Comparing Mouse and Human Tissue-Resident γδ T Cells

Circulating immune cell compartments have been extensively studied for decades, but limited access to peripheral tissue and cell yield have hampered our understanding of tissue-based immunity, especially in γδ T cells. γδ T cells are a unique subset of T cells that are rare in secondary lymphoid organs, but enriched in many peripheral tissues including the skin, uterus, and other epithelial tissues. In addition to immune surveillance activities, recent reports have revealed exciting new roles for γδ T cells in homeostatic tissue physiology in mice and humans. It is therefore important to investigate to what extent the developmental rules described using mouse models transfer to human γδ T cells. Besides, it will be necessary to understand the differences in the development and biogenesis of human and mouse γδ T cells; to understand how γδ T cells are maintained in physiological and pathological circumstances within different tissues, as well as characterize the progenitors of different tissue-resident γδ T cells. Here, we summarize current knowledge of the γδ T phenotype in various tissues in mice and humans, describing the similarities and differences of tissue-resident γδ T cells in mice and humans.

Heilig and Tonegawa's nomenclature proposed in 1986 segregated mouse gd T cells into six distinct subsets: Vg1, Vg2, Vg4, Vg5, Vg6, and Vg7 (3). Meanwhile, the human g chain locus consists of four subgroups; VgI includes Vg2, 3, 4, 5, and 8. Among the three other Vg subgroups, only Vg9 (from the VgII group) is functional when using the nomenclature of Lefranc and Rabbitts (4). Besides, gd T cells are reported to bridge the gap between innate and adaptive immune responses in mice and humans. Although gd bearing cells were shown to constitute a minor proportion of peripheral T lymphocytes, their coevolution with ab T cells and B lymphocytes revealed nonredundant functions.
gd T cells mostly reside within tissues, particularly in epithelial layers, where they might play tissue-protective or inflammatory roles (5). Experiments in mice have demonstrated that gd T cells are predominantly tissue-resident immune cells (6,7). From further mouse studies, it is nonetheless becoming increasingly clearer that the gd T pool residing in a given tissue is the result of the wave of development from fetal to adult life, referred to as layered ontogeny (8). Nevertheless, how the ontogeny of gd T cells differs between tissues remains obscure. Although the origin of tissue-resident gd T cells in humans is technically challenging to address, there is evidence that the local gd T cells pool can partially be replenished by infiltration and in situ differentiation of circulating naïve gd T cells (9).
Reflecting their tissue residency and the impact of the microenvironment on gd T cell function, recent studies have revealed profound tissue-specific transcriptional signatures for human (9) and mouse gd T cells (10). Accumulating evidence suggests that gd T cells are shaped by the microenvironment and exert tissue-specific functions depending on the signals they receive. This review summarizes recent studies on the tissuespecific features of gd T cells across organs in mice and humans. We discuss the phenotypic differences that contribute to distinct gd T cell profiles in different tissues, highlighting the similarities and differences between mice and humans. Understanding how various tissue microenvironments impact gdT cells is important for improving therapeutic strategies in pathologies that affect specific tissues.

gd T CELLS DEVELOPMENT IN MICE
ab T cells and gd T cells arise from a common progenitor known as a double-negative cell (DN; lacking CD4 and CD8 expression) in the thymus (11). gd T cells that develop without preprogramming in the thymus and receive the TCR signal in the periphery develop as adaptive types, whereas gd T subsets that receive the signal in the thymus are innate types, and those which receive the TCR signal in the periphery but during an early phase of life get converted into innate-like gd T cells (12).
During the development of mouse gd T cells, gd T cells are the first T cells to develop in the mouse embryonic thymus and appear as early as embryonic day 15 of gestation. These cells express a monoclonal Vg5Vd1 T cell receptor (TCR) and are always located in the skin epidermis. A few days later, by an oligoclonal Vg6Vd1 TCR-expressing population, entered multiple peripheral locations, including the tongue, dermis, uterus, testis, abdominal cavity, adipose tissue, and meninges. Semi-invariant Vg4 + gd T cells also develop within this time range, and these cells are associated with Vg6 + cells that have the same functional characteristics. Such Vg4 + gd T cells are home to the lungs, the dermis of the skin, and lymph nodes. Subsequent perinatal Vg7 + gd T cell waves enter the intestine, followed by polyclonal Vg1 + and Vg4 + gd T cell populations, which are more systematically distributed, including peripheral lymphoid organs, where they exhibit adaptive behavior when activated (revised Figure 1A).
On the other hand, mouse gd T cells can commit to effector cytokine production during thymic development; two main functional subsets have been extensively described: IFN-gproducing gd T cells and IL-17-producing gd T cells. (i) IFN-gproducing gd T cells express surface markers, such as CD45RB and CD27. Subpopulations include the fetal and perinatally derived Vg5 + dendritic epidermal T cells (which is called DETC), which are home to the skin, and the postnatally generated cells that express more polyclonal gd T cell receptor (TCRs) (mostly Vg1 + or Vg4 + ) and localize to lymphoid tissues. (ii) IL-17-producing gd T cells lack CD27 expression and include the fetal derived monoclonal and/or oligoclonal Vg6 + T cells that are home to the tongue, dermis, uterus, testis, adipose tissue, and brain meninges, and the Vg4 + IL-17-producing gd T cells that express multiple semi-invariant TCRs and are home to the lung, dermis, and lymph nodes. The development of mouse gd T cells and their subsets depends critically on IL-7 and IL-15 (5). The growth of dermal gd T cells preferentially requires IL-7, whereas IL-15 is mandatory for the generation of gd TCR-expressing intraepithelial lymphocytes (IELs) (13). IL-7 signaling promotes the development of IL-17-producing gd T cells, whereas IL-15 and IL-2 induce IFN-g secretion. Besides, various cytokines have been reported to affect the differentiation of effector gd T cells. IL-12 and IL-18 promote IFN-g production, while IL-1b and IL-23 drive them towards IL-17-producing cells (14).
In summary, these very curious 'waves' of mouse gd T cell development ensure that most peripheral tissues are effectively colonized by long-lived gd T cells (Revised Figure 1A) that are ideally placed to play important roles in situ.

gd T CELLS DEVELOPMENT IN HUMANS
Unlike murine gd T cells, human gd T cells are usually sub-divided based on the use of one of two variable regions of TCR-d chains, which is Vd1 or Vd2. The Vg9 and Vd2 variable (V) gene segments are the first g/d chains to undergo rearrangement in development, detected in the fetal liver from as early as at weeks 5~6 of gestation (15) and in the fetal thymus after 8 weeks of gestation (16). By midgestation (20~30 weeks), Vg9Vd2 + T cells dominate the gd repertoire (Revised Figure 1B). Vd2 is the largest subset of circulating human gd T cells in the blood, which gets rapidly recruited to the mucosal surface to participate in the clearance of localized infection (17). Functionally, Vd2 + T cells exist as naive (CD45RA + CD27 + ), central memory (CD45RA − CD27 + ), effector memory (CD45RA − CD27 − ), and terminally differentiated (CD45RA + CD27 − ) populations (18). By contrast, human Vd1 + subsets are the major gd T cells population in the intestine and skin, whereas Vd3 + subsets are enriched in the liver and gut.
Several features of the Vg9Vd2 + compartment suggest similarities to mouse gd T-cell subsets (19). First, the early fetal wave of Vg9Vd2 + production, with the semi-invariant Vg9Vd2 + TCR repertoire, mirrors early waves of semi-invariant mouse gd T cells. Second, the semi-invariant mouse population expresses Vg4 sequences of restricted length and diversity, analogous to public human Vg9 sequences (20,21). Third, consistent with related immunobiology, butyrophilins (BTN3A1 and BTN3A2/ 3) are important for Vg9Vd2 + T cell recognition (22). However, while some semi-invariant mouse gd T cell populations can become hyporesponsive to TCR triggering following initial strong TCR signaling during development (23), apparently, this does not apply to human Vg9Vd2 + T cells. Notably, Vg9Vd2 + T cells remain responsive to both pyrophosphate antigens (pAg) and anti-CD3 stimulation, a feature that underlies their potential use in several cancer immunotherapy applications (24), and they also exhibit the potential for further TCR-mediated plasticity (25).
In summary, gd T cells comprise distinct functional subpopulations. Current views in the field suggest that the functional potential of mouse gd T cells is related to the use of Vg, while the functional potential of humans is related to the use of Vd (26). When assembling TCRs, human gd T cells express seven bona fide Vg genes but only three Vd genes (27).

gd T Cells in the Skin
gd T cells localized to the skin are mainly involved in maintaining tissue homeostasis and epithelial repair, maintaining epithelial barriers, and contributing to innate immunity. However, the gd T subsets in mouse and human skin differ.

gd T Cells in Mouse Skin
The skin is composed of two major compartments, the epidermis and the dermis, that are populated in the steady-state by distinct gd T cell subsets. Intraepithelial Vg5 + and Vg6 + gd T cells are present in the dermis (28). In wild-type mice, the epidermal T cell compartment is dominated by a highly specialized gd T cell subset termed dendritic epidermal T cells (DETCs) (29). DETC precursors that express a canonical Vg3Vg1 TCR are the first T cells to develop in the mouse thymus. Vg3 + thymocytes are generated only during the early fetal stages of thymic development from E13 to E18 and migrate to the epidermis, where a defined homeostatic density is maintained throughout life by self-renewal (30). Moreover, SKINT1 was shown to couple thymic selections of DETC precursors to their functional programming as IFN-g producers (31). SKINT1, a mousespecific member of the butyrophilins (BTNs) family that is exclusively expressed in the thymic epithelium and the epidermis, was shown to be essential for thymic selection and skin-specific homing of Vg5Vg1 T cell (32).
When the skin is damaged or infected, the gd T cells that function in the epidermis of mouse skin are the epidermislocalized Vg5 + DETCs whose dendritic morphology enables them to contact several adjacent cells simultaneously, such as keratinocytes, Langerhans cells and melanocytes, which increase their own susceptibility to tissue stress and pathology (33). The maintenance of steady-state numbers of DETC is dependent on epithelial cell-derived IL-15, insulin-like growth factor I (IGF1) produced by DETC itself, and through the transcription factor aryl hydrocarbon receptor (AHR) ligand (2). Wendy and colleagues have found that the lack of DETCs in Tcrg -/-(which means the mice lack all gd T cell subsets) mice also results in increased keratinocyte apoptosis due to a deficiency of insulinlike growth factor 1 (IGF1) (34). Although DETCs are thymically programmed to produce IFN-g rather than IL-17 in wild-type mice, DETCs on a skint-1-deficient background are primarily committed toward an IL-17 effector phenotype (35,36). IL-17 release by DETCs can promote DNA repair following exposure to UV radiation and protect the skin against potential opportunistic infections by releasing keratinocyte-derived antimicrobial peptides (37,38). However, in the models of psoriasis and dermatitis, IL-17 is detrimental and is produced by dermal Vg4 + and Vg6 + gd T cells rather than by DETCs. Paradoxically, another study showed that IL-17-producing gd (gd17) T cells have a beneficial role in steady-state skin physiology, and gd17 T cells are also necessary for skin homeostasis (Revised Figure 2).

gd T Cells in Human Skin
The composition of T cell subsets in the skin differs between mice and humans. There is no direct equivalent of DETCs in human skin as the immune cell composition of the epidermis is subject to species-specific differences (2). In human skin, gd T cells dominate in both the dermis and the epidermis, but gd T cells are present in both compartments (2).
In humans, the subset of gd T cells localized in human skin is Vd1 + gd T cells, which express oligoclonal clonal sequences distinct from circulating gd T cells (39). Unlike mouse skin epidermal T cells that only contain DETCs, the human epidermis contains both ab T cells and gd T cells, and Vd1 + gd T cells are localized in both the epidermis and dermis (40). Similar to DETCs, human epidermal T cells produce keratinocyte growth factor (KGF) and insulin-like growth factor 1 (IGF1) and promote wound healing upon activation. It can be seen that DETCs can be regarded as a conserved expression in mouse and human skin and have similar functions, but there are differences in the subgroups of gd in different species. First, the human gd T cells subsets rarely secrete IL-17, which was quite different from the mouse gd T cells subset in the skin. Second, the human gd T cells subset in the skin is Vg1 gd T cells verse DETCs in the mouse skin. Third, the mechanism of how human gd T cell protects from infection is also different from that of the mouse gd T cells.
In summary, although the role of DETCs in wound healing in mice has been demonstrated, the functions and roles of human epidermal gd T cells are just beginning to be elucidated (33) (Revised Figure 2). There is an urgent need to explore human gd T cell functions in future work.

gd T Cells in Mouse Lungs
Considerable numbers of Vg4 + and Vg6 + gd T cells are present in mouse lungs, but their effect on lung tissue physiology is unclear (28). When lung infection occurs, Vg1 + , Vg4 + , and Vg6 + T cells proliferate in the lung, and Vg4 + gd T cells secrete CXC chemokine ligand 2 (CXCL2; also known as MIP2) and TNF to promote neutrophil recruitment (41). The secretion of IL-17 by gd T cells may be the main mechanism involved in lung immunity. Studies have shown that infected dendritic cells, through IL-23, can increase the production of IL-17 by Vg4 + and Vg6 + T cells and promote granuloma formation. IL-17 production by lung-resident Vg4 + T cells can also be increased upon secondary attack (42).

gd T Cells in Human Lungs
In the human lung, both Vd1 + gdT cells and Vd2 + gdT cells play vital roles. However, the mechanisms of these two subsets in specific diseases and the comparison of the immune effect need further research. During lung infection, Vg9Vd2 gd T cells are aggregated to produce IL-17 and IFN-g, the former being the most important cytokine in TB protection (43). Vg9Vd2 gd T cells specifically recognize the phosphoantigen (E)-4-hydroxy-3methylbutylpyrophosphate (HMB-PP), which is abundantly produced by Mycobacterium tuberculosis, and this selective immunity elicits rapid and long-lasting memory, rapidly producing more IL-17 and IFN-g upon pathogen-specific rechallenge, enhancing bacterial clearance (44). In advanced nonsmall cell lung cancer, Vd1 gd T cells and Vd1-Vd2-gd T cells are the main subpopulations of gd T cells in the lung, and higher levels of intratumoral Vd1 gd T cells is a poor prognosis factor (45). Due to the lack of methods to expand Vd1 gd T cells in lung cancer in vitro, we have not been able to clarify the role of Vd1 gd T cells in the lung (46) (Revised Figure 2).

gd T Cells in Mouse Uterus
Mouse Vg6/Vd1 cells are closely associated with the epithelial tissue of the female reproductive tract and account for a major proportion of gd T cells in uterine tissue (47). Unlike other subpopulations, Vg6/Vd1 cells contain a typical Vg6 TCR amino acid junction. A recent study has reported that the percentages of gd T cells were significantly higher in the uterus than in peripheral blood, and most gd T cells in mouse uterus were distributed in the endometrium (48). Further studies indicated that the majority of gd T cells in the uterus were memory cells with higher expression of CD44 and CD27 but lower expression of CD62L and CCR7 compared to those in the blood (48). In addition, mouse gd T cells in the uterus were tissue-resident memory gd T cells expressing CD69 and expressed high levels of CCR6, GranzymeB, and CD107a. Moreover, gd T cells in the uterus were activated and fully expressed transcription factor RORgt. After a short time of activation, mouse gd T cells in the uterus significantly expressed high levels of IL-17 but not IFN-g, promoting the invasion of murine trophocytes.

gd T Cells in the Human Uterus
In healthy pregnant women, there was an accumulation of Vd1 + circulating cells, in contrast to women with recurrent abortions where the Vd2 + circulating cells dominated (47). The ratio of activated gd TCR + cells was significantly increased in normal pregnancies compared to that of recurrent abortions (48). A bias towards circulating Vd1 + gdT cells seemed to be required for a successful normal pregnancy. However, the precise role of circulating gd T cells in pregnancy is not yet completely established. Although convenient to study the gd T cells subsets during pregnancy in the peripheral blood, it hardly to study that how the circulating Vd1 + cells might simply be a spilling over from the fetus-maternal interface.

CONCLUDING REMARKS
Recent reports have undoubtedly revealed significant tissue-specific functions of gd T cells. We highlight the distribution, features, and specific markers of distinct subsets of murine and human gd T cells (Revised Table 1). In humans, gd T cells in blood display a quiescent state and migratory behavior reminiscent of naiüve T cells. By contrast, gd T cells in peripheral organs make up a spectrum of Vd2 PBMCs -Unique Feature:Activated Vd2gd T cells acquire APC properties (such as antigen presentation, costimulation and expression of adhesion molecules MHC-II, CD80 and CD86) -As circulating gd T cells, it also possesses cytotoxicity, cytokine and chemokine production and modulation capabilities against infected or tumor cells NKG2D, Toll-Like Receptor, CD45

Vd3
PBMCs(very few), Liver Increasing CD1d recognition and kill CD 1d target cells, releasing Th1, Th2 and Th17 cytokines, and inducing dendritic cells to become APCs, when stimulated by mitogens and IL-2.
CD56, CD161, HLA-DR, NKG2D activation states that differ depending on the organ. Although human gd T cells deserve more research, mouse gd T cells display tissue-specific degrees of IFN-g and IL-17A production that appear to be regulated by factors present in the tissues, such as cytokines. gd T cells subsets in different organs show variable means of sensing the microenvironment, particularly regarding cytokines. Finally, the tissue-specific functions of gd T cells, in terms of tissue retention and response to chemokines/cytokines, are not only related to the organ but also to species. Further elucidation of gd T cell-mediated tissue immunity, particularly in humans, will be necessary to improve the development of tissue-specific immunomodulatory drugs to be used, for example, in inflammatory conditions and cancer.

AUTHOR CONTRIBUTIONS
GQ and SW helped in drafting the manuscript. ZZ and DJ helped draw the image in Figures 1, 2. JL and AL conceptualized and revised the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by a grant from the National Natural Science Foundation of China (31900657 to JL).