Objectives: Studying immune tolerance induced by HLA-G in kidney allograft acceptance may help understanding of its mechanisms, hoping in the future to boaster it and decrease the immuno­suppressive drugs given that are well known to have serious adverse effects.

Materials and Methods: The current study sought to evaluate soluble HLA-G in 3 groups: kidney transplanted patients with no rejection episodes, transplanted patients with biopsy-proven renal rejection, and healthy age-matched non­trans­planted individuals.

Three groups were studied: kidney transplanted patients with no rejection episodes (n = 43); transplanted patients with biopsy-proven renal rejection (n = 27); healthy, age-matched, non­transplanted individuals as controls (n = 42). Soluble HLA-G level was measured in the serum by a quantitative sandwich enzyme linked immuno­sorbent assay.

Results: sHLAG level was significantly higher in the transplanted patients compared with the control. Prograf and not cyclosporine or Rapamune had positive effects on sHLAG levels. Patients with chronic rejection had a significant lower level of sHLAG compared with a graft stable group. No effect of donor type, infection or duration post­transplant, on sHLAG levels was found.

Conclusions: The results of the current study are consistent with previous studies addressing the role of sHLAG in inducing immunotolerance postkidney transplant. The findings from the current study on the chronic rejection group, supports the on-going research of having a treatment with HLA-G/or derivate, which may constitute in the future a novel efficient anti-graft rejection therapy.

Key words : Immunotolerance, Kidney transplant, Tolerogenic molecule

Introduction

Human leukocytes antigen G (HLA-G) is an immune molecule also called tolerogenic molecule found to be associated with better graft acceptance.¹ It was discovered in trophoblastic tissue,² and is expressed at high levels by this tissue.³ The nonclassical HLA-G molecule is distinguished from classical HLA-class 1 by different features and properties, among them is its restricted tissue distribution and its biological properties leading to immune tolerance.^4-6

Rejection of an allograft is a complex process with a cascade of events involving many cells, among them natural killer cells (NK) and T-cytotoxic cells, which play a prominent role in the cascade. Indeed, there is an immunologic effect of HLA-G on NK cells functions.^7,8

While NK cells are involved mainly with innate immunity, the immunosuppressive properties of HLA-G also can be directed against adaptive immune responses. 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 the cytotoxic response of CD8+ T cells needed for antigens expressed by these target cells.^9,10 Soluble HLA-G also has 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.¹¹ Furthermore, in vitro studies have demonstrated that soluble HLA-G5 inhibits CD4+ and CD8+ T-cell proliferation after an allogeneic response induced by T-cell receptor activation, by binding to ILT2 receptors and arresting cell cycle progression. The effects of HLA-G also may be enhanced by up-regulation of inhibitory receptors in CD4+ cells via interactions with these inhibitory receptors and activation of a positive feedback loop.¹²

In addition to protecting against NK cells and T cells, HLA-G has been shown to inhibit antigen presenting cell (APC) function, by a mechanism that mediates evasion of the adaptive immune response. Interactions between recombinant HLA-G complexes and the ILT4 receptor on human dendritic cells, in vitro, results in impaired dendritic cell maturation, characterized by reduced cell surface expression of MHC-II and costimulatory molecules, known to be induced by the maturation stimulus. The HLA-G/ILT4 interaction also has been shown to reduce the ability of dendritic cells in inducing allogeneic T-cell proliferation.⁵

Human leukocyte antigen G has been reported to affect the transplant course by 2 mechanisms: First, HLA-G may inhibit both NK cell-and CD8+ T-cell–mediated cytolysis. Second, HLA-G may suppress the CD4+ T-cell–allo-proliferative response. Both mechanisms show that HLA-G molecules affect the main effector cells involved in graft rejection.¹⁰ The role of soluble (s)HLA-G in allograft tolerance may involve peripheral deletion of alloreactive T cells after allotransplant in vivo. In heart, liver, and combined liver-kidney transplant patients, increased sHLA-G has been associated with decreased acute rejection episodes, decreased chronic rejection, and better transplant outcomes.^9,13 During a renal trans­plant, increased sHLA-G has been associated with a decreased frequency of HLA IgG antibodies.^10,14

According to Crispim and his colleagues treatment with tacrolimus (Prograf) but not with cyclosporine is associated with increased expression of HLA-G and absence of rejection.¹² This indicates that increased HLA-G expression after kidney transplant may be related to the immunosuppressive treatment to prevent rejection. Glucocorticoids enhance the level of HLA-G transcripts in cultured trophoblast cells, which were one of the first cells reported to produce HLA-G. Besides treatment, the genetic background of donor and recipient and the presence of inflammatory mediators present in the graft milieu also may be responsible for HLA-G modulation.^1,12,13,15 The significance of sHLA-G in immunotolerance is a subject of ongoing research.¹⁶

Research objectives
The hypothesis of this study is that HLA-G is up-regulated in patients with a successful renal allograft. In contrast, in patients with frequent episodes of immunologically mediated (biopsy-proven) rejection, HLA-G is not up-regulated.

Studying immune tolerance induced by HLA-G in allograft acceptance may help to better understand the mechanisms involved. Understanding these immune suppressive mechanisms might allow us to manipulate them to boost immunosuppression and decrease the dosage of immunosuppressive drugs and their adverse events.

Materials and Methods

Study design
This prospective study was done on kidney transplanted patients in the Nephrology Transplant Unit, Salmaniya Medical Complex, Ministry of Health, Kingdom of Bahrain. This study is a population-based case-control study. This design basically included selected kidney transplanted subjects based on their graft stability status, either stable or having graft rejection, testing the role of sHLA-G in graft tolerance.

Subjects of the study
Exclusion criteria
Individuals having the following conditions were excluded from the study: autoimmune disease, malignancy, pregnancy, or allergy. In addition, pediatric transplanted cases (< 15 y) were excluded from this study.

Study groups
Three groups were studied:

• Group 1: kidney-transplanted patients with no rejection episodes (n = 56).
• Group 2: transplanted patients with biopsy-proven renal rejection (n = 27) Three of them had acute rejection while the rest had chronic rejection.
• Group 1 and 2 were further divided, according to duration of transplant into: less than 5 years, 5 to 10 years, and more than 10 years. Moreover, subdivision according to the immunosuppressive drug used, cyclosporine, rapamycin, or tacrolimus (Prograf), was made. Group 3: healthy age-matched nontransplanted individuals were used as controls (n = 43).

Methods
Five mL blood was collected from patients and controls, and the serum was separated within 15 minutes of collection. The serum samples were frozen at (-80ºC) until the time of assay. A quantitative Sandwich enzyme linked immuno­sorbent (ELISA) assay (Uscn Life Science Inc., Wuhan, China), was used. Assay steps were followed according to the manufacturer’s instructions.

Statistical analyses
Descriptive statistics were performed to compare the various parameters between the different groups. Statistical analyses also were done with Microsoft Excel 2007 and Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 15.0, IBM Corporation, Chicago, IL, USA). Data are expressed as means ± SD, percentage, range and median. Statistical significance of differences was analyzed with the chi-square test.

Ethical/research approval
Transplanted patients and healthy controls recruited in the study were asked to complete and sign an informed consent form agreeing to participate. After explaining the purpose of the study and its implications, they were asked to fill a standard questionnaire form. Approval of the Salmaniya Medical Complex and Ministry of Health research committees was obtained. All of the protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration.

Results

The study population included 3 groups; transplanted patients with stable graft, transplanted patients with graft rejection, and a healthy control group with no history of transplant. One hundred and twenty samples were investigated for sHLAG measurement. The age of subjects ranged from 17 to 70 years. Further subgrouping was done according to gender, immunosuppressive medication (rapamycin [RAPA], cyclosporine [CSA], Prograf), donor type (living related [LR], living nonrelated [LNR], deceased donor [DD]), posttransplant period (less than 5 years, from 5 to 10 years, and more than 10 years] and presence of infection (urinary tract infection [UTI], cytomegalovirus [CMV], hepatitis B virus [HBV], and hepatitis C virus [HCV]); details are shown in Table 1.

To explore a relation of sHLAG level with the graft stability or graft loss, the groups were compared simultaneously; the following results were found:

• Level of sHLAG was significantly higher (P < .001) in the total transplanted patients (n = 77) tested (19.6 ± 31.4 U/mL) compared to the control group (n = 43) (5 ± 10.2 U/mL) (Figure 1).
• Patients with chronic rejection (CR) (n = 18) (9.99 ± 9.67 U/mL) had a significant lower level of sHLAG (P = .047) compared with the graft stable group (n = 56) (20.3 ± 34.2 U/mL) (Figure 2).
• Patients with chronic rejection (CR) (n = 18) (9.99 ± 9.67 U/mL) had a lower level of sHLAG compared with patients with acute rejection (n = 3) (64.3 ± 23.9 U/mL), yet the difference was not significant (P = .061).

In an attempt to explore a relation of sHLAG level with the immunosuppressive medication used, whether RAPA, CSA or Prograf, a comparison was made in the graft stable group (n = 56), between the different groups.

No significant difference was found (P = .350) on sHLAG concentration, when comparing patients on RAPA (n = 16) (10.7 ± 15.7 U/mL) with patients on CSA (n = 16), (17.6 ± 29.9 U/mL). Similarly, no significant difference was found (P = .123), on sHLAG concentration, when comparing patients on RAPA (n = 16) (10.7 ± 15.7 U/mL) with patients on Prograf, (n = 16) (21.5 ± 22.2 U/mL). Also, no significant difference was found (P = .647) on sHLAG concentration, when comparing patients on CSA (n = 24), (17.6 ± 29.9 U/mL) with patients on Prograf (n = 16), (21.5 ± 22.2 U/mL).

Interestingly, the level of sHLAG was significantly higher (P = .02) in the graft stable group on Prograf (n = 16) (21.5 ± 22.2 U/mL) compared with the control group (n = 43) (5 ± 10.2 U/mL) (Figure 3).

On the contrary, no significant difference was found (P = .099) in sHLAG concentration, when comparing patients either CSA (n = 24) (17.6 ± 29.9 U/mL) or RAPA (n = 16) (10.7 ± 15.7 U/mL) with the control group (5 ± 10.2 U/mL).

Exploring the effect of each drug alone, a comparison was made for sHLAG level, for patients with stable graft and patients with chronic rejection, using the same immunosuppressive drug; the following results were found:

• Prograf in graft stable group (n = 16) versus CR group (n = 8) No significant difference was found (P = .053)
• CSA in graft stable group (n = 24) versus CR group (n = 6)No significant difference was found (P = .232)
• RAPA in graft stable group (n = 16) versus CR group (n = 3) No significant difference was found (P = .094).

In summary, Prograf, and not CSA or RAPA had a positive effect on sHLAG levels according to results of the current study.

No significant difference was found (P = .384) in sHLAG concentration according to the type of donor, whether LNR (n = 45; 21.4 ± 37.3 U/mL), or LR (n = 29; 15.5 ± 20.6 U/mL), in the examined transplant recipients.

Similarly, no significant difference was found (P = .522) in sHLAG concentration according to presence of infection, (n = 8; 15.4 ± 17 U/mL) or no infection (n = 69; 20.1 ± 32.7 U/mL), in the transplanted recipients [n = 77].

Regarding the effect of gender, no significant difference was found (P = .647) in sHLAG concentration when men (n = 39; 18.6 ± 27.3 U/mL) were compared with women (n = 17; 24.3 ± 47.1 U/mL), in the graft stable transplant recipients. Similarly nonsignificant findings were found in the CR group (n = 18) (P = .782), when sHLAG concentration of men (n = 9; 9.33 ± 9.79 U/mL) was compared with women (n = 9; 10.7 ± 10.1 U/mL).

Regarding sHLAG level according to posttrans­plant duration, no significant difference was found in sHLAG concentration when comparing the 3 periods less than 5 years (n = 28), between 5 to 10 years (n = 31), and more than 10 years n = 18 as shown below.

(A) Less than 5 years (n = 28) versus from 5 to 10 years (n = 31)
No significant difference in sHLAG concentration was found (P = .884) when comparing the period less than 5 years (19.8 ± 29.7 U/mL) and from 5 to 10 years (21.1 ± 38.8 U/mL).

(B) Less than 5 years (n = 28) versus more than 10 years (n = 18)
No significant difference in sHLAG concentration was found (P = .585) when comparing the period less than 5 years (19.8 ± 29.7 U/mL) and more than 10 years (15.9 ± 18.5 U/mL).

(C) From 5 to 10 years (n = 31) versus more than 10 years (n = 18)
No significant difference in sHLAG concentration was found (P = .528) when comparing the period from 5 to 10 years (21.1 ± 38.8 U/mL) and more than 10 years (15.9 ± 18.5 U/mL).

In summary the result of the current study did not show any effect of donor type; infection, or duration posttransplant, on sHLAG levels.

Discussion

The current study explores the role of soluble HLAG [sHLAG] in kidney transplant recipients as immunotolerant tools of the immune system. Three different groups were studied; patients with stable graft, those with graft rejection, and healthy controls with no history of kidney transplant. Soluble HLA-G concentration levels were measured in sera.

To evaluate whether clinical parameters affect sHLAG levels, we evaluated some transplant factors include whether the kidney was from (donor origin related [living related] vs nonrelated donor [nonliving related or deceased donor]), and infectious factors (BK virus, cytomegalovirus, hepatitis B virus, and hepatitis C virus). The results of our study revealed that neither the type of donor nor the presence of infection had an effect on serum sHLAG concentration; this was also reported by others.17-19 Regarding gender, we found no significant difference between male and female patients sHLAG concentrations.

To study the effect of duration posttransplant on HLAG, we divided the patients according to the period posttransplant into 3 subgroups: less than 5 years, from 5 to less than 10 years, and more than 10 years. No significant difference was found.

There was a significant higher sHLAG level found when comparing either of the transplanted group with the control group; this finding supports the role played by HLAG expression in graft immunotolerance as previously highlighted by others. Soluble HLA-G is expressed not only in patients with stable grafts, but in cases of acute or chronic graft rejection; this has been found in other studies.^{8,13 20,21} In addition, we found a significant difference between graft stable and graft rejection groups; same finding was previously reported by others.¹² Nevertheless multifactor effects occur in the event of graft acceptance, like genetic background of donor and recipient and the presence of inflammatory mediators present in the graft milieu, which may affect HLA-G modulation. In the studied graft stable group, we observed 2 patients with high sHLAG concentration, [200 and 133 U/mL] while the mean for the whole group was 20.3 U/mL, 1 was exposed to an occupational chemical exposure accident while the other is an Indian hypertensive patient using herbal drinks; this raises the possibility of different environmental and nutritional factors that may affect HLAG expression; thus, future studies are required to understand this.

Moreover, we found no significant difference in the level of sHLAG between patients on different immunosuppressive drugs (RAPA, cyclosporine, and Prograf), yet when comparing patients on Prograf versus controls, a significantly higher level was observed; the role of Prograf in enhancing the HLAG expression was reported previously.¹² Also corticosteroids have been reported to enhance production of soluble HLA-G,13 yet none of our patients received it when tested.

Soluble HLA-G5 has been shown to suppress T-cell functions and induces regulatory T cells.22 Additionally,²³ it was demonstrated that sHLA-G5 secreted by adult bone marrow-derived mesen­chymal stem cells are responsible for the immuno­modulatory effects; blocking experiments using neutralizing anti-HLA-G antibody demonstrate that HLA-G5 contributes first to the suppression of allogeneic T-cell proliferation and then to the expansion of CD4+CD25highFOXP3+ regulatory T cells.²³ In the current study, the level of sHLAG in the peripheral blood appeared to be influenced by kidney transplant and by the immunosuppressive medications.

Conclusions

In conclusion, the results of the current study are consistent with previous studies addressing the role of sHLAG in inducing immunotolerance postkidney transplant.

In summary, the following were found:

• sHLAG was significantly higher in all transplanted patients compared to the control group.
• Prograf but not CSA or RAPA had a positive effect on the sHLAG level.
• Patients with CR had a significantly lower level of sHLAG compared with graft stable group.
• No effect of donor type; infection or duration posttransplant, on sHLAG levels.

The findings from the current study on the chronic rejection group, support the ongoing research of having treatment with HLA-G1 or HLA-G5 and T-reg, which may constitute a novel efficient antigraft rejection therapy in the future; yet sHLAG measurement must be improved and thus hopefully it may be used in the future to monitor progress of transplant patients. Indeed the recent article published 2014, addressing the role of HLA-G dimers in the prolongation of kidney allograft survival supports this concept.¹⁶

References:

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