Roles of Virtual Memory T Cells in Diseases

Abstract

Memory T cells that mediate fast and effective protection against reinfections are usually generated upon recognition on foreign Ags. However, a “memory-like” T-cell population, termed virtual memory T (T_VM) cells that acquire a memory phenotype in the absence of foreign Ag, has been reported. Although, like innate cells, T_VM cells reportedly play a role in first-line defense to bacterial or viral infections, their protective or pathological roles in immune-related diseases are largely unknown. In this review, we discuss the current understanding of T_VM cells, focusing on their distinct characteristics, immunological properties, and roles in various immune-related diseases, such as infections and cancers.

INTRODUCTION

Classically, it was believed that CD8⁺ T cells comprise populations of naive CD8⁺ T (T_N) cells, effector T (T_EFF) cells, and memory T (T_MEM) cells, which remain quiescent while awaiting Ag-specific activation (1). A recent report described a CD8⁺ T-cell population in unmanipulated mice, which could rapidly respond to innate or TCR-mediated stimuli, and showed similar immunological characteristics with conventional T_MEM cells (2). Remarkably, these cells achieved “memory-like” phenotype, and responded rapidly as T_MEM cells, without activation by previously encountered Ags (3). Based on these characteristics, this subset of CD8⁺ T cells is considered “memory-like” and has been termed virtual memory (T_VM) cells (4). It was considered that microbiota-derived Ags might be responsible for T_VM-cell development, but this theory was disproved by the finding that T_VM cells exist at equal frequency in secondary lymphoid organs from both germ-free and feral mice (4,5).

T_VM cells express high levels of CD44, CD122, and Eomesodermin (Eomes), which are known to regulate the fate and function of CD8⁺ T_EFF and T_MEM cells (6) (Fig. 1). For many years, T_VM cells were misclassified as central memory T (T_CM) cells due to their high CD44 and CD62L expression levels, and the absence of specific markers to distinguish T_VM cells from T_MEM cells (7). T_VM cells rarely express CD49d, which is only upregulated in T_MEM cells after TCR stimulation by its cognate Ag (4). T_CM and T_VM can be distinguished based on CD49 level. Previous publications have described these memory-like cells using a variety of names—most commonly T_VM cells, memory precursor T (T_MP) cells, and innate memory T (T_IM) cells (2). Among them, T_IM cells develop from naïve CD8⁺ T cells in the thymus, abundant with IL-4 secreted by PLZF⁺ cells including αβ iNKT cells and γδ NKT cells (8,9,10). T_VM cells that originated from specific precursors in the thymus develop in the periphery and appear in mice shortly after birth (11). Since T_IM, T_VM, and T_MP cell populations cannot be separated phenotypically in the periphery, they are currently referred to as “virtual memory” cells from a functional point of view (3).

Figure 1. Characteristics of T_VM cells. T_VM cells originate from either IL-4 dependent T_IM cells or CD5^hiCD8⁺ T cells. T_VM cells maintain their population dependent on IL-15. With the high expression level of Eomes at the transcriptome level, T_VM cells highly express CD44, CD122, and CD5. However, CD49d, a marker for cognate Ag experience, is downregulated in T_VM cells. T_VM cells have various cytokine responsiveness. For example, IL-12/18 stimulates T_VM cells and triggers the production of proinflammatory cytokines including IFN-γ production, and IL-15 induce NKG2D-dependent innate-like cytotoxicity. T-bet and NKG2D levels varies between T_IM and T_VM.

DC, dendritic cell.

In this review, we present the recent knowledge regarding T_VM-cell populations, and discuss their distinct characteristics, immunological properties, and roles in various immune-related diseases.

CHARACTERISTICS OF T_VM CELLS

In mice, depending on the strain, different cytokines play crucial roles in the generation of T_VM cells. In BALB/c background mice, T_VM-cell development is absolutely dependent on IL-4, as shown by the finding that helminth infection does not induce an increase of T_VM cells in IL-4 knockout (KO) or IL-4R KO mice (12,13). On the other hand, helminth infection could increase the frequency of T_VM cells in C57BL/6 mice lacking IL-4 or IL-4R (12,14). It was demonstrated that T_VM-cell expansion during helminth infection in C57BL/6 mice was dependent on IL-15, and concluded that IL-4 was not a direct driver of T_VM cell proliferation in C57BL/6 mice with helminth infection (15). Unexpectedly, IL-4 exposure during development and homeostasis reduces CD8⁺ T cells inherent ability of CD8⁺ T cells to generate IFN-γ rapidly but increase a potent proliferative response to lymphocytic choriomeningitis virus (LCMV) infection in BALB/c mice (16). These findings imply that immune response to LCMV is induced by IL-4 conditioning, which also enhances the quantity but not the quality of memory-like CD8⁺ T cells (16).

Furthermore, the TCR repertoire of T_VM cells reveals a TCR bias (11,17,18). Supporting the notion that T_VM differentiation is TCR-dependent, T_VM cells reportedly exhibit elevated amounts of Nur 77 in humans and CD5 in mice, which are surrogate markers of TCR signal intensity and indicators of increased TCR self-reactivity (19,20). It is believed that high self-peptide reactivity during T_VM cell growth is responsible for the elevated cytokine sensitivity of T_VM cells in the blood, which is at least partly due to the expression of Eomes (3). A previous study demonstrated that increased TCR responsiveness to self-ligands leads to Eomes upregulation in T_VM cells during thymic maturation (11). Additionally, it has been shown that Eomes binds to the il2rb promoter, thereby activating it and increasing CD122 expression (21). Therefore, it appears that the elevated reactivity of self-peptide MHC in T_VM cells leads to upregulation of Eomes, which causes increased CD122 expression, driving the IL-15 sensitivity and dependency of T_VM cells. Type I IFN signaling also reportedly increases Eomes expression in T_VM cells (22). In fact, IFN signaling led to Eomes-dependent increases of both peripheral T_VM cells and thymic T_IM cells, while IFNAR^−/− mice exhibited drastically decreased T_VM cells (22).

T_VM cells are sensitive to cytokines including IL-12, IL15, and IL-18 (23,24) (Fig. 1). Previous reports showed that stimulation of T_VM cells with IL-12 and IL-18 can lead to IFN-γ production in an Ag-independent manner (23,25). As previously mentioned before, T_VM cells induce bystander activation in a manner dependent on IL-15 (23,26). When Ag-irrelevant T_VM cells having TCRs specific to irrelevant Ag were adoptively transferred into IL-15 KO mice and then challenged with ovalbumin-expressing Listeria monocytogenes (LM-OVA), the transferred T_VM cells displayed significantly lower expression levels of NKG2D, granzyme B, and IFN-γ, compared to the wild-type (WT) mice transferred with same cells (23).

T_VM cells express NK cell receptors (NKRs), and seem to constitute a cell subset that is functionally different from the CD8⁺ T cells that display NK cell markers with aging and during infection (23,27,28). Compared to NKR⁻CD8⁺ T cells, human CD8⁺ T cells that express NKRs (e.g., NKG2A, KIR2DL, and KIR3DL) produce lower effector cytokine levels in response to TCR-mediated stimulation (29). However, KIR/NKG2A⁺ CD8⁺ T cells can recognize MHC class I-deficient target cells, and trigger TCR-independent cytotoxicity via CD16 ligation and increased degranulation (30). Consequently, KIR/NKG2A⁺ T cells seem to exhibit decreased TCR-mediated responses, together with increased innate responsiveness on a functional level. Although NKG2D activity on T_MEM and T_VM cells is a sign of senescence and TCR-mediated malfunction, it paradoxically promotes improved innate response. Increased granzyme and perforin production following NKG2D interaction on memory phenotypic CD8⁺ T cells has been used to detect TCR-independent cytotoxicity (31,32). In cases where inhibitory Ly49 is expressed on memory phenotype T cells, this molecule reduces TCR-mediated T-cell activation without reducing responsiveness to IL-15 (33). Collectively, these results illustrate the reciprocal TCR-mediated and innate-like functionality of T cells.

ROLE OF T_VM CELLS IN AGING

Unlike T_N cells, T_VM cells in young mice have the ability to produce cytokines and proliferate rapidly upon TCR stimulation or cytokine stimulations (27). However, the proportion of dysfunctional T_VM cells increases with age (27). A study in young and aged germ-free C57BL/6 and BALB/c mice revealed that the frequency of peripheral T_VM cells increases with age, regardless of genetic background (5). Moreover, it has been suggested that various hygienic conditions, such as cohousing laboratory mice and feral mice, showed minimal effects on the T_VM cell numbers in the blood (5). These results imply that there is a common homeostatic mechanism exists during aging, which is independent of genetic background and commensal microbiota.

The long-term maintenance of T_VM cell function in aged nonimmunized mice is of interest to understand the characteristics of T_VM cells. A previous study demonstrated that T_VM cells from aged OT-I or WT mice exhibited a number of characteristics not seen in younger mice (34). Aged T_VM cells showed a robust response to homeostatic cytokines, but also a selective reduction of their ability to replicate in response to TCR signals. Notably, this impairment was found to be associated with increased apoptosis in response to peptide stimulation, but decreased apoptosis in response to homeostatic cytokines in aged T_VM cells compared to aged T_N cells (34). Interestingly, in contrast to the response in young mice, among aged mice, the de novo response to influenza virus was dominated by T_VM cells, consistent with the age-related increase of T_VM cells (35). Aging-related changes in T_VM-cell function and frequency may also be explained by increases of cytokine levels (including IL-6, IL-15, and IL-18) with age (36,37).

According to a previous study, helminth infection does not stimulate the proliferation of aged T_VM cells, which is likely at least partly because aged mice exhibit an impaired Th2-related immune response to helminth infection (38). They also showed that T_VM cells from aged mice display an intrinsic defect in cytokine sensing. These findings indicate that the absence of T_VM cell expansion following helminth infection is explained by defective intrinsic T_VM-cell cytokine responsiveness, in conjunction with dysregulated helminth infection responses in aged mice. Furthermore, it was discovered that while young T_N and T_VM cells showed extensive proliferation, aged T_VM cells, not aged T_N cells, showed a severely diminished proliferative capacity primarily due to decreased cell division upon CD3 stimulation (27). Upon evaluating the proliferation of young and aged T_N and T_VM cells in response to IL-15 stimulation, they observed vigorous proliferation of only the young and aged T_VM cells (27). This indicated the independent regulation of TCR- and cytokine-dependent proliferation responses in T_VM cells. Further experiments involving adoptive transfer in mice revealed that young T_N and T_VM cells transferred to an aged environment acquired a proliferative defect and reduced functionality. Moreover, when aged T_N and T_VM cells were transferred to a young environment, more T_VM cells were recovered than T_N cells (27). This suggests that aged T_VM cells exhibit enhanced survival, possibly due to increased expression of receptors for homeostatic cytokines and integrins (23). Evaluation of exhaustion-related signatures revealed that age-related changes in T_VM cells were indicative of immune senescence, not indicative of T-cell exhaustion (27), which suggest that the inflammaging features of elderly individuals permits T_VM cells to survive relatively well, because T_VM cells respond better to cytokines than to TCR-mediated stimulation.

Collectively, the accumulated evidence regarding T_VM cells in the aging process suggests that these cells are highly dependent on innate-like immune responses, which continue to act efficiently via cytokines, rather than on Ag-specific responses, which become impaired.

ROLE OF T_VM CELLS IN INFECTION

Like innate cells, T_VM cells reportedly play a protective role in first-line defense to viral, bacterial, and parasitic infections (12,13,35,39) (Fig. 2). In the past, NK and NKT cells had been thought to be the major IFN-γ producers by pathogen-derived inflammatory triggers (40). However, a previous study identified a population of IFN-γ–secreting CD8⁺ T cells in the spleen and lymph nodes of LPS-injected mice, indicating that other immune cells are capable of early IFN-γ production (41). Moreover, it was shown that IFN-γ production by this CD8⁺ T-cell population was restricted to CD44^hi cells, which was independent of MHC class I (41). Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) can stimulate inflammatory cytokine production by various innate immune cells, including macrophages and dendritic cells that can produce IL-12 and IL-18 during the early phase of an infection. IFNα/β, IL-12, and IL-18 derived from macrophage and dendritic cells can indirectly stimulate Ag-independent IFN-γ production by memory-like CD8⁺ T cells (4,41,42). T_VM cells constitutively express both IL-12 and IL-18 receptors (23), suggesting a mechanism through which T_VM cells can respond quickly to IFN-γ production in the early stages of infection. Moreover, a recent study revealed that in vitro stimulation with IL-12 and IL-18a yielded a higher frequency of IFN-γ–producing T_VM cells in C57BL/6 mice compared to BALB/c mice, due to the greater IL-18 receptor expression in T_VM cells of C57BL/6 mice (5).

Figure 2. Roles of T_VM cells in infectious diseases and cancers. T_VM cells have diverse immunological roles. T_VM cells show protective roles in situations of infections with virus, bacteria, and helminth in a bystander manner. T_VM cells proliferate with cytokine stimulation and have cytotoxic effects. In particular, bystander activated T_VM cells mediate cytolytic activity in a NKG2D-dependent manner. In case of chemotherapy in cancer patients, T_VM cells expand and form a tumor-associated T_VM cell environment with higher clonality. Cancer cells with impaired MHC class I molecules undergo apoptosis with increased cytotoxicity of T_VM cells.

Lee et al. (43) demonstrated that in the case of viral infection, IL-4 induced a large number of innate CD8⁺ T cells that produced high levels of both IFN-γ and TNF. Upon LCMV Clone 13 infection, these innate CD8⁺ T cells completely controlled the viremia and were dependent on IL-4, as IL-4 KO mice were incapable of clearing the virus (43). T_VM cells also play a role in bacterial infections, such as LM, and Yersinia pseudotuberculosis (12). Additionally, during the acute parasitic infection with Trypanosoma cruzi, thymic cells enriched with T_IM cells exhibit a substantial capacity to produce IFN-γ in response to stimulation with IL-12 and IL-18, and adoptive transfer of these cells can protect T. cruzi-infected mice (39). Hussain et al. (15) demonstrated that IL-15 is critical for the helminth-induced induction of T_VM cells in C57BL/6 mice, which was solely driven by proliferation of existing T_VM cells, with minimal contribution from naive cell differentiation. Moreover, compared with other CD8⁺ T cells, the T_VM-cell population proliferated the most in response to helminth infection and IL-15 (15). However, it has also been reported that the high CD5 expression of T_N cells with IL-15 stimulation and helminth infections can convert T_N cells into T_VM cells in BALB/c mice (13). Therefore, the possibility of a species-specific effect must be considered.

As previously stated, T_VM cells tend to respond to cytokines during infections. Nonetheless, these cells possess a fully functional TCR repertoire. It was demonstrated that OTI T_VM cells can protect against LM-OVA infection by decreasing bacterial colony-forming units in the spleen (23). Moreover, a comparable protection level was observed in a TCR transgenic mouse model that does not recognize bacteria in an Ag-specific manner, indicating that T_VM cells can mediate immune-protection against bacterial infection, regardless of the presence of their cognate Ag (23). In the Ag-independent context, the effector function of T_VM cells is largely dependent on IL-15. In IL-15^−/− mice as recipients, only Ag-specific T_VM cells conferred a protective activity, whereas Ag-nonspecific T_VM cells exhibited compromised functional capacity (23). Taken together, these findings suggest that T_VM cells can rapidly respond in an Ag-specific or Ag-nonspecific manner in inducing immune response.

Quinn et al. (27) demonstrated that T_VM cells can give rise to T_EFF cells in response to TCR stimulation. However, T_VM-derived T_EFF cells produced predominantly IFN-γ, whereas T_N-derived T_EFF cells were more multifunctional by producing a broader spectrum of cytokines (27). Furthermore, T_VM-cell-derived T_EFF cells become short-lived effector cells, while T_EFF cells derived from T_N cells are more likely to be differentiated into stable T_MEM cells (25,44). When a previous report evaluated the secondary immune responses by Ag-specific T_MEM and T_VM cells, both subsets expanded equally, but T_VM cells produced significantly more T_CM cells than T_MEM cells (25). In addition, Hou et al. (45) demonstrated that CCR2⁺ T_VM cells played a dominant role in providing early protection, while CCR2⁻ T_VM cells had a greater capacity to produce resident memory T (T_RM) cells. These findings indicate that T_VM cells can respond in a TCR-specific or non-specific manner in inducing effector functions such as rapid IFN-γ production during the early phase of infections, and can also respond to a secondary challenge by predominant differentiation into T_CM or T_RM cell populations.

ROLE OF T_VM CELLS IN CANCER

In recent years, there has been growing interest in cellular immunotherapy as a means of harnessing the immune system to fight cancers, and in the production and anticancer effect of cancer Ag-specific T cells (46,47,48). However, cancers can evade these cells by downregulating or losing MHC I Ag presentation, making them less stimulating or even invisible to CD8⁺ T cells, without impairing their growth or metastatic potential (49). Therefore, we need to focus on the anticancer activities of non-cancer-specific T cells. It have been shown that T_VM-like cells exhibit increased production of inflammatory cytokines in the absence of Ag immunization (31,50). However, these studies could not determine whether these cells were indeed T_VM cells, because of not using the phenotypic markers that currently define T_VM cells.

Since defining T_VM-cell propulsion, researchers have recently examined its role in cancer immunology (Fig. 2). Miller et al. (11) examined the presence of T_VM clones in tumors and draining lymph nodes of prostate adenocarcinoma-bearing mice. After co-transfecting these mice with polyclonal T_VM and T_N cells for 4 months, the author discovered that T_VM cells constituted a substantial proportion of the CD8⁺ T cells that infiltrated the tumors. The TCR repertoire of T_VM cells was significantly distinct from that of T_N cells, and was highly conserved among cancer-bearing mice. They further demonstrated that prostate cancers drove the recurrent enrichment of tumor-associated T_VM clones, which were uncommon in the periphery, but selectively enriched in prostate tumors. This means that T_VM cells quantifiably contribute to the immune infiltration of tumors.

Wang et al. (51) demonstrated that chemotherapy resulted in a large increase of T_VM cells in tumors. In vitro treatment of tumor cells with cytarabine or doxorubicin activated T_VM cells, causing them to produce large amounts of granzyme B, which killed target cells. It was also shown that treating tumors with chemotherapy activated T_VM cells in a humanized mouse model, in a manner that did not involve MHC class I in the tumors (Fig. 2).

T_VM CELLS IN HUMANS

As specific markers of Human T_VM cells, KIRs and/or NKG2A surface markers with effector memory T cells re-expressing CD45RA phenotypes and Eomes expression have been suggested, which were identified in cord blood and peripheral blood (30). T_VM cells have high cytotoxic potential due to their high perforin and granzyme B content, and the innate-like effector function of T_VM cells in cord blood has been demonstrated by their IFN-γ secretion in response to IL-12 and IL-18 (30). Similar to in the mouse model, that express Eomes and CD122 tend to be affected by cytokines such as IL-4, IL-15, and type I interferon in terms of homeostasis and development of human T_VM cells (52).

Recent studies have examined the phenotype of human T_VM cells in detail. A RNA-seq analysis of CD8⁺CD45RA⁺NKG2A⁺ cells and CD8⁺CD45RA⁺pan-KIR2D⁺KIR3DL1/DL2⁺ cells revealed that these 2 cell populations have distinct characteristics (28). In particular, CD8⁺KIR⁺ cells have been shown to share characteristics with regulatory CD8⁺ T cells, including higher CD122 expression (53). Similarly, another RNA-seq analysis of CD8⁺KIR⁺ T cells confirmed that KIR⁺CD8⁺ T cells are the functional equivalent of mouse Ly49⁺CD8⁺ T cells, and play a regulatory role in autoimmune disorders (54). Schattgen et al. (55) reported that, compared to KIR2D⁻NKG2C⁻CD8⁺ T cells, a significant portion of KIR2D⁺NKG2C^+/−CD8⁺ cells are Helios⁺ cells, supporting the demonstration that KIR⁺ T_VM cells have a regulatory function.

Confirming the function of KIR/NKG2A⁺Eomes⁺ T_VM cells, when chemotherapy-treated human lymphoma cells were co-cultured with CD8⁺ T cells from healthy donors, granzyme B increased along with the proportion of KIR/NKG2A⁺ Eomes⁺ T_VM cells (51). Additionally, KIR/NKG2A⁺Eomes⁺ T_VM cells have been discovered in solid tumors, such as ovarian and breast cancer (52). Jin et al. (56) found that KIR/NKG2A⁺Eomes⁺ T_VM cells were also increased in HIV patients receiving antiretroviral therapy, and exerted suppressive action in a KIR receptors dependent manner. These findings show that human T_VM cells play roles in various disease conditions.

CONCLUDING REMARKS

T_VM cells have long been confounded with T_CM cells, such that their function within the immune system has not been fully understood. Over recent years, there has been growing interest in the discovery of human T_VM cells with features resembling mouse T_VM cells. Important new challenges are to learn to distinguish between the roles of T_VM cells and Ag-specific cells, and to understand the significance of the T_VM-cell population in various disease processes.

Abbreviations

Eomes

Eomesodermin

KO

knockout

LCMV

lymphocytic choriomeningitis virus

LM-OVA

ovalbumin-expressing Listeria monocytogenes

NKR

NK cell receptor

T_CM

central memory T

T_EFF

effector T

T_IM

innate memory T

T_MEM

memory T

T_MP

memory precursor T

T_N

naive CD8+ T

T_RM

resident memory T

T_VM

virtual memory T

WT

wild-type