In the Right Place at the Right Time: Trm Cells in Immunity to Cancer

Abstract

Tissue resident memory (Trm) cells have recently emerged as essential components of the immune response to cancer. Here we highlight new studies that demonstrate how CD8⁺ Trm cells are ideally suited to accumulate in tumors and associated tissues, to recognize a wide range of tumor antigens, and to persist as durable memory. We discuss compelling evidence that Trm cells maintain potent recall function and serve as principal mediators of immune checkpoint blockade therapeutic efficacy in patients. Finally, we propose that Trm and circulating memory T cell compartments together form a formidable barrier against metastatic cancer. These studies affirm Trm cells as potent, durable, and necessary mediators of cancer immunity.

Introduction

Trm cells are a non-recirculating memory T cell lineage that resides durably in tissues [1,2]. Originally identified for their potency against viral reinfection [3], Trm cells have more recently become recognized for their role in opposing cancer [4–7]. Trm cells are classically defined by their expression of tissue retention markers CD103 and CD69 [1,8] together with a lack of egress-associated molecules including CD62L, CCR7, S1PR1 [2]. Expression of CXCR6, FABP, PD-1, CTLA-4 and CD39 also identifies Trm populations in normal tissues and in tumors, although expression of these molecules and their isoforms can vary in different tissues [9–12]. Transcriptionally, Trm cells in cancer are also defined by high expression of ZFP683 (HOBIT), RUNX3, FOS, BHLHE40 and NR4A-family members, as well as low KLF2 and EOMES expression [2,5,13–16]. Over the past several years, Trm cells with these features have been identified infiltrating a broad range of solid tumors [11,17].

Here we highlight recent studies that demonstrate how CD8⁺ Trm cells are ideally suited to form and persist in tumors and associated tissues, to maintain effector function, and to respond to immune checkpoint blockade (ICB) therapy. These studies elevate Trm cells as critical players in the anti-tumor response, revealing their role as broadly distributed mediators of cancer immunity.

The right place: Trm cells in tissues and tumors

Recent studies have shown that tumor growth alone is sufficient for tumor Ag-specific Trm generation in tumors and across peripheral tissues [18]. However, robust protective Trm populations can also be induced therapeutically. Tissue localized [19] and systemic vaccinations [20] elicit Trm responses, which can be further augmented by adjuvant treatments such as anti-CTLA-4 [18,20]. Our studies showed that depletion of regulatory T cells is a key driver of melanoma-specific Trm generation in tumor-bearing hosts, inducing transcriptionally distinct Trm populations across skin, lungs, liver, and lymph nodes (LNs) [21]. Thus, Trm cells can be generated within tumors and across tissues in response to tumor growth and therapeutic interventions (Fig. 1A).

Figure 1. Overview of Trm cell persistence and function in immunity to cancer.

(A) Tumor-Ag-specific Trm populations form across tissues where tumors grow and metastasize, with clonal counterparts in the blood; “?” denotes the unknown relationship between these compartments. (B) Multiple mechanisms enforce Trm cell persistence, retention, and fitness in the tumor microenvironment (TME). (C) Immune checkpoint blockade (ICB) therapy promotes clonal expansion and effector molecule production by Trm cells in tumors.

In contrast to pre-malignant lesions [22], a wide range of human solid tumors contain tumor infiltrating lymphocyte (TIL) populations with pronounced Trm characteristics [11]. This is consistent with the findings that features inherent to the TME fortuitously promote Trm generation. TGF-β can be produced and used in an autocrine or paracrine fashion by Trm cells, with integrin αv-expressing cells affording TGF-β transactivation in skin [23]. Tumor cell-expressed αv integrin similarly supports Trm differentiation [24]. Accordingly, alterations in the microbiome that impair TGF-β production in tumors were shown to reduce Trm formation [25]. Effects of TGF-β are highly tissue dependent [26] and alone insufficient for the full Trm transcriptional program [27], although human T cells cultured with TGF-β under hypoxic conditions expressed prominent Trm characteristics [28]. This is congruent with findings that T cell-intrinsic hypoxia-inducible factor (HIF) activity promotes the generation of cytolytic Trm-like (CD103⁺CD69⁺) TILs in mouse melanoma [29]. These results have important therapeutic implications, as knockout of a HIF negative regulator improved anti-tumor function and Trm formation by chimeric antigen receptor (CAR)-T cells [29].

CXCR6 expression has also emerged as a cell-intrinsic mediator of Trm retention and function in the TME. In human ovarian cancer, CD8⁺CD103⁺ Trm-like TILs express high levels of CXCR6 [30,31], and CXCR6 deficiency impairs intratumoral T cell retention, anti-tumor function, and responsiveness to anti-PD-1 therapy [30,32]. Vaccine-induced CXCR6-deficient T cells are similarly impaired in their recruitment to tumors [33]. Mechanistically, we showed that CXCR6 mediates melanoma-specific Trm interactions with CXCL16-expressing dendritic cells (DCs) and is required for Trm maintenance and associated melanoma protection in the skin [34]. CXCR6-CXCL16 interactions also enable IL-15 trans-presentation by DCs to CD8 T cells in tumors [35] allowing delivery of survival signals for T cell maintenance. The importance of Trm-DC interactions is underscored by findings that DCs are associated with elevated Trm proportions in human cancers [36–38]. Thus, numerous features of the TME are conducive to Trm cell occupancy (Fig. 1B).

Tumor antigen specificity

Tumor-infiltrating Trm cells recognize a wide range of tumor antigens (Ags). In oral cancer and breast cancer, CD103⁺CD69⁺ TIL populations were found to recognize cancer testes Ags MAGE-1 and NY-ESO-1, respectively [39,40]. In melanoma survivors with vitiligo, Trm cells in tumors were clonally matched to those in skin, inferring specificity for melanoma/melanocyte shared Ags [15]. Using neo-Ag prediction for human non-small cell lung cancer (NSCLC), studies recently revealed that Trm populations recognize a range of mutation-associated neoantigens [14]. Moreover, Trm cells in tumors can recognize viral Ags, including tumor-associated viral Ags, such as Hepatitis B in hepatocellular carcinoma (HCC) [41]. However, TILs also recognize solid tumor-irrelevant viruses, such as influenza and Epstein bar virus [9,14,40,42], and such cells can be repurposed for immunotherapy [42]. The T cell exhaustion marker CD39 is useful for distinguishing tumor Ag-specific Trm cells from such bystander cells in tumors [9,14,40]. Compared with tumor specific T cells, bystanders also have lower expression of Trm markers [14,40], suggesting that the Trm phenotype and transcriptional state is enforced by Ag recognition in the TME. Whereas persistent TCR signaling is dispensable for Trm maintenance in viral infection models [43,44], its role in maintaining Trm responses to cancer remains unknown.

Transcriptional heterogeneity and markers of exhaustion

In accordance with ongoing TCR engagement, CD8 Trm cells in tumors often express markers of exhaustion/activation. In NSCLC, CD103⁺CD8⁺ TILs express more PD-1, TIM-3, and CD39 than their CD103-negative counterparts [45]. However, Trm cells are transcriptionally heterogeneous within tumors and across human cancer types. Single cell RNA sequencing of CD8 T cells in HCC revealed five separate Trm-like clusters, with only a minor subset expressing exhaustion markers PDCD1, HAVCR2 (TIM-3), and ENTPD1 (CD39) [41]. Similarly, Trm in melanoma formed three Trm-like sub-clusters one of which expressed negative checkpoints (PDCD1, CTLA4, and HAVCR2), and another of which was enriched for effector markers (GZMA, TNFA, and IFNG) [15]. A similar Trm cluster expressing IFNG and other effector cytokine transcripts was also identified within HBV Ag-specific T cells from HCC [41]. In oral head and neck squamous cell carcinoma (HNSCC), two major ITGAE (CD103) and ZFP638 (HOBIT) expressing Trm clusters were identified, only one of which expressed PDCD1 [39].

While it is tempting to use the above markers to infer cellular function or exhaustion, caution is needed. Indeed, studies using gene signatures derived from viral infection models revealed that TILs with features of exhaustion also co-express programs of activation [36]. While some studies show that Trm-like TILs can express the canonical exhaustion marker TOX [15,40], others show that TOX is lower on Trm cells than their terminally exhausted (Tex) counterparts [46]. TIL clonotype and differentiation trajectory analyses show that Trm cells exist in a functional effector state but can also differentiate into TOX⁺ Tex populations [31]. This is consistent with a model in which Trm cells can adopt either functional or exhausted states in tumors.

Cytokine production, stemness, and persistence: hallmarks of resident memory in cancer

Recent ex vivo and prognostic studies have provided compelling evidence that CD8 Trm cells can mount functional recall responses against cancer. Luminal breast cancer-derived CD39⁺ Trm cells produced robust IFN-γ, TNF-α, and IL-2 upon restimulation [47]. Accordingly, restimulated CD103⁺ CD8 T cells from gastric cancer produced more IFN-γ and TNF-α than their CD103-negative counterparts [48]. In HCC, Trm-like cells were also clearly distinct from more exhausted TILs that produced lower levels of effector cytokines [49]. In line with superior cytokine production, Trm-associated phenotypes and gene signatures are associated with improved prognosis across a wide range of human cancers including bladder [50], liver [41], ovarian [31], melanoma [15,21,36], and breast [40,47]. In HNSCC, the presence of Trm in HPV-negative tumors rescues prognosis to levels observed for HPV-positive patients [51]. In melanoma, an IFNG-expressing Trm subset was more prognostic than a TOX-expressing Trm subcluster [15], consistent with findings that IFN-related gene signatures associate with improved prognosis and the presence of Trm cells [36,50]. Combined with prior studies in mouse models [52], these newer studies provide convincing evidence that Trm cells can serve as functional mediators of immunity in humans.

Stemness and durable persistence of the anti-tumor Trm compartment has now also been demonstrated. Whereas the stemness marker TCF-1 has not generally been considered a marker of Trm cells, a subset of sorted CD8⁺CD103⁺CD69⁺ TILs from ovarian cancer were found to express TCF-1/TCF7 as well as IL7R, and LEF1 [31]. Trajectory analysis suggested that these stem-like Trm cells give rise to effector-like Trm cells, with a stem-like Trm signature optimally predicting improved survival in ovarian cancer patients [31]. Such stemness may also explain how Trm clonotypes can persist. In melanoma long-term survivors, we observed that clonotypes identified in early tumor biopsies persisted for up to 9 years in skin, where they exhibited Trm transcriptional properties [15]. Indeed, the presence of “late memory” and “Trm” transcriptional signatures in melanoma patient specimens were most strongly associated with patient survival [36]. Collectively, these studies provide compelling evidence that Trm cells are a functional memory compartment that can confer durable immunity in cancer patients.

The right time: Trm cell function during immune checkpoint blockade (ICB) therapy

Despite their prognostic value, the presence of Trm cells in progressively growing tumors infers their susceptibility to immunosuppressive factors in the TME. Indeed, PD-1/PD-L1 blocking antibodies can act both directly and indirectly to augment Trm cell function. Proliferation and cytokine production by triple negative breast cancer (TNBC)-derived CD39⁺CD8 Trm cells was rescued by the addition of anti-PD1 during ex vivo restimulation [40]. Cytokine production from gastric cancer-derived CD103⁺CD8⁺ T cells was also greatly augmented by anti-PD-L1 treatment in vivo. Interestingly, anti-PD-L1 decreased FABP4/5 expression by gastric tumor cells while simultaneously increasing its expression on Trm cells, thus favoring fatty acid uptake by Trm cells in the TME [48]. Recently it was shown that anti-PD-1/CTLA-4 dual checkpoint blockade preferentially enhances the expansion and killing capacity of CD8⁺ Trm-like cells, but not their exhausted counterparts [46]. Thus negative checkpoint blockade appears to preferentially rescue Trm function in tumors.

These studies are consistent with findings that Trm-like TIL presence is predictive of patient response to immunotherapy. A large study of NSCLC and bladder cancer patients showed that CD103⁺ Trm cells in tumors predicted anti-PD-L1 treatment outcomes [53]. Similarly, high CD39⁺CD103⁺PD1⁺ Trm-like TILs at baseline predicted significantly lower risk of recurrence in the adjuvant immunotherapy setting for melanoma [54]. Even more compelling are findings of preferential Trm cell expansion in patients who respond to anti-PD-1 therapy. In melanoma and oral cancer patients receiving anti-PD-1, Trm-like TILs expanded during treatment [36,39]. In contrast, ICB treatment did not expand non-Trm TILs, suggesting that Trm cells are uniquely poised to respond to ICB therapy [39]. These studies convincingly demonstrate that Trm cells mount recall responses during ICB therapy (Fig. 1C).

Trm cells as a component of systemic memory to cancer

While Trm cell localization across tumors and tissues positions them to respond to disseminated cancers, circulating memory (Tcirm) cells also play a key role in this process. In contrast to melanoma protection in the skin and lymph nodes, we found that protection against metastatic-like melanoma in the lungs is afforded by Tcirm cells [21]. Interestingly, studies in patients indicate a link between Trm and Tcirm populations, with numerous clonotypes shared between these compartments [15,39]. Although the relationship between these populations is not yet completely understood, it is known that circulating memory T cells can differentiate into Trm cells in mouse models [6,55]. In patients, neoadjuvant ICB treatment resulted in the expansion of tumor Ag-specific clonotypes in blood [14,39], and these cells shared transcriptional features of TILs (e.g. CXCR6) [39]. Moreover, in gastric cancer patients, ICB treatment induced expression of CD103 on Tcirm cells in blood [56]. It is intriguing to speculate that tumor-specific Trm cells from tumors or tissues might replenish the circulating memory response, particularly considering the recent discovery of such “outside-in” Trm cell responses to peripheral viral reinfection [44,57].

Importantly, the identification of Trm clonal matches across tumor, lymphoid tissues, blood and normal tissues, has revealed that Trm populations form truly systemic memory against cancer. Human melanoma and breast cancer-involved LNs were recently shown to contain Trm populations [21,36,40]. In mice, we demonstrated that Trm cells are protective against melanoma rechallenge in tumor-draining LNs [21]. Trm clonotypes from tumors have similarly been identified across patient-matched normal lung [14,45], healthy liver [41,49], and skin [15]. Together with Tcirm, such broadly disseminated and functional Trm responses constitute a formidable barrier against metastatic cancer.

Conclusions

Trm cells thrive in tissues and tumors as functional memory T cells that are poised for response to ICB therapy (Fig. 1). Additional work is needed to better understand Trm recall capacity, locational dynamics, and plasticity in relation to Tcirm populations. Further study of Trm features within CD4 T cell compartments is also warranted. Therapeutic generation of Trm populations for adoptive therapy is currently in its infancy. However, based on this compelling evidence of Trm function against cancer, future advances must focus on generating these potent, tissue-wide memory T cell compartments.

HIGHLIGHTS.

• Trm cells are ideally suited to persist in solid tumors and associated tissues

• Trm cells recognize a wide range of tumor antigens

• Heterogenous subpopulations of Trm cells occupy human tumors

• Tumor-specific Trm cells are functional and long-lived

• Trm cells are uniquely poised to respond to immune checkpoint blockade therapy