Anti-viral protective capacity of Tissue Resident Memory T cells

Abstract

It has become increasingly clear that a subset of T cells which persist at diverse infection sites, known as tissue-resident memory T cells (T_RM), can mediate efficacious protective immunity against many types of viral infections. Recent studies have elucidated the mechanisms by which T_RM coordinate enhanced viral clearance in different sites through rapid production of effector cytokines and cytolytic mediators, in situ expansion, differentiation to circulating effector cells, and immune cell recruitment. This tissue localized response also includes enhancement at the local lymphoid sites which contribute to fortifying T_RM-mediated protection. Understanding how these responses occur in a tissue-wide context will provide key insights for development of vaccines and therapeutics.

Introduction

Tissue resident memory T cells (T_RM) have emerged in recent years as a key mediator of immune protection against a wide range of microbial threats. T_RM are a transcriptionally distinct subset of memory T cells and reside in nearly all tissues examined in mice and humans such as barrier sites (lung, skin, intestines, and female reproductive tract (FRT)), primary and secondary lymphoid organs, and other non-lymphoid tissue (brain, liver, pancreas, kidneys, salivary glands). In contrast to effector memory T cells (T_EM) and central memory T cells (T_CM), which circulate between blood, peripheral tissue, and/or secondary lymphoid organs (SLO), T_RM are functionally characterized as a non-circulating subset retained in specific tissue sites [1,2]. The combined function of circulating and tissue-resident T cells coordinate for immune-mediated protection, with additional subsets such as CX3CR1^int peripheral memory T cells (T_pm) contributing to peripheral non-lymphoid tissue surveillance [3].

Following infection or site-specific immunization, T_RM are generated in diverse tissue sites and particularly in mucosal and barrier sites [4], reflecting the extensive exposure to different types of pathogens throughout one’s life. T_RM in mice and humans are typically identified by a distinct transcriptional profile and expression of surface proteins such as CD69, CD103, CD101, CD49a and CXCR6 [2,5,6]. Expression of these T_RM signature markers can vary between sites and may control differential T_RM localization within a tissue site such as the lung [7,8]. Such heterogeneity among T_RM suggests that they are adapted to their tissue site either through their residence in specific tissue niches or their interaction with epithelial cells and other resident immune cells such as macrophages. The maintenance and function of T_RM may therefore differ between sites.

Both CD4⁺ and CD8⁺ T_RM can be generated following infection and have been shown in animal models to mediate efficacious protection in multiple types of virus infection models including respiratory, mucosal, cutaneous and systemic viruses (Table 1). While the presence of T_RM in these models is sufficient for protection, the mechanisms by which T_RM direct protective responses is less clear. Previous work has demonstrated the importance of effector cytokines during rapid in situ responses [9,10]; however, more recent studies have revealed the complexity of T_RM-mediated responses, including their role in tissue surveillance, in situ proliferation and expansion, differentiation and interactions with cells within tissue niches and neighboring lymphoid sites are critical for tissue-localized protection. In this review we discuss recent findings which advance our understanding of T_RM protective responses and their translation to human health and disease.

Table 1.

Tissue and viral infection models of CD4⁺ and CD8⁺TRM-mediated protection

Tissue Location	Subset	Viral infection model*
Lung	CD4	Influenza [33,51], Coronavirus [35]
	CD8	Influenza [28,34,52,53], RSV [54]
Skin	CD8	HSV [14,44] VACV [55,56]
FRT	CD4	HSV [46]
	CD8	HIV [57], HSV [58,59], VACV [9]
Brain	CD8	LCMV [31], VSV [30], WNV [60]
Salivary Gland	CD8	CMV [61]

^*RSV: respiratory syncytial virus; HSV: Herpes simplex virus; VACV: vaccinia virus; WNV, West Nile virus; VSV: Vesicular stomatitis virus

T_RM-mediated tissue surveillance

T_RM reside within tissue niches without recirculating into blood or lymph. Specific tissue retention of T_RM has been demonstrated in mouse models using parabiosis [11], protection from intravascular antibody labeling [12,13] and tissue transplantation [14]. In humans, long term retention of CD4⁺ and CD8⁺ T_RM in mucosal sites in vivo has recently been demonstrated in transplanted organs (lungs and intestines) [15–17]. Although T_RM remain in tissues, they can exhibit mobility within their tissue niche as a part of their sentinel function. T_RM motility has been found to be highly tissue dependent, in which differences in tissue architecture may account for discrepancies in local T_RM migratory patterns [18,19]. In the lung, the T_RM surface marker CD49a mediates T_RM motility and patrol function, which is essential for heterosubtypic immunity against influenza virus challenge [8]. CD8⁺ T_RM in the skin slowly (~1–2 μM/min) but efficiently crawl within the epidermis and exhibit dendritic morphologies in order to expand their patrol function while scanning for antigen [20,21]. In both the female reproductive tract (FRT) and skin, CD8⁺ T_RM motility was significantly reduced upon antigen stimulation [22,23]. In humans, ex vivo culture and imaging studies of skin tissue showed that like their murine counterparts, human CD8⁺ T_RM actively migrate within both dermal and epidermal skin layers and similarly arrest their motility upon antigen stimulation [24]. These studies indicate an active role for T_RM in tissue homeostasis.

Although T_RM patrol their tissue niche, they are thought to permanently reside within the tissue site without migration into blood or lymph. However, new analysis of human blood revealed the presence of CD4⁺ CD103⁺ memory T cells that were both functionally and transcriptionally similar to CD4⁺ CD103⁺ T_RM found in the skin [25]. Using a xenograft model of human skin transplanted onto murine hosts, human CD4⁺CD103⁺ cutaneous T_RM were able to reseed distal skin sites, suggesting that T_RM may exhibit some capacity for bloodborne migration [25]. Whether this property is specific to skin T_RM in humans remains to be determined as lung-derived donor T cells were not detected in the blood of lung transplant recipients, even at early times post-transplantation [15].

While T_RM emergence in circulation during steady state may be variable, activation of T_RM may drive their release into the periphery. Two recent studies in mice found that T_RM contributed significantly to the circulating T cell pool following virus challenge. Upon reactivation in VSV infection models, TCR transgenic OT-I CD8⁺ T_RM in lymphoid and peripheral sites differentiated into effector and T_CM cells re-entered the circulating pool [26] (Fig. 1). Moreover, these ex-T_RM circulating memory T cells retained a T_RM-like transcriptional signature, including expression of the T_RM-specific transcription factor Hobit, and could re-differentiate into T_RM upon appropriate cytokine stimulation [26,27]. Together, these findings indicate that T_RM may have the capacity to further differentiate into tissue homing effector or memory T cells.

Figure 1. Mechanisms for T_RM- mediated protection to secondary viral challenge.

T_RM mediate their protective capacity through a variety of mechanisms. (A) During maintenance, T_RM patrol their tissue niche until pathogen encounter, at which point (B) they rapidly release cytokines and cytolytic mediators for direct anti-viral effects on infected cells. (C) T_RM also undergo rapid proliferation in situ which amplifies their response. (D) T_RM both at the site of infection and in lymphoid sites can also differentiate into circulating memory T cells that enter the site of infection and may be predisposed to regenerate T_RM. (E) T_RM produce chemokines to further recruit circulating memory and effector T cells

T_RM –mediated effector responses

T_RM are situated at the site of infection and often near the cellular targets of specific viruses, such as the clustered positioning of influenza-specific lung T_RM close to airway epithelial cells [13]. In this way, T_RM are poised to rapidly respond to pathogens through direct release of cytotoxic mediators, cytokines, and chemokines (Fig. 1). T_RM-derived IFN-ɣ in particular has been shown to broadly enhance tissue tissue-wide antiviral responses such as the upregulation of type I IFN signaling pathway factors and enhanced leukocyte recruitment to the site of infection [9,10,28–30]. Murine infection models have demonstrated that protection by CD4⁺ and CD8⁺ lung T_RM and brain T_RM to local viral challenge was associated with enhanced production of IFN-ɣ [30–34]. Moreover, inhibiting IFN-ɣ signaling significantly impaired lung T_RM-mediated protection in coronavirus and influenza infection models [34,35]. Together, these studies indicate that IFN-γ can promote or facilitate site-specific protection by TRM in diverse sites.

Human T_RM are similarly enhanced in their production of pro-inflammatory cytokines and rapid IFN-γ production. Human lung CD4⁺ and CD8⁺ T_RM express elevated levels of preformed transcripts for IFN-ɣ and granzyme B, indicating a poised state for rapid recall responses [5,36]. Human brain CD69⁺ and CD103⁺ CD4⁺ and CD8⁺ T_RM also produce high levels of multiple pro-inflammatory cytokines, such as IFN-γ and TNF-α [37]. Actinomycin D- mediated inhibition of de novo transcription by human lung CD4⁺ CD103⁺ T_RM dramatically reduced IFN-ɣ production, indicating that a significant portion of superior T_RM cytokine production was due to a more accessible IFN-ɣ locus [38]. T_RM effector activity also strongly depends on its localization to a particular tissue niche. In mouse lungs, CD8⁺ T_RM localized to airways produced significant IFN-ɣ compared to their parenchymal counterparts [34]. Conversely, human skin CD49a⁺ CD8⁺T_RM localized to the epithelial layer exhibited enhanced IFN-ɣ production and high constitutive expression of perforin and granzyme B compared to CD49⁻ CD8⁺T_RM [39]. A recent study showed that clearance of HSV infection in human skin by TRM was associated with production of IFN-γ and granzyme B at the local site of viral reactivation [40]. Mathematical modeling of T cell-mediated protection against HSV in the skin revealed that both local production of cytokines and recruitment of peripheral T cells were likely required [40]. Together these mouse and human studies identify a role for in situ inflammatory function by TRM, but this may not be sufficient for full in situ clearance; other mechanisms involving recruitment and interaction with immune cells also play important roles.

In situ T_RM proliferation

In addition to direct production of effector cytokines and recruitment from the periphery, T_RM proliferation also contributes to the expanding immune effector pool in the tissue niche (Fig. 1). Recent studies have found that T_RM rapidly proliferate after reactivation in a variety of tissues, including the lung, skin, and FRT [22,23,41]. Using parabiotic mice, in situ cellular expansion after peptide and pathogen challenge was determined to be dominated by host cell responses, suggesting that circulating T cells were not significant contributors, but did not rule out rapid migration to the tissue by host T cells [22]. However, there is evidence in other studies that T cells from the circulation contribute to the generation of de novo T_RM. Repeated pathogen or antigen exposures result in net increases in T_RM numbers [42–44] with newly generated T_RM not necessarily derived from existing T_RM. In addition, a recent study of skin T_RM in a peptide challenge model did not find evidence to support in situ T_RM proliferation as the dominant source of T_RM expansion, as this increase in T_RM numbers post-challenge was eliminated by systemic anti-CD8 antibody treatment [43]. During heterosubtypic recall responses to influenza challenge in the lung, CD4⁺ and CD8⁺T_RM exhibit proliferative expansion and there was a significant increase in lung niche T cells that derived from T_RM and from newly generated effector cells primed in the lung-associated lymph node [45]. These findings suggest that tissue-localized immunity directed by T_RM may also have a significant circulating component from the associated lymphoid tissue.

T_RM interactions with the local tissue environment

Recent studies have highlighted the interactions of T_RM with other cells within the local tissue environment in order to promote protective responses. Tissue macrophages interact with both CD4⁺ and CD8⁺ T_RM to promote T_RM clustering and patrol function [46,47]. Depletion of macrophages disrupted T_RM migration in salivary glands, which follow the macrophage topology to migrate within the epithelium [47]. How T_RM are activated in situ, and by which tissue-localized accessory cells, has not yet been fully elucidated. In a murine model of influenza infection, CD8⁺lung T_RM were found to be rapidly primed by both hematopoietic and non-hematopoietic cells suggesting that in addition to professional antigen-presenting cells, epithelial and/or endothelial cells may also initiate TRM responses [48]. Dendritic cells in particular have been recently shown to enhance tissue-localized T cell responses. Primary influenza infection in mice triggers an increased number of conventional DCs to populate local lung-associated lymph nodes and these increased cDC numbers persist over months in vivo [45]. During a recall response to influenza challenge, cDC-mediated activation of T cells promotes rapid expansion and homing to the lung tissue niche, enhancing T_RM -mediated protection [45]. These findings suggest that the interactions of T_RM within the local environment, including the tissue-associated lymph nodes is crucial for optimizing protection.

The in situ function of T_RM involving expansion, recruitment and local effector function is essential for rapid pathogen clearance, but can also promote local tissue damage and immunopathology. T_RM appear to have intrinsic mechanisms for regulation. Inhibitory proteins including PD-1, CTLA-4, and Tim-3 are highly expressed in mouse and human T_RM [5,6] and may function to limit their effector and cytolytic activity. In a mouse model of persistent infection, PD-1 signaling by CD8⁺ T_RM was associated with increased control of persistent virus and lower inflammation [49]. In tissue sites which are particularly vulnerable to inflammation-mediated damage, endogenous T_RM express high levels of PD-1 and interact with PD-L1⁺ macrophages which can limit their functional potency in situ, as shown in a recent study in human pancreas [50]. It will be important to define the mechanisms by which T_RM are adapted to promote the appropriate balance of protection and immunoregulation which appears to be a universal hallmark of T_RM and associated with their long-term persistence in tissue sites.

Concluding Remarks

Tissue resident memory T cells are a protective correlate for site-specific viral infections in diverse tissues. As such, T_RM are important targets for generation in vaccines and for promoting immunity in ongoing infections. However, the different mechanisms for their protective responses are not fully elucidated and are important to define for targeting the pathways involved in promoting pathogen elimination, and preserving tissue integrity. Recent findings have shown that T_RM are versatile in their immune effector functions and also possess intrinsic regulation and inhibitory activity. T_RM can also proliferate in situ and in some cases, may contribute to the circulating T cell pool, further expanding upon the highly diverse repertoire of protective mechanisms employed by T_RM. In addition, T_RM can interact with other immune cells both within the tissue and promote enhanced communication with local lymphoid sites. Continued progress in our understanding of the mechanisms underlying their protective responses is critical to developing vaccines and treatments against current and future pathogenic threats.

Figure 2. Lung T cell-mediated recall responses involve in situ expansion and enhanced migration from lung-associated lymph nodes.

During a recall response, lung T cells rapidly expand in number in response to respiratory virus, which is due to in situ proliferation of T_RM as well as the rapid ingress of peripheral T cells that are rapidly activated in the local draining lymph node due to increased numbers of lymph node dendritic cells, leading to enhanced effector T cell generation and migration to the lung.