Human Leukocyte Antigens and Immune Tolerance

Abstract

Immune tolerance is a known immune cascade of events by which our immune system can regulate its function, avoiding unwanted immune response reactions to immune privilege sites in our body. The role of HLA-G in fetal–maternal immune tolerance to prevent the embryo from being rejected can be applied to the process of transplantation as well as other clinical applications. The gut is also an important site of immune tolerance, with the constant assault of food antigens and its billions of resident microbes. In transplantation, the level of expression of HLA-G in the graft tissues correlates with organ acceptance and controls the recipient’s immune response. Furthermore, tumor immune escape is associated with both the expression of immune checkpoint molecules on peripheral immune cells and soluble forms of the human leukocyte antigen-G (HLA-G) in the blood, which is consequently discussed as a clinical biomarker for disease status and outcome of cancer patients. Future studies are needed to explore more immune tolerance pathways for HLA-G and to apply and use this in transplantation to prevent rejection and treat miscarriage cases and autoimmune diseases. In addition, therapies to block HLA-G in malignant diseases are exciting and need more clinical trials. This chapter addresses and reviews the published articles related to the advances in HLA G and immune tolerance.

1. Introduction

Immune tolerance is a known immune cascade of events by which our immune system can regulate its function, avoiding unwanted immune response reactions to immune privilege sites in our body. This regulation takes various mechanisms both at the molecular as well as cellular level, involving signaling, cell-to-cell interaction release of cytokines, and chemokines. Two interesting articles, addressing this topic were published in 2020, one on the immunology of the pregnant female [1] while the other on neuro-immunology [2].

2. The gut as a site of immune tolerance

With the constant assault of food antigens and its billions of resident microbes, the gut is an important site of immune tolerance. The mechanism and pathway between the immune system and the gut microbiota are still not totally revealed; interesting two articles were recently published addressing novel findings in this aspect; an article published titled “Novel antigen-presenting cell imparts Treg-dependent tolerance to gut microbiota” by Akagbosu et al. [3], their studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programs. Another study titled: “ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut” by Lyu et al. [4]; their results define a paradigm whereby ILC3s select for antigen-specific RORγt + T reg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health. Moreover, a review article on the same aspect was recently published titled “Localization and movement of T regs in the gastrointestinal tract” by Harad et al. [5].

3. Pregnancy and semi-allogeneic fetus acceptance

Pregnancy is a metabolic and immune challenge for the mother who, in her womb, has to adapt a semi-allogeneic fetus, whose 50 percent of antigens are of paternal origin; thus, the fetus should be viewed as an allograft and should be rejected via T cell-mediated, MHC-restricted mechanisms. The fetus does not come into direct contact with maternal tissue, but the embryonic trophoblast forms the interface between the maternal and fetal compartments and, thus, is the site of fetal antigen presentation [6]. Specific immune protective mechanisms are involved in establishing the active multifactorial maternal–fetal tolerance to the semi-allogenic fetus, where HLA-G plays an important role [7].

4. HLA-G and regulatory T cells in pregnancy

It has been demonstrated that mHLA-G expressed on trophoblasts is one of the key factors in regulating cytokine balance by shifting the Th1/Th2 balance toward Th2 polarization, a favorable medium for maintaining pregnancy [8]. The effects of HLA-G are highly concentration-dependent, and HLA-G-producing cells are located in the placental bed. This implies that sHLA-G might reduce the ability of T cells to function effectively in the pregnant uterus but is less potent in the periphery or away from the uterus [9]. HLA-G induces regulatory T cells by differentiating naïve T cells into CD4⁺ CD25⁺ or by forming temporary HLA-G⁺ suppressor cells. Moreover, “Foxp3⁻ suppressor T cells” are produced that function in the presence of IL-10. Also, CD4⁺ CD25⁺ Foxp3⁺ and Th3 IL-10⁺ TGF-β⁺ are induced from naïve T cells when HLA-G inhibits the maturation of DCs. Maternal APC (macrophages and dendritic cells) are scattered all over the human decidualized endometrium during all stages of pregnancy. Studies showed that dendritic cells (DC) change to DC-10 by s HLA-G and IL-10and thus, induce production of type I T_regs cells (Tr1) followed by suppression of cytotoxic T cell responses by secreting IL10 and TGF-β. This plays an important role in maintaining maternal tolerance [10].

5. HLA-G molecule

In 1987, Geraghty and their Colleagues were the first who discover the HLA-G molecule, an 8–10 amino acid peptide, located within MHC loci at human chromosome 6q21.3 [11] and was extensively documented as a major potential promoter of tolerance at the human maternal–fetal interface; the expression of HLA-G was first described in trophoblasts [4]. HLA-G has 8escribed alleles and 3151 base pairs in [12]. The HLA-G molecule has special characteristics which make it different from other HLA class I molecules; it has restricted tissue distribution and lower polymorphisms. The HLA-G mRNA, encodes seven HLA-G isoforms: HLA-G1, HLA-G2, HLA-G3, and HLA-G4 membrane-bound proteins, and HLA-G5, HLA-G6, and HLA-G7 soluble proteins. Moreover, HLA-G membrane-bound isoforms have a shortened cytoplasmic tail which delays antigens recycling [13].

6. HLA-G polymorphism

HLA –G polymorphism is reduced with only 9 different HLA-G protein variants encoded by 28 alleles, of which 23 correspond to substitutions in the coding sequence. Not only polymorphism in non-coding regions affects HLA-G gene expression but also HLA G genotypes. Haplotype UTR-1 is associated with higher s HLA-G levels while haplotype UTR-5 or UTR-7 are associated with less s HLA-G levels [14].

7. Normal and pathological tissue distribution of HLA-G

It was initially thought that HLA-G under normal status is not expressed except by the fetal tissue cytotrophoblast, at the fetal–maternal interface, and in transplanted patients [15]. Further studies showed that in non-pathological (physiological) conditions HLA-G is also expressed in HLA class I-positive tissues such as oocytes, embryos, amnion, adult thymic epithelial cells, cornea, and nail matrix, which are considered immunologically privileged sites and in cytokine activated monocytes. Furthermore, HLA-G is expressed in pathological conditions, like some tumors, such as melanoma, colon carcinoma, lung carcinoma, ovarian carcinoma, gastric carcinoma, endometrial carcinoma, renal cell carcinoma, mesothelioma, breast carcinoma, trophoblastic tumors and hematopoietic tumors (hematologic malignancies, such as acute myeloid leukemia, chronic lymphocytic leukemia, and lymphoma) and may represent an escape mechanism from anti-tumoral immune responses [16]. HLA-G may be induced in other cell types during pathological processes, which include inflammatory disorders (e.g., skin inflammations and muscle inflammation), viral infections, HIV infection, by non-rejected allografts, and autoimmune diseases such as multiple sclerosis [MS] [17, 18].

8. Receptors for HLA-G

Unlike classical MHC-I proteins responsible for antigen presentation, HLA-G does not appear to play a role in activating the immune response. Instead, HLA-G exerts its inhibitory function against NK cells, T lymphocytes, and antigen-presenting cells (APCs) through direct binding to the inhibitory receptors. Three different inhibitory HLA-G receptors have been identified. These include the immunoglobulin-like transcript 2 (ILT-2 (LILRB1/CD85j)), which has been detected in monocytes, macrophages, dendritic cells, B cells, as well as subsets of T cells and NK cells; the immunoglobulin-like transcript 4 (ILT-4 (LILRB2/CD85d)) expressed by APC, namely monocytes, macrophages and dendritic cells, and the killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4) (CD158d) which is mainly expressed by NK cells. Their expression might be increased by HLA-G binding itself. In addition, HLA-G has been shown to ligate the CD8 co-receptor expressed by certain T cells and NK cells [13].

The ITL2 and − 4 receptors are now known as LILRB1 and LILRB2 (leukocyte inhibitory receptors) [19]. Both receptors have broad specificities and bind classical MHC-I molecules in addition to HLA-G, however binding to HLA-G with a higher affinity than that with which they bind classical MHC-I proteins, indicating that ILT (LILR)/HLA-G interactions play a major role in controlling NK cell, T cell, and APC activity. Both receptors also have a higher affinity for HLA-G dimers, which are linked by an intermolecular disulfide bond. However, the affinity is different and dependent on the form (monomer, dimer). Shiroishi et al. demonstrated that HLA-G dimers induce more efficient ILT2-mediated signaling than monomers. A soluble form of HLA-G5 could form a disulfide-linked dimer with the intermolecular Cys42-Cys42 disulfide bond. In addition, the membrane-bound form of HLA-G1 can also form a disulfide-linked dimer on the cell surface of the Jeg3 cell line, which endogenously expresses HLA-G [20]. An important difference between the ILT2 and − 4 receptors is that HLA-G must associate with β2M to bind to the former. Nonetheless, both possess inhibitory properties and modulate the immune response accordingly. The KIR2DL4 receptor, unlike ILT2 and − 4, binds exclusively to HLA-G and not to classical MHC-I molecules. It has been shown to possess both inhibitory and stimulatory properties. As a result of the nature of KIR2DL4, the immunosuppressive effects of HLA-G have mostly been described through mechanisms involving the ILT2 and − 4 receptors [19].

9. HLA-G and immune tolerance

HLA-G induces immune tolerance by different mechanisms, mainly by regulation of cytokine production, suppression of CTL and NK cell killing activity and viability, inhibition of proliferation and induction of a suppressive phenotype in T helper cells, and alteration of DC stimulatory capacity and maturation of this lineage. Moreover, APCs transfected with HLA-G can prevent the proliferation of CD4 + T cells and drives an immunosuppressive profile where the cells produce high levels of TGF-β1 [21]. During these processes, HLA-G enhances the expression of Th2 anti-inflammatory cytokines, including IL-4, IL-10, and IL-13, and decreases Th1 pro-inflammatory cytokines, including IL-2, TNF-a, and IFN-g [22].

10. HLA-G and NK cell interactions

Many studies were done on the immunological effect of HLA-G on NK cell functions [23]; their results showed that HLA-G-positive target cells are protected from NK cytolysis through interaction with killing inhibitory receptors (KIR2DL4 and ILT2) HLA-G1 membrane-bound or soluble HLA-G protein [24]. ILT-2 is expressed on both NK and T cells, while KIRs, belongs to the immunoglobulin superfamily (Ig-SF), p49 Ig-SF KIR, and CD94/NKG2A inhibitory receptor, which belongs to the C-type lectin superfamily. Those receptors interact with HLA-E, which consequently inhibits NK lysis.

11. HLA-G and T cell function

Impairment of CD4+ and CD8+ T cell function has been well documented. Direct evidence is illustrated by the fact that HLA-G1, when transfected into target cells, blocks cytotoxic responses of CD8+ T cells specific for antigens expressed by these target cells [25]. Furthermore, soluble HLA-G has also been shown to induce apoptosis in CD8+ T cells by interacting with CD8, leading to Fas ligand (FasL) upregulation, FasL secretion, Fas/FasL interaction, then apoptotic signaling [26]. Furthermore, in vitro studies have demonstrated that soluble HLA-G5 inhibits CD4+ and CD8+ T Cell proliferation following an allogeneic response induced by T cell receptor activation, by binding to ILT2 receptors and arresting cell cycle progression.

A small fraction of CD4+ and CD8+ cells from peripheral blood have been found to stably express HLA-G and show less proliferation to allogeneic stimuli compared to their HLA-G- counterparts. These CD4 + HLA-G+ and CD8 + HLA-G+ cells represent novel Treg cell subsets, different from the traditional CD4 + CD25 + Foxp3+ population, and are independently able to suppress lymphocytic proliferation of both CD4 + HLA-G- and CD8 + HLAG- populations. Resting and activated CD4 T cells can rapidly acquire HLA-G1 through membrane exchange with HLA-G+ APC. Following the acquisition of membrane-bound HLA-G, these effectors can inhibit allo-proliferative responses. While these cells differ from CD4 + CD25 + Foxp3+ Treg cells, they temporarily function as such through the HLA-G1 they acquire but do not constitutively express. Acquisition of HLA-G1 by CD4+ T cells might explain how a few HLA-G+ cells can protect against immune aggression toward HLA-G target cells in the local milieu. There is, thus, considerable evidence demonstrating that in addition to directly inhibiting effector CD4+ and CD8+ T cells, HLA-G can generate populations of Treg cells to suppress these effectors [27].

12. Inhibition of antigen-presenting cell function

Interactions between recombinant HLA-G complexes and the ILT4 receptor on human dendritic cells, in vitro, resulting in impaired dendritic cell maturation characterized by reduced cell surface expression of MHC-II and co-stimulatory molecules typically induced by the maturation stimulus. The HLA-G/ILT4 interaction has also been shown to reduce the ability of dendritic cells to induce allogeneic T cell proliferation [19].

Recombinant HLA-G complexes have been shown to impair dendritic cell maturation, induce T cell energy, diminish CD4+ and CD8+ T cells responses, and generate Treg cells in ILT4 transgenic mice compared to their non-transgenic counterparts. Thus, transgenic animal models have demonstrated how HLA-G can impair APC maturation and, consequently, diminish their ability to activate T cells [28].

13. HLA-G in solid organ transplantation

HLA-G expression was detected in different solid organs after transplantation. In heart, liver, and combined liver-kidney transplant patients, increased sHLA-G has been associated with decreased acute rejection episodes, decreased chronic rejection, and a better transplant outcome [25]. HLA-G polymorphisms are associated with rejection or acceptance. Several clinical studies showed that the expression of HLA-G has a protective role, induction of immune tolerance, and subsequent graft acceptance in transplantation. Moreover, studies showed that donor and recipient genotypes could influence the local HLA-G expression in the transplanted organ, as well as the activity of the host immune system response [22].

An interesting study from Japan conducted on 40 kidney transplant patients studied HLA-G expression on proximal tubular epithelial cells, and they concluded that HLA-G expression might confer long-term renal preservation effects in renal transplanted allografts [29].

Interestingly, another study showed that plasma levels of sHLA-G significantly decreased during the first year after renal transplantation and that lower levels of sHLA-G were found in recipients with post-transplant diabetes mellitus or obesity carrying the HLA-G14bpins/ins or HLA-G + 3142G/G genotypes [30].

Moreover, looking at HLA G genotypes, a study reported that HLA-G 3’-UTR variants are promising genetic predisposition markers both in donors and recipients that may help to predict susceptibility to either viral infectious complication of BKV or allograft rejection in kidney transplant [31].

Moving to heart transplants, a study evaluating HLA-G polymorphisms and cell-mediated rejection development, conducted on 55 recipients, concluded that HLA-G + 3196 G allele was identified as a risk factor for cell-mediated rejection diagnosis; according to their study, HLA-G may have a role in therapeutic/diagnostic strategies against heart transplant rejection [32].

A recent observational study on heart transplants, including 59 patients, concluded that soluble HLA-G levels decreased over the first year after a heart transplant. Also, higher HLA-G expression was associated with a higher frequency of infections but not with the burden of acute rejection or the development of coronary allograft vasculopathy, neither with the long-term patient or graft survival [33].

Regarding lung transplant, a single-center study examined 11 HLA-G SNPs in 345 consecutive recipients and 297 donors of a first bilateral lung transplant; specific donor SNPs were associated with mortality risk after lung transplantation, while certain donor–recipient SNP pairings modulated chronic lung allograft dysfunction risk. Trans-bronchial biopsies demonstrated predominantly epithelial, and therefore presumably donor-derived, HLA-G expression in keeping with these observations. This study is the first to demonstrate the effect of donor HLA-G SNPs on lung transplantation outcomes [34].

14. HLA-G and tumors

Tumor immune escape is associated with both the expression of immune checkpoint molecules on peripheral immune cells and soluble forms of the human leukocyte antigen-G (HLA-G) in the blood, which is consequently discussed as a clinical biomarker for disease status and outcome of cancer patients.

Regarding HLAG and T cell mechanism, a study demonstrated that priming of PBMC with sHLA-G1 protein before 48 h activation resulted in enhanced frequencies of ILT-2 expressing CD8C T cells, and in upregulation of immune checkpoint molecules CTLA-4, PD- 1, TIM-3, and CD95 exclusively on ILT-2 positive CD8C T cells. In contrast, when PBMC were primed with EV (containing HLA-G1 or not), upregulation of CTLA-4, PD-1, TIM-3, and CD95 occurred exclusively on ILT-2 negative CD8C T cells. Taken together, their data suggest that priming with s HLA-G forms induces a pronounced immunosuppressive/exhausted phenotype and that priming with sHLA-G1 protein or extra vesicular vesicles (EV) derived from HLA-G1 positive or negative SUM149 cells affects CD8C T cells complementary by targeting either the ILT-2 positive or negative subpopulation, respectively, after T cell activation. They report that they provide the first evidence that immune modulation by soluble HLA-G might involve IC molecules toward an immune suppressor phenotype. They conclude HLA-G functional analysis needs to be thoroughly performed in cancer patients [35].

Furthermore, Kataoka et al. published an interesting article addressing the killer immunoglobulin-like receptor 2D L4 (KIR2DL4) in pregnancy and in cancer metastasis by regulating mast cells [36]. Moreover, they added that stimulation of KIR2DL4 by HLA-G may enhance both conditions and may be useful in suppressing allergic reactions mediated by mast cells.

Regarding laryngeal carcinoma, a study suggested that HLA-G alleles may participate in LSCC pathophysiology; and The −14/−14 and − 14/+14 alleles may affect the biological function of the expressed HLA-G protein. Thus, their presence may act as a genetic risk factor that may predispose them to LSCC pathogenesis [37].

15. Therapy trials for s HLA-G for immune tolerance

Moving to therapeutic developments for immune tolerance, Radi et al. addressed in their review article “[Opinion on Immune Tolerance Therapeutic Development”] the major challenges to developing tolerance-inducing pharmaceutical drugs, including the selection of appropriate disease models to establish efficacy, adequate, and acceptable in vitro and in vivo safety assessments, relevant biomarkers of human safety and efficacy, and finally, some regulatory guidelines to successfully develop immune tolerance therapeutics [38].

Indeed multi-centric clinical trials are needed for HLA-G application in therapy. One line is to have s HLA-G as a therapy for repeated miscarriage cases, in the transplantation field, and in some autoimmune diseases. On the other side, therapies to block or downregulate the HLA-G molecule are needed in cases of tumors, thus enabling the immune system to fight those tumors and get rid of the tumor cells [13].

16. HLA G ongoing research

HLAG yet from the clinical diagnostic feasibility measuring it in serum is more practical as a non-invasive tool. Indeed using sHLAG to monitor post-transplant immune tolerance would be an easy way to monitor the possibility of losing such tolerance, thus alerting the clinician of a rejection episode before it occurs. Many researchers are working in this line, hoping to use sHLAG measurement as a diagnostic and monitoring laboratory tool in different clinical conditions [39]. A recent article by [40] addressed the role of the HLA G gene and its expression in the genesis of recurrent miscarriage. Another publication from India, addressed the role of stem cell transplant outcomes [41]. Measurement of sHLA-G plasma levels might be a good marker of efficient implantation after IVF [13]. Moreover, an excellent review article recently published in Frontier in Immunology reviewed the role of HLA G in organ transplantation [22].

Indeed the future is for immunotherapy and making use of the naturally occurring immune tolerance phenomenon that is, by time, more and more understood, whether in the gastrointestinal tract with the un-harmful gut microbiota or with the fetus during pregnancy, hopefully, can be achieved with a transplanted organ or as a therapy for cases of repeated abortion, autoimmune diseases and even in malignancy. Measuring T reg and s HLAG may be monitoring laboratory markers soon for several clinical conditions. In my opinion, the need for the development of new tools to analyze in-depth the HLA-G tumor neo-expression patterns, opening the way for the generation of new monoclonal antibodies and cell-based immunotherapies is urgent as mentioned by Loustau, et al. in their review [42].

17. Conclusion

The role of HLA-G in fetal–maternal immune tolerance to prevent the embryo from being rejected can be applied to the process of transplantation. In transplantation, the level of expression of HLA-G in the graft tissues correlates with organ acceptance and controls the recipient’s immune response.

Future studies are needed to explore more immune tolerance pathways for HLA-G and to apply and use this in transplantation to prevent rejection and treat miscarriage cases and autoimmune diseases. In addition, therapies to block HLA-G in malignant diseases are exciting and need more clinical trials.