Role of pathogenic T-helper cells-17 in chronic antibody-mediated rejection in renal allograft recipients

Brijesh Yadav; Narayan Prasad; Vinita Agrawal; Manoj Jain; Vikas Agarwal

doi:10.4103/ijot.ijot_149_20

ORIGINAL ARTICLE

Year : 2022 | Volume : 16 | Issue : 1 | Page : 88-95

Role of pathogenic T-helper cells-17 in chronic antibody-mediated rejection in renal allograft recipients

Brijesh Yadav¹, Narayan Prasad¹, Vinita Agrawal², Manoj Jain², Vikas Agarwal³
¹ Department of Nephrology and Renal Transplantation, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
² Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
³ Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission	05-Dec-2020
Date of Acceptance	17-Oct-2021
Date of Web Publication	31-Mar-2022

Correspondence Address:
Prof. Narayan Prasad
Department of Nephrology and Renal Transplantation, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow - 226 014, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijot.ijot_149_20

Abstract

Background: Both T-cell and B-cell activities are interlinked. The role of a new subset of T-helper cells (Th17), which is thought to be more pathogenic than other effector T cells, is not much studied in cases of chronic antibody-mediated rejection (CABMR). Therefore, we aimed to determine the circulating frequency of Th17, Pgp⁺ Th17, Pgp⁺ CD4T cell, and serum interleukin (IL)-17A cytokine level in patients of CABMR and stable graft function (SGF). Materials and Methods: We had recruited 42 patients of biopsy-proven CABMR (n = 32) and SGF (n = 10). The frequency of Pgp⁺ CD4Tcells, Pgp⁺ Th17 and Th17 cell was quantified in circulating blood by flow cytometry, and IL-17A level was determined in serum by ELISA techniques. Results: We observed that the frequency of Th17 cell (2.30 ± 1.15 and 4.46 ± 2.05; P = 0.003), Pgp⁺ CD4T (15.73 ± 4.38 and 25.27 ± 5.15; P < 0.001), and Pgp⁺ Th17 (0.78 ± 0.65 and 2.19 ± 0.93; P < 0.001) was higher in CABMR as compared to that of SGF. The ratio of Th17/CD4T cell (0.077 ± 0.045 and 0.14 ± 0.06; P = 0.004), and Pgp⁺ CD4T/CD4T cell (0.51 ± 0.14 and 0.81 ± 0.17, P < 0.001) was also higher in CABMR as compared to SGF. Serum cytokine IL-17A level was (42.39 ± 13.25 and 66.97 ± 18.27; P < 0.001) pg/ml was also higher in CABMR than that of SGF. The frequency of Pgp⁺ Th17 cell was positively correlating with 24-h proteinuria (r = 0.309; P = 0.04), serum creatinine (r = 0.423; P = 0.005), and negatively correlating with eGFR (r = −0.468; P = 0.002). Similarly, the frequency of Pgp⁺ CD4T cell was positively correlating with 24-h proteinuria (r = 0.351; P = 0.023), serum creatinine (r = 0.310; P = 0.04), and negatively correlating with eGFR (r = −0.414; P = 0.006). Conclusions: Higher Pgp⁺ TH17 cell is associated with CABMR, proteinuria, and graft dysfuntion.

Keywords: Chronic antibody mediated rejection, interleukin-17, permeable glycoprotein, T-helper cells-17

How to cite this article:
Yadav B, Prasad N, Agrawal V, Jain M, Agarwal V. Role of pathogenic T-helper cells-17 in chronic antibody-mediated rejection in renal allograft recipients. Indian J Transplant 2022;16:88-95

How to cite this URL:
Yadav B, Prasad N, Agrawal V, Jain M, Agarwal V. Role of pathogenic T-helper cells-17 in chronic antibody-mediated rejection in renal allograft recipients. Indian J Transplant [serial online] 2022 [cited 2022 May 25];16:88-95. Available from: https://www.ijtonline.in/text.asp?2022/16/1/88/342430

Introduction

Chronic antibody-mediated allograft rejection (CABMR) is a major cause of graft loss in late post-transplant period. Patients with CABMR respond poorly to conventional immunosuppressive therapy and rapidly progress to end-stage graft failure.^[1],[2] The graft injury in CABMR is associated with preform or de novo donor-specific antibody (DSA) and nonspecific antibodies as well.^[3],[4] The de novo DSA appears despite immunosuppression. Sub-optimal dosing and nonadherence of calcineurin inhibitors (CNIs) lead to increased de novo antibody formation.^[5],[6] CNIs act by inhibiting calcineurin and transcriptional activation of cytokine genes interleukin (IL)-2, a factor responsible for the conversion of B cells to plasma cells and the formation of antibodies. IL-2 transiently upregulates expression of IL-2R, through which it induces the terminal growth and differentiation of activated B cells into plasma cells producing antibodies.^[7]

Both T-cell and B-cell activities are interlinked. The role of a new subset of T-helper cells (TH17), which is thought to be more pathogenic than other effector T cells, is not much studied in cases of CABMR. TH17 cells can function as B-cell helpers and induce a strong proliferative response of B cells in vitro. TH17 cells also trigger antibody production with class switch recombination in vivo. Transfer of Th17 cells into cell receptor α–deficient mice, which lack endogenous T cells, induces a pronounced antibody response with preferential isotype class switching to IgG1, IgG2a, IgG2b, and IgG3, as well as the formation of germinal centers. Conversely, blockade of IL-17 signaling results in a significant reduction in both the number and size of germinal centers.^[8] There is a possible link of Th 17 cells in the formation of antibodies in renal transplant recipients with immunosuppression inhibiting T cells such as steroids and CNIs.

Th17 cells secret pro-inflammatory cytokines IL-17, tumor necrosis factor-α (TNF-α), and IL-21. IL-17A induces proliferation and recruitment of neutrophils and macrophages in renal allograft tissue.^[9],[10] A few experimental studies also showed an association of IL-17 in cardiac and pulmonary fibrosis.^[11],[12] A study showed increased expression of Th17 cells on tubular epithelial cells in case of acute antibody-mediated rejection in renal transplantation patients.^[13] The dysregulation of TH17 in early graft dysfunction during the early posttransplant period in patients on CNIs has also been reported.^[14],[15]

It has been observed that permeable glycoprotein (P-gp) expression on Th17 cells increases the pathogenicity of TH17 cells in autoimmune diseases.^[16] CNIs are the substrate of P-gp, a transmembrane glycoprotein expressed on lymphocytes which removes the drug from cytoplasm to extracellular space and limits the bioavailability of the drug to the cells.^[17] It has been reported that increased P-gp expression on lymphocytes leads to poor prognosis and hasten graft rejection.^[17] In this study, we aimed to demonstrate the role of Th 17 cells, IL-17 cytokines, and P-gp expressing Th17 cells in patients with CABMR as compared to that of patients with stable graft function (SGF).

Materials and Methods

Patients recruitment

We had recruited 42 live related renal allograft recipients (n = 10 SGF and n = 32 CABMR). SGF was defined by <10% cortical surface area showing IF/TA on histology, stable serum creatinine level for 6 months, absence of proteinuria, and no DSA in the serum of recipients. CABMR was defined by the presence of morphologic evidence of antibody-mediated rejection, C4d⁺ in PTCs, the presence of circulating DSA along with multi-layering, and duplication of capillary basement membranes along with interstitial fibrosis and tubular atrophy on light microscopy as per Banff 2017 criteria^[18] The eGFR was determined by the chronic kidney disease epidemiology formula.^[19] Patients with a history of acute rejection, delayed graft function, more than one transplant, and any active infection were excluded from the study.

Blood collection

Five-milliliter venous blood was withdrawn (1 ml in heparinized vials for Th17 cell immunophenotyping, 4 ml in plain vials for serum isolation for IL-17A cytokine level determination by ELISA).

Permeable glycoprotein expressing Th17 cell immunophenotyping

Cell stimulation

One milliliter heparinized whole blood was diluted with 1 ml of complete RPMI-1640 (Sigma-Aldrich, St. Louis) media. Blood cells were stimulated with Phorbol 12-myristate 13-acetate (20 ng/ml; Sigma-Aldrich, St. Louis) and Ionomycin (1 μg/ml; Sigma-Aldrich, St. Louis) for 5 h and in last 2 h of stimulation; 2 μm of Monensin (BD Biosciences, San Diego, CA, USA) was also added to inhibit intracellular transport of cytokines outside of the cell as optimized in our lab.^[20]

Cellular staining

For surface staining, 20 μl of FITC conjugated mouse anti-human CD4 and 5 μl of Phycoerythrin-Cyanine-7 (PE-Cy7) conjugated mouse monoclonal anti-human P-gp (Biolegend CA, USA) antibody were added and incubated for 30 min in the dark at room temperature. After surface staining, RBCs were lysed with BD FACs lysing solution. The cells were washed with PBS followed by fixation and permeabilization with Cytofix/Cytoperm kit (BD Pharmingen) as per the manufacturer's instruction. For intracellular IL-17A cytokine staining, 5 μl of phycoerythrin (PE) conjugated mouse monoclonal anti-human IL-17A, the antibody was incubated for 30 min in darkness at room temperature. Isotype-matched antibodies were used as controls in each experiments. Minimum 10,000 lymphocytes gated cells were acquired on BD FACS Calibur machine (Becton Dickinson, Mount View, CA, USA) for each sample and analyzed with Kaluza version 2.1 software. Results were expressed as the percentage of positive cells.

Gating strategy

First 10,000 lymphocytes cells were analyzed (Cell in Gate-A and Representative flow cytometry [Figure 1]a) and then CD4⁺ T cell (Cell in Gate-A and Representative flow cytometry [Figure 1]f) were analyzed and in CD4 cell, CD4⁺IL-17A⁺ was considered Th17 (cell in Gate-C), Pgp⁺CD4⁺T (Cell in Gate-D) cells were analyzed. Furthermore in CD4 cells, Pgp⁺IL-17⁺ cells (cell in quadrate E++)(Triple positive CD4⁺Pgp⁺IL-17⁺) were considered as Pgp⁺Th17 cell. Further, to confirm the count and intensity of respective markers expression a histogram was plotted for individual marker (IL-17A⁺, Gate-F), (Pgp⁺, Gate-G) and a combine overlay for Pgp⁺ IL-17⁺ cell [[Figure 1]m and [Figure 1]p] was plotted.

Figure 1: (a-p) Representative flowcytometry panel in CABMR and SGF patients. SGF: Stable graft function, CABMR: Chronic antibody mediated rejection

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Analysis of cytokines in serum

Serum was separated from plain vials blood by centrifuging at 1500 rpm for 5 min. The serum was stored in −80°C till the ELISA experiment setting.

Cytokine interleukin-17A levels of serum

Soluble serum cytokines level was quantified by commercially available human IL-17A duoset ELISA kit (R&D system, Minneapolis, USA) following kit's protocol. The lower detection limits for the assays was15.6 pg/ml.

Statistical analysis

Data were expressed as mean ± standard deviation. Data were analyzed using SPSS statistical software 20.0 (SPSS, Chicago, IL, USA). The mean values in different groups were compared with the Man-Whitney U test for continuous variables, and the Chi-square test was used for categorical variables. Pearson correlation and multivariat analysis was applied for correlation and reassociation analysis. P ≤ 0.05 was considered as statistically significant.

Declaration of patient consent

The patient consent has been taken for participation in the study and for publication of clinical details and images. Patients understand that the names, initials would not be published, and all standard protocols will be followed to conceal their identity.

Ethics statement

This study was approved by the institutional ethics committees (IEC.2012-117-PhD-63) and the study was performed with ethical standards laid down in the declaration of Helsinki and Istanbul.

Results

Demographic and clinical characteristics of patients

Patients of both groups SGF and CABMR were similar in demographic and biochemical parameters except serum creatinine, posttransplant biopsy interval, BUN, and 24-h urine protein were significantly higher in CABMR group as compared to that of SGF [Table 1] and eGFR was significantly lower in CABMR group as compared to that of SGF.

Table 1: Demographic and clinical characteristics of patients

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Histological injury scores

Patients with CABMR had significantly higher grades and cumulative injury scores for the peritubular capillaries, glomerulus, tubules, interstitial inflammation, fibrous intimal thickening as compared to that of SGF [Table 2].

Table 2: Histological lesions scores were analyzed by two histopathologists blindly following banff scoring criteria 2017

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Frequency of circulating Pgp⁺T cell subset in stable graft function and chronic antibody mediated rejection

The frequency of CD4⁺ Th cell was similar in between CABMR (31.54% ± 4.10%) and SGF (31.24 ± 4.96; P = 0.84) [[Figure 2]a and representative flow cytometry [Figure 1]b and [Figure 1]g]. The frequency of Th17 (CD4⁺IL-17A⁺) cell was significantly higher in patients of CABMR (4.46 ± 2.05) as compared to that SGF (2.30 ± 1.15; P = 0.003) [[Figure 2]b and representative flow cytometry [Figure 1]c and [Figure 1]h]. The ratio of Th17/CD4⁺Th cell was higher in patients of CABMR (0.14 ± 0.06) as compared to that of SGF (0.077 ± 0.045; P = 0.004) [Figure 2]c.

Figure 2: (a-f) Frequency of different Pgp⁺ T cell subset in patient of CABMR and SGF. SGF: Stable graft function, CABMR: Chronic antibody mediated rejection

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Similarly, the frequency of Pgp⁺CD4⁺ Th cell was significantly higher in patients of CABMR (25.27 ± 5.15) as compared to that of SGF (15.73 ± 4.38; P < 0.001) [Figure 2]d and representative flow cytometry [Figure 1]d and [Figure 1]i]. The ratio of Pgp⁺CD4⁺Th cell/CD4⁺ Th cell was higher in patients of CABMR (0.81 ± 0.17) as compared to that of SGF (0.51 ± 0.14; P < 0.001) [Figure 2]e. The frequency of Pgp⁺Th17 (Pgp⁺IL-17A⁺CD4⁺ Th; triple positive) cell was significantly higher in patients of CABMR (2.19 ± 0.93) as compared to that of SGF (0.78 ± 0.65; P < 0.001) [[Figure 2]f and representative flow cytometry [Figure 1]j and [Figure 1]e]. Further, to look the number of positive cell and their intensity a histogramme was plotted for IL-17A⁺ [histogramme [Figure 1]k and [Figure 1]n], P-gp⁺ [Figure 1]l and [Figure 1]o, and an overlay of P-gp⁺IL-17A [double positive cell, [Figure 1]m and [Figure 1]p] was plotted.

Serum level of interleukin-17A

The serum soluble IL-17A level was significantly higher in patients of CABMR (66.97 ± 18.27) as compared to that of SGF (42.39 ± 13.25; P < 0.001 pg/ml) [Figure 3].

Figure 3: Serum IL-17A level in CABMR and SGF patients. SGF: Stable graft function, CABMR: Chronic antibody mediated rejection, IL-17A: Interleukin-17A

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Correlation between Kidney graft function parameters and circulating Pgp⁺T cells subsets

The frequency of Pgp⁺Th17 cell was positively correlating with 24-h proteinuria (r = 0.309; P = 0.04), serum creatinine (r = 0.423; P = 0.005), and negatively correlating with eGFR (r = −0.468; P = 0.002). Similarly, the frequency of Pgp⁺CD4 T cell was positively correlating with 24 h proteinuria (r = 0.351; P = 0.023), serum creatinine (r = 0.310; P = 0.04), and negatively correlating with eGFR (r = −0.414; P = 0.106) [Table 3]. On multivariate analysis, only Th17 (β = 3.02, 95% confidence interval [CI] 0.15–2.4, P = 0.027) and Th17/CD4⁺T (β = −2.7, 95% CI − 72.15 to −2.80, P = 0.035) cell were significantly associated with serum creatinine.

Table 3: Correlation between kidney graft function parameters and circulating Th17 and Pgp⁺Th cell subsets

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Discussion

In this study, we have observed that the frequency of P-gp expressing Th17 cell and IL-17A cytokine levels was significantly higher in patients with CABMR as compared to that of SGF. The donor kidney persistently stimulates the recipient's immune system to induce an inflammatory response. The increased frequency of CD4⁺IL-17⁺Th cells, and increased IL-17 cytokines in peripheral blood in our finding suggest that inflammatory milieu leads to increased P-gp expression on CD4⁺ T cell subset.^[21] In addition, inflammatory cytokines stimulate endothelial cell to express chemokine receptors helps in infiltration of other immune cells in graft, leading to allograft injury.^[20],[22] It is also possible that higher P-gp expression exacerbated inflammatory milieu as P-gp removes cytokines from the cells.^[23]

Th17 is an inflammatory cell associated with chronic inflammatory responses,^[24] which secrets IL-17A, IL-21, and TNF-α cytokines. IL-17 has been reported to mediate inflammation and fibrosis in the heart,^[11] lung,^[12] and liver^[25] in experimental studies. Inflammation and fibrosis are common histopathology findings in CABMR, and IL-17 has been reported to be responsible for fibrosis and progressive decline in graft function in renal allograft recipients.^[26] IL-21 cytokine of Th17 also induces differentiation of naïve B cell into antibody-secreting plasma B cell,^[27] which is not an uncommon event in the transplant setting, however, this aspect was not studied in the present study.

Recently, a study revealed that P-gp expressing Th17 cells is more aggressively pathogenic and relatively refractory to glucocorticoids therapy.^[16] We have also observed in our study that patients with CABMR had a significantly higher level of P-gp expressing Th17 cells as compared to that of SGF patients. Chung et al., in their study, showed thatTh17 cells are not suppressed with CNI in vitro.^[14] It is possible that increased expression of P-gp on Th 17 cells removes CNI from these cells. An increased P-gp expression increases the threshold dose of immunosuppressive and favors the antigenic Th17 cell proliferation with CNI. Furthermore, P-gp expels other proliferative cytokines IL-2 in circulation, which again enhances alloreactive T cell proliferation and inflammatory milieu favoring the development of pathogenic Th17 cell.^[28],[29] P-gp acts like a double edge sword; at one edge P-gp acts like an efflux pump which expels xenobiotics and toxins from cytoplasm to intracellular space, thus protecting the cells from these offending agents,^[16] while at another edge, it limits the availability of drugs to the site of action resulting into a poor response to therapy. P-gp is expressed on lymphocytes cells to avoid oxidative stress and drug toxicity.^[30] Increased expression of P-gp on T helper cells are associated with poor response to therapy in diseases such as nephrotic syndrome, rheumatoid arthritis, and systemic lupus erythematosus.^[23] We have already shown in our previous study that P-gp expression on lymphocytes increases during relapse and decreases during remission of nephrotic syndrome patients.

We have also shown the association of MDR-1 gene polymorphism with steroid responsiveness in nephrotic syndrome patients. CNIs (Tacrolimus and Cyclosporine) are P-gp substrates as well as inhibitors. CNIs inhibit expression of P-gp in many T-cell mediated diseases resulting in remission of the disease; moreover, in renal transplant patients, CNIs also induce expression of P-gp to avoid nephrotoxicity.^[30]

Conclusions

The frequency of Th-17 cells and its cytokines was higher in CABMR as compared to that of SGF. Moreover, P-gp expressing Th17 cells were also higher in CABMR as compared to that of SGF in renal allograft recipients which suggest an association of these cells with CABMR.

Limitation and strength of study

The present study is an observational, prospective study. The patients were categorized into two groups CABMR and SGF based on biopsy findings. The study highlights the link of TH17 cells and antibody formation. It also demonstrates the pathogenic role of P-gp expressing Th17 cells in CABMR. Anti-IL-17 therapy may be a therapeutic option in these groups of patients, in the absence of any definitive therapy to retard the progression of the diseases. The study is limited to a small number of patients with SGF due to performance of biopsy after clinical sign indication as the protocol of our center. Further many of CABMR patients had tubulitis, indicating an involvement of T-cell in rejection process.

Acknowledgment

Brijesh Yadav is a “DST-INSPIRE” fellow, Department of Science and Technology, New Delhi, Govt. of India and worked under supervision of Prof. Narayan Prasad. The study has been completed with Intramural research grant of the institute.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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