Immune tolerance during the female reproductive cycle and pregnancy is required for fertility. Successful ovulation, fertilization, and pregnancy rely on an efficient regulatory mechanism that prevents immune responses to sperm cells, oocytes, and placental cells, which express various antigens (1–6). Several female pathologies are linked to immune tolerance dysfunctions, including preeclampsia, recurrent miscarriages, and autoimmune ovarian failure. Pre-eclampsia, which accounts for about 20% of maternal mortality, is associated with a dominant Th1 inflammatory response resulting from impaired function of cells with regulatory activities (7–10). Recurrent miscarriages, which affect about 1-5% of women, are associated with reduced regulatory T cells (11, 12). More recently, autoimmune ovarian dysfunction, a disorder affecting female patients with autoimmune disorders, has been associated with immune tolerance alterations (13–15). These disorders highlight that an imbalance in the immune tolerance equilibrium in the female reproductive system (FRS) can cause severe gynecological and obstetrical complications.

Suppression of immune responses by cells with regulatory activities is one of the central mechanisms for induction and maintenance of self-tolerance (16). CD4⁺CD25⁺ T regulatory cells (CD4⁺Tregs) are the most extensively studied suppressor cells regarding reproductive failure and reproductive immune tolerance (17–19). Furthermore, estrogen and seminal fluid drive the expansion of CD4⁺Tregs, enhance their suppressive function (11, 21, 22), and regulate their activities in the FRS. However, recent data finds that a subset of T cells with unique potent regulatory functions and dominance in the reproductive system (20, 21), known as the double negative T regulatory cells (DNTregs), also play crucial roles in regulating immune responses (21–24). This current review focuses on understanding the characteristics and function of DNTregs as potential regulators of reproductive female immune tolerance and their potential as a novel therapeutic option for reproductive failure, including those associated with autoimmunity. Specific details of DNTregs on non-reproductive autoimmune conditions such as graft versus host disease (GvHD) and cancer are covered elsewhere (25–28). We will provide the context for the interplay of DNTregs in reproductive function. We will also discuss and compare the presence, function, and relevance of CD4⁺Tregs and DNTregs in reproductive immune tolerance. Finally, this review will address how DNTregs represent a potentially viable therapeutic option in female reproductive disorders, including autoimmune conditions.

Immune cells modulate several aspects of the female reproductive function, including folliculogenesis, ovulation, implantation, pregnancy, and labor (29, 30). At distinct points in the reproductive cycle, different immune cells are identified throughout the FRS. Immune cells occur in high frequencies within the upper FRS (the ovaries, oviduct, uterus, and endocervix) and occur less in the lower FRS (ectocervix and vagina) (31). The FRS is an exceptional immunological site, and the distribution of immune cells facilitates tolerance to allogeneic sperm and the semi-allogeneic fetus and placenta (11, 32, 33).

During the reproductive cycle, steroid hormones and immune cells regulate ovulation, fertilization, and implantation in a cyclic fashion and tandem (34–36). Fluctuations of estradiol and progesterone drive relevant processes within the uterus and ovary, including cyclic recruitment of immune cells (37). Within the FRS, immune cells are primarily under ovarian hormone regulation (31, 36, 38) and contribute to the modulation of the reproductive cycle and fertility (39). For instance, T cells (and immune cells in general) express sex-steroid receptors (40–43), and the high infiltration of CD8⁺ T cells into the regressing corpus luteum (CL) coupled with the expression of cytolytic proteins corresponds to decreased progesterone and estradiol concentrations during the period of luteal regression in the ovaries (44). Furthermore, alterations in the immune system affect ovarian function in both animals and humans (45). For instance, blockade of gonadotropin-releasing hormone (GnRH), which centrally regulates the hypothalamic-pituitary-ovarian axis (46), decreases regulatory T cells’ proliferation and thymic mass (47).

Moreover, estrogen deficiency corresponds with increased peripheral cytotoxic T cells and CD8⁺/CD4⁺ T cell ratios. Furthermore, the rise in circulating estrogen levels is associated with increased CD4⁺Treg populations (48). These findings indicate that the reproductive hormones tightly regulate immunological changes in the ovary and endometrium (49).

Most mature αβ T cell receptor (TCR)⁺-T cells in normal mice and humans express either the CD4 or CD8 coreceptor molecules. However, approximately 1–5% of the peripheral T cell population that expresses CD3 but neither the CD4 nor the CD8 coreceptor is termed, CD4 and CD8 double-negative T (DNT) cells. DNT cells were identified in spleen cells from irradiated mice over 40 years ago, but at the time, they were referred to as natural suppressor cells (50, 51) with a null phenotype in neonatal mice (52). In 1984 Oseroff et al. reported that DNT cells do not express the T cell marker CD90/Thy-1, the surface immunoglobulin (Ig), the myeloid marker CD11b/MAC1, the macrophage marker F4/80, or the monocyte specific esterases (52). Their study reported that DNT cells were similar to natural killer (NK) cells based on their combined lack of antigen-specificity and coreceptor CD4 and CD8 molecules (52, 53). However, later reports found that DNT cells inhibited T cell response to alloantigen (54, 55) in an antigen-specific manner (24). Similarly, early reports on this subject suggested that the suppressive function of DNT cells may not depend on proliferation, as suppression was maintained after exposure to high levels of radiation (56). This finding partly explained why DNT cells exert potent suppressive functions despite low numbers. Furthermore, early research also suggested that these unique cells were dependent on T cells in the spleen (50) and that their function becomes dominant when CD4⁺, CD8⁺ T cells, and B lymphocytes are unable to function effectively (56). However, DNT cells with regulatory functions (DNTregs) were not classified as such until 2000 (24).

Naturally occurring T cell maturation and differentiation into CD4⁺, CD8⁺, and CD4⁺Tregs require thymic development (57, 58) though peripheral or induced CD4⁺Tregs can develop from peripheral naïve conventional T cells (59). However, the origin of DNTregs is still a matter of debate. Several reports suggest that DNT cells originate from the thymus either as DNT cells or from single positive (SP) T cells under regulation by sex steroids (21, 60). However, researchers also found that large DNT cell populations can develop, mature, and gain regulatory function in the absence of a thymus or outside the thymic microenvironment (61), just like the peripheral CD4⁺Tregs. In support of the latter, other studies found that DNT cells can develop and mature in the bone marrow (62), liver (63), nasal-associated lymphoid tissues (NALTs) (64), and, as concerns this review, in the FRS (20). Furthermore, studies that suggest CD4⁺ or CD8⁺ precursors for DNT cell maturation indicate a requirement for the thymus at least for their initial development (65–67). The conclusions for DNTs' origin from CD8⁺-derived DNT cells were based on the observation that antigen encounter decreases CD8⁺ populations and increases DNT cell numbers in vitro (68, 69) or in vivo (70, 71). However, recent in vivo data revealed that the development and function of DNT cells could occur in the absence of CD8⁺ cells, indicating that DNT cells can develop from a cell lineage independent of CD8 expression (61), consistent with an earlier report (52). The increase in DNT cells in the study describing decreased CD8⁺ populations upon antigen encounter may have been due to direct activation and expansion of a pre-existing population of DNT cells. Other studies propose that DNT cells in lpr mice (which have null allele for Fas (CD95) (72)) may occur from CD8⁺T cell precursors in the periphery (73, 74) and that DNTs may also occur from CD4⁺ T cell precursors on stimulation of CD4⁺T with allogeneic DCs in the presence of IL-15 or IL-2 (22). Taken together, DNT cells may develop from a separate lineage. However, there may be some degree of peripheral differentiation from peripheral single positive T cells, and further studies are required to determine their precise origins.

It is well accepted that estrogen drives the expansion of the CD4⁺Treg compartment by inducing their proliferation and promoting suppressive functions (17–19). Interestingly, estrogen also drives the accumulation of DNTs during pregnancy (106). Moreover, like estrogen, seminal fluid also modulates Tregs in the FRS (107–109) and contributes to maternal-fetal tolerance (108, 110).

The roles of CD4⁺Tregs in the FRS, including maternal-fetal tolerance, have been extensively explored and reviewed elsewhere (111–116). We will therefore focus on how DNTregs compare with CD4⁺Tregs in the FRS. CD4⁺Tregs undergo profound changes in the ovary and uterus during the reproductive cycle. In healthy women, an increase is observed during the follicular phase of the menstrual cycle reach maximum numbers during the late follicular phase. This process correlates with increased circulating estrogen levels (11, 117). Moreover, endometrial CD4⁺Tregs expand during the proliferative phase of the cycle (11), which corresponds to the follicular phase in the ovaries. However, as women with reproductive failure experience a dramatic reduction in the population of CD4⁺Tregs (11), it is yet unknown whether a similar phenomenon occurs with DNTregs (117, 118). (119)Since estrogen regulates CD4⁺Tregs and DNTs in the FRS, the function of DNTregs in the FRS may be similar to CD4⁺Tregs. However, establishing precise DNT functions in the FRS and how the populations differ during the female reproductive cycle requires further research. In addition, it is also unknown if seminal fluid modulates DNTreg expansion just as it does CD4⁺Tregs in the FRS and requires further research.

CD4⁺Tregs are also crucial to pregnancy maintenance, as changes to the population of regulatory cells may have severe consequences for fertility (118, 119). Several lines of evidence have described both the systemic and decidual expansion of CD4⁺CD25⁺Foxp3⁺ Treg populations in the first two trimesters of pregnancy (120–122).These findings indicate that CD4⁺Tregs are necessary for reproductive immune tolerance. Since both CD4⁺Tregs and DNTregs have been detected in the non-pregnant endometrium during the reproductive cycle, and reproductive hormones modulate CD4⁺Tregs, we also postulate that similar mechanisms modulate DNTregs during non-pregnant and pregnant conditions. Though the literature on the role of DNTregs in the reproductive system pre- and post-fertilization is scanty compared to that of CD4⁺Tregs, it is clear, based on reports on other tissues, that DNTregs play significant regulatory roles similar to CD4⁺Tregs. Indeed, reports indicate consistently large numbers of DNTregs in the FRS compared to other organs (20, 21, 123). The large numbers of DNTregs found in the uterus, gut, and kidney suggest a preference for highly vascularized regions, particularly mucosal tissues (20, 124). Johansson and Lycke observed that DNTreg numbers in the uterus did not vary in non-pregnant mice, after insemination, and on day-4 pregnancy. They, however, state that this does not exclude a function for these cells during pregnancy (20). That the number of DNTregs did not decrease during early mouse pregnancy may suggest both a relevance or irrelevance for these cells in maintaining maternal-fetal tolerance. A possible explanation for the lack of increase observed during early mouse pregnancy may be related to the finding that during the first trimester, pro-inflammatory cells and cytokines are more relevant as these cells assist the blastocyst to successfully penetrate or ‘implant’ the endometrium (125). However, regulatory cells remain present within the endometrium to keep the pro-inflammatory cells in check and will expand during the second trimester and then decrease during the latter part of the third trimester (125). On the other hand, since CD4⁺Tregs are reported to increase in the first two trimesters of pregnancy (121, 122) and a study reports that DNTregs do not increase or decrease during early mouse pregnancy (20), these findings may also suggest that DNTregs may not play a significant role during early pregnancy and instead may be more relevant as immune response regulators throughout pregnancy duration (and in non-pregnant conditions) (116). However, further studies are required to confirm the role of DNTregs in the different phases of pregnancy. Indeed, a report found that the suppressive capacity of CD4⁺Tregs in peripheral blood and decidua is similar among non-pregnant, pregnant, and patients with recurrent spontaneous abortion (RSA) (121), which could be extrapolated to DNTregs as both cells are recruited and regulated by sex steroids. Moreover, a review by Chapman et al. suggests a possible function of DNTregs during pregnancy which involves maintaining trophoblast and blood vessel integrity and promoting angiogenesis (21).

Taken together, DNTregs can contribute to establishing immune tolerance within the endometrium in preparation for fetal allograft, a concept consistent with their reported suppressive role in preventing rejection of donor-specific allografts (126, 127). While available data on DNTs in different phases of the reproductive cycle are scarce, current knowledge on the presence of DNTs in the FRS and during pregnancy suggests a prominent role for these cells in the FRS that may involve immune regulation and providing support to CD4⁺Tregs which should be explored further.

Tolerance to peripheral T cells is necessary to prevent autoimmune damage to self, and regulatory T cells generally act to suppress responses to self and allogeneic antigens (23). Human pregnancy has been described as a condition that requires maintenance of maternal immune tolerance to a semi-allograft fetus comprised of paternally derived antigens. Though the mechanisms involved in sustaining maternal immune tolerance to the semi-allogeneic fetus are still poorly understood, a report suggests that spontaneous abortion is due to the allo-rejection of the fetus by the mother (121). Clearly, a crucial role of regulatory cells in pregnancy involves the mediation of maternal tolerance of the fetus (122). In addition, DNTregs induce donor-specific transplantation tolerance to MHC-mismatched allografts (24). Subsequent reports confirmed that DNTregs induce tolerance to skin and islet transplantation (22, 128), and cardiac xenografts in a donor-specific manner (129) and (122) support a potential role for DNTregs present in the FRS in maintaining reproductive immune tolerance. Studies in several murine models have demonstrated that DNTregs suppress CD4⁺ and CD8⁺ T cell-mediated allogeneic and xenogeneic immune responses and the response to self-antigens (126, 129, 130). DNTregs prevent allograft rejection (131), GvHD (132, 133) and modulate the severity of autoimmune diseases such as autoimmune diabetes (134, 135). However, CD4⁺Foxp3⁺Tregs and human DNTregs appear to require activation by allogeneic antigen-presenting cells (APCs) or anti-CD3/anti-CD28 antibodies to induce their regulatory potential. DNTregs also reversibly suppress the proliferation of responder T cells via cell contact-dependent mechanisms (136) which will be discussed subsequently. Furthermore, DNTregs can function as anti-tumor effector cells that mediate nontumor antigen-restricted immunity while maintaining immune regulatory functions (27). These features position DNTregs to effectively function in the different reproductive phases, maintaining remodeling processes and ensuring successful placenta invasion of the endometrium. It is important to note that the precise roles of DNTregs in maternal immune tolerance are yet to be fully established and requires further research. However, we have indicated potential roles of these cells in maternal immune tolerance based on their immune regulatory roles in other tissues and systems.

The influx of immune cells into the FRS at different cycle stages, particularly during the ovulatory and endometrial process, indicates the importance of immune cells with regulatory functions in maintaining tolerance from pre-conception to implantation and pregnancy (119, 191, 192). While the information on DNTregs within the FRS is still growing, this review highlights the capacity and functions of DNTregs, a unique and remarkable regulatory cell subset complimentary to CD4⁺Tregs, and their potential in supporting FRS functions. DNTregs possess unique features for enhanced specificity, plasticity, effective killing, and suppression while evading hostile microenvironments. These features and the findings that DNTregs preferentially home to mucosal sites and are additionally dominant in the FRS, position DNTregs as novel regulators with extraordinary potential to become the next targeted cellular therapy, particularly for autoimmune female reproductive disorders. That DNTregs can be successfully expanded from healthy individuals and utilized as an off-the-shelf therapy enhances their relevance and provides an emerging shift in considering regulatory cell subtypes for autoimmune conditions. via

EB conceptualized and wrote the article; JV participated in writing and reviewing the article; HY participated in conceptualizing, reviewing, and writing the article. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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