brain share similar expression of several chemokine and cytokine receptors. Fig. 5 shows a heat-map analysis of genes that are similarly expressed between the memory T cells isolated from the brain but which differ from the splenic memory T cells. In comparison with the splenic memory T cell pool, the memory T cell isolated from the brain displayed upregulation of several chemokine genes including CCL3, CXCL10, CCL4 and downregulation of others including CX3Cr1 and CCL9. It is noteworthy both CXCL10 (23) and CCL3 (24) have been previously linked to the recruitment of T cells into the brain. There are also several migration molecules that are differentially expressed between the two brain populations (Fig. 6). These include sphingosine-1-phosphate receptor 1 (S1P1R), CCR7, and CXCR4. Notably, S1P1R is involved in regulation of T cell egress from lymph nodes. Expression of CD69 has been shown to suppress the function of S1P1R and thereby reduces T cell migration out of the lymph node in response to an S1P gradient (25). CD69 expression has been documented on Trm cells isolated from different tissues (4, 13). Thus, in addition to a role in circulating T cell egress, S1P1R also may be involved in tissue egress as suggested by earlier reports (26).

FIGURE 5.
FIGURE 5.

Heat map of brain memory OT-I signature genes. Microarray analysis of brain OT-I.CD103+, brain OT-I CD103−, and spleen OT-I.CD103− memory T cells presented as a heat map of the 50 genes that have the greatest difference in expression when comparing brain (both CD103+ and CD103−) to spleen populations (presented as normalized log2 intensities).

FIGURE 6.
FIGURE 6.

Gene expression profile comparing brain CD103+ and brain CD103− resident memory T cells. Microarray analysis of brain OT-I.CD103+ and brain OT-I CD103− memory T cells presented as a heat map of the 50 genes with the greatest difference in expression (presented as normalized log2 intensities).

These array data indicate that brain CD103+ Trm cells are a distinct memory T cell population displaying a unique molecular signature.

Discussion

Memory CD103+ CD8 T cells that persist within the brain after an acute virus infection are bona fide Trm cells. We base this classification on several functional and phenotypical characteristics including i) they are a self-sustaining T cell population that persists independently of the circulating memory T cell pool; ii) they are resident within the tissue and do not recirculate; iii) they express the integrin CD103; and iv) they do not survive well after dissociation from the tissue (8). However, to date there was no molecular genetic profile defining these peripherally deposited memory T cells. We have now determined the molecular signature of a Trm cell population.

Genetic profiling revealed that CD103+ Trm cells express low levels of the transcription factors Tcf-1 and eomes in comparison with the other memory T cell subsets analyzed. Both transcription factors have been demonstrated to be important in differentiation and persistence of memory CD8+ T cells (27, 28). Zhou et al. (27) show that Tcf-1 deficiency impaired central memory T cell differentiation, and Tcf-1–deficient memory T cells were progressively lost over time. This memory T cell decay was most striking when analyzing memory T cells within the spleen, and, notably, when other organs were assessed (i.e., liver and lung), the decay was not as pronounced. This implies that Tcf-1 may only be important in the persistence of circulating memory T cells and not memory T cells lodged within peripheral tissues. It is noteworthy that Zhou et al. (27) also performed a transcriptome analysis of Tcf1-deficient memory T cells. They show that Tcf1-deficient T cells express lower levels of eomes, CCR7, and SELL and higher levels of granzyme A and B compared with wild-type memory T cells. Notably, brain CD103+ memory T cells express low levels of Tcf1 and the subsequent genetic profile associated with Tcf-1 deficiency including low levels of eomes, CCR7, SELL, and high levels of granzyme A and B. Furthermore, studies by Jeannet et al. (29) demonstrate that Tcf-1–deficient memory T cells are impaired in their ability to expand upon secondary challenge. Brain CD103+ T cells fail to undergo recall expansion after dissociation from the tissue in which they reside, and this lack of expansion may be associated with the Tcf-1 deficiency these cells display.

Our microarray analysis reveals that brain CD103+ Trm cells express high levels of several inhibitory receptors including CTLA-4 and PD-1 (30). It is interesting to speculate that expression of these inhibitory receptors by CD103+ Trm cells is in an effort to maintain peripheral tolerance. Trm cells are a highly sensitive, activated T cell population that resides within a variety of peripheral tissues. Expression of these inhibitory receptors may serve as a means to prevent this memory T cell population from accidentally being activated and unnecessarily attacking self. Brain CD103+ Trm cells are not exhausted or senescent, and in this study we show that during a local bacterial infection, these cells are able to provide substantial protection. Furthermore, it has been shown that brain CD103+ Trm cells can make cytokines and are cytotoxic (8). Hence, during a localized infection, Trm cells must be released from this inhibitory state as these cells are clearly functional during a secondary bout of infection.

Brain CD103+ Trm cells express elevated levels of the anti-viral protein IFITM3 (31). The constitutive expression of anti-viral proteins may protect Trm from pathogens that they will undoubtedly encounter as a consequence of being situated within the peripheral tissue. Resistance to virus infection is an extremely beneficial attribute for a memory T cell population localized to common portals of pathogen entry to possess. This finding further supports the notion that Trm cells develop characteristics that allow them to better survive and function within their local environment. It remains to be determined whether Trm cells in other tissues express a similar signature to that of brain Trm cells or whether Trm cells in different tissue microenvironments express unique genetic profiles.

We have previously reported that the brain CD103+ Trm cells represent a T cell pool that has encountered cognate Ag locally within the brain microenvironment, most likely during the acute stages of the infection. This local Ag presentation promoted CD103 expression and the retention of these memory T cells within the brain tissue (8). This is in contrast to the CD103− memory T cell population, which we suspect represents a T cell population that was recruited to the brain during the acute infection but fails to attain local Ag stimulation. These CD103− memory T cells do persist within the brain tissue, albeit poorly compared with their CD103+-expressing counterparts. Functional assays highlight further differences between these two cell populations with brain CD103− memory T cells undergoing better ex vivo recall expansion in comparison with brain CD103+ Trm cells (8). We have now identified several hundred genes that are differentially expressed between brain CD103+ and brain CD103− memory T cells. Hence, this local “programming” of Trm cells not only influences CD103 expression and thus T cell retention but also initiates the expression of a series of genes that likely influence T cell survival, function, and maintenance within the tissue microenvironment. These data highlight that there is vast complexity within the memory T cell pool, and even memory T cells occupying the same niche have strikingly different genetic profiles.

Gene expression profile analysis indicates that CD103+ Trm cells are a distinct memory T cell population displaying a unique molecular signature, which likely results in optimal survival and function within their local environment. In summary, these studies provide a base work with which to begin dissection of the factors that influence Trm cell function, survival, and maintenance. This knowledge will ultimately open the way for novel vaccination strategies that promote Trm cell formation.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

This work benefited from data assembled by the ImmGen consortium (32).

Footnotes

  • This work was supported by the Howard Hughes Medical Institute and by National Institutes of Health Grant U19 AI083019 (to M.J.B.). L.M.W. is supported by an Overseas Biomedical Fellowship from the National Health and Medical Research Council of Australia.

  • The data sets presented in this article have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE39152.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    7-AAD
    7-aminoactinomycin D
    eomes
    eomesodermin
    FDR
    false discovery rate
    i.c.
    intracranially
    i.n.
    intranasal(ly)
    IFITM3
    IFN-induced transmembrane protein 3
    LN
    lymph node
    MDS
    multidimensional scaling
    p.i.
    postinfection
    S1P1R
    sphingosine-1-phosphate receptor 1
    Tcf-1
    T cell factor 1
    Tcm
    central memory
    Tem
    effector memory
    Trm
    tissue resident memory
    VSV
    vesicular stomatitis virus.

  • Received May 8, 2012.
  • Accepted July 29, 2012.

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