Steroid free immunosuppression is associated with enhanced Th1 transcripts in kidney transplantation

Accepted Manuscript Steroid free immunosuppression is associated with enhanced Th1 transcripts in kidney transplantation

Petra Hruba, Irena Tycova, Eva Krepsova, Eva Girmanova, Alena Sekerkova, Janka Slatinska, Ilja Striz, Eva Honsova, Ondrej Viklicky PII: DOI: Reference:

S0966-3274(16)30143-5 doi: 10.1016/j.trim.2017.03.001 TRIM 1083

To appear in:

Transplant Immunology

Received date: Revised date: Accepted date:

6 November 2016 26 March 2017 28 March 2017

Please cite this article as: Petra Hruba, Irena Tycova, Eva Krepsova, Eva Girmanova, Alena Sekerkova, Janka Slatinska, Ilja Striz, Eva Honsova, Ondrej Viklicky , Steroid free immunosuppression is associated with enhanced Th1 transcripts in kidney transplantation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Trim(2017), doi: 10.1016/j.trim.2017.03.001

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ACCEPTED MANUSCRIPT Steroid free immunosuppression is associated with enhanced Th1 transcripts in kidney transplantation Petra Hruba1, Irena Tycova1, Eva Krepsova1, Eva Girmanova,1 Alena Sekerkova3, Janka Slatinska2, Ilja Striz3, Eva Honsova4 and Ondrej Viklicky1,2.

Transplant Laboratory; 2Department of Nephrology, Transplant Center, Institute for Clinical and

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Experimental Medicine, Prague, Czech Republic; 3Department of Clinical and Transplant Immunology,

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Institute for Clinical and Experimental Medicine, Prague, Czech Republic 4Department of Clinical and

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Transplant Pathology; Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

Professor of Medicine

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Ondrej Viklicky, M.D., Ph.D.

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Address for Correspondence:

Department of Nephrology, Transplant Center

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Institute for Clinical and Experimental Medicine

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Videnska 1958/9, 14021 Prague, Czech Republic E-mail: [email protected]

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ACCEPTED MANUSCRIPT Authors’ contributions: O.V. and P.H. participated in designing the research, P.H., I.T., E.K., E.G., J.S., A.S., I.S. and E.H. participated in performing the research, P.H., I.T. and O.V. participated in analyzing

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data and P.H. and O.V. participated in writing the manuscript.

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ACCEPTED MANUSCRIPT Abbreviations: HLA, human leukocyte antigen PBMCs, peripheral blood mononuclear cells

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POD, post-operative day PRA, historical panel reactive antibodies

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TCMR, T cell mediated rejection

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ACCEPTED MANUSCRIPT Abstract Background: Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. Methods: In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early

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withdrawal and center standard of care treated patients were monitored for 12 months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance,

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were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3-months while lymphocyte subpopulations were

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analyzed by flow-cytometry within the follow-up.

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Results: Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B (GZMB) transcript at POD 14 and perforin 1 (PRF1) transcript at POD 7. The higher interleukin 2 (IL-2) expression at POD 7 was detected only in the steroid avoidance group.

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Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14 days was observed in the

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months biopsies among groups.

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standard of care group only. There were no differences in analyzed intrarenal transcripts in 3-

Conclusions: The enhanced expression of some of Th1 associated transcripts and limited effects

rejection.

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on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute

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Keywords: Kidney transplantation; steroid elimination; tolerance; rejection, ADVANCE trial

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ACCEPTED MANUSCRIPT

1. INTRODUCTION Corticosteroids are routinely used as a component of maintenance immunosuppression in kidney transplant recipients. Since corticosteroids have several side effects, many attempts were done to withdraw corticosteroids at a specified time post-transplant (1-3) or avoid them at all (4). Most published data have shown that steroid avoidance at transplantation or early withdrawal of

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steroids after transplantation is associated with increased risk of acute rejection but without adversely affecting patient or graft survival (2, 5-7) while some studies did not find any significant

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difference in acute rejections or graft loss (8). ADVANCE, a large, prospective, multicenter trial with more than 1000 enrolled kidney transplant recipients, has very recently shown that steroid

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avoidance is associated with plus 5% (p=0.001) more acute rejection as compared to 10-day steroid withdrawal arm while all patients received basiliximab induction and tacrolimus/MMF long term

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immunosuppression (9).

The mechanisms of how steroids prevent early acute rejection development remain illdefined and in vivo data are lacking. In vitro steroids exert their immunosuppressive effects by

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inhibiting Th1 cytokine production and by enhancing the production of Th2 cytokines (10, 11). Many rejection-associated markers were described so far, including GZMB, PRF1 (12, 13), C3orf23, MAN1A1 (14), FOXP3 (15) and inflammatory cytokines TGFB1, IFNG and IL-2. Very recently, several B-

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cell related markers of operational tolerance were also identified (16, 17) and these markers were

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shown to play a role also in rejection free patients (18). In this single center observational study we evaluated molecular and cellular markers

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associated with rejection and operational tolerance in both ADVANCE trial arms (9) of locally enrolled patients and compared them with markers measured in the standard of care treated patients.

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2. MATERIALS AND METHODS 2.1. Study design and patients Twenty three kidney transplant recipients who received kidney transplantation between 2011-2013 at the Institute for Clinical and Experimental Medicine in Prague, Czech Republic and participated on large randomized, open-label, prospective, multicenter clinical study (ADVANCE, ClinicalTrials.gov; NCT01304836) were randomly assigned to the steroid 10-day withdrawal or steroid avoidance arm with only perioperative steroids bolus (9). Fourteen patients were randomized into steroid avoidance arm and 9 patients into steroid 10-days withdrawal arm. As a control group, patients who received center standard of care immunosuppression with basiliximab induction, tacrolimus once daily, mycophenolate mofetil and long term steroids were selected based on 5

ACCEPTED MANUSCRIPT identical inclusion criteria as for ADVANCE study (n=10) (9). All patients participated in local prospective immune-monitoring and biobank study. Immune monitoring of peripheral blood was performed prospectively in all patients regularly before transplantation and then in POD 7, 14, 90 and 365. Intragraft gene expression was measured at 3-months protocol biopsies in a patient subgroup (steroid avoidance, n=6; steroid withdrawal, n=8 and standard of care, n=5).

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Main pre-transplant donor´s and recipient´s characteristics were not significantly different

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among steroid avoidance, steroid 10-day withdrawal and standard of care treated group (Table 1). Inform consent: All patients signed informed consent with the participation in local immune

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monitoring study (ethic approval no. G 10-04-11) and patients who were enrolled also in ADVANCE

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study (ethic approval no.1771/10) signed both informed consents.

2.1.2. Biopsies

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Renal biopsies were obtained under ultrasound guidance (Toshiba, Power Vision 6000) using a 14-gauge Tru-Cut needle (Uni-Cut Nadeln, Angiomed, Germany). Most of the renal tissue was processed for conventional histology. Histological examination was interpreted according to the 2013

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Banff working classification criteria (19). The residual portion (2mm) of the cortical zone of the renal

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tissue was immediately placed in RNA later (Ambion Corporation, Austin, TX), snap frozen and stored at -80 °C until RNA extraction. Intragraft gene expression was measured in patients on steroid free regimens

(steroid

avoidance,

n=6

and

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withdrawal,

n=7)

and

steroid-based

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immunosuppression (standard of care, n=5).

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2.1.3. Flow cytometry and isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Lymphoprep™ (Axis-Shield, Oslo, Norway) from peripheral blood anticoagulated with ethylenediaminetetraacetic acid (EDTA). PBMCs (100 µl approximately 1 x 106) were labeled with fluorochrome-conjugated (FITC– fluorescein isothiocyanate, PE-phycoerythrin, ECD-Phycoerythrin-Texas-Red, APC-Allophycocyanin, PC5-Phycoerythrin-Cyanin 5, PC7-Phycoerythrin-Cyanin 7) monoclonal antibodies diluted in phosphate-buffered saline-bovine serum albumin buffer for 20 min at room temperature in the dark. The specific antibody panels used consisted of anti-CD4-PC7 (clone: SFC112T4D11) anti-CD25-PC5 (clone: B1.49.9) anti-CD127-PE (clone: R34.34) (Beckman Coulter, Brea, CA). Intracellular FoxP3 6

ACCEPTED MANUSCRIPT staining of Tregs was performed as described by the manufacturer (Human Regulatory T Cell Staining Kit, eBioscience, San Diego, CA). Extracellular staining of freshly prepared and isolated PBMCs was carried out using CD4-FITC (clone: RPA-T4), CD25-APC (clone: BC96), antibodies prior to intracellular staining with anti-Foxp3-PE (clone: PCH101). NK cells were labelled with CD45-FITC (clone: B3821F4A), CD16-PE(clone: 3G8), CD56-PE (clone: N901), CD3- PC5 (clone: UCHT1), T lymphocytes with CD45-FITC (clone: B3821F4A), CD3-PC5 (clone: UCHT1) and B lymphocytes with CD45-FITC

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(clone: B3821F4A), CD19-ECD (clone: J3-119), CD3-PC5 (clone: UCHT1) (Beckman Coulter, Brea, CA). Following staining procedure, samples were analyzed using an FC 500 flow cytometer

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(Beckman Coulter) and the data were processed by C × P and Kaluza software (Beckman Coulter, Brea, CA). Individual subsets were defined as follows: Tregs as CD4+CD25+FoxP3+ and

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CD4+CD25+CD127−, NK cells as CD45+CD3-CD16/CD56+, T lymphocytes as CD45+CD3+ and B lymphocytes as CD45+CD19+CD3-. Flow cytometric analyses were performed with at least 100 gated

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events.

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2.1.4. RT-qPCR analysis

For gene expression analysis RNA was isolated from peripheral blood, collected at POD 0, 7, 14, 90 and 365 using the PAXgene Blood RNA kit (Qiagen, Hilden, Germany) or from 3-months

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protocol biopsies using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany) according to manufacturer´s instructions. RNA was quantified by measuring absorbance at 260 nm on the ND-

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1000 Spectrophotometer (NanoDrop Technologies). RNA was reverse transcribed using Superscript Reverse transcriptase II (Invitrogen). The synthetized cDNA was subjected to RT-PCR analysis. Quantitative RT-PCR was performed using a custom-made

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Taqman low density array (Applied Biosystems) to analyze 31 genes (BTLA-Hs00699198_m1, C3orf23Hs00603313_m1, CD79B-Hs00236881_m1, CD247-Hs00167901_m1, FCRL1-Hs00364705_m1, FCRL2FOXP3-Hs00203958_m1,

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Hs00229156_m1,

GATA3-Hs00231122_m1,

GZMB-Hs01554355_m1,

HS3ST1-Hs01099196_m1, IFNG-Hs00989291_m1, IGLL1-Hs00252263_m1, IL2-Hs00174114_m1, IL10Hs00961622_m1, MAN1A1-Hs00195458_m1, MS4A1-Hs00174849_m1, PNOC-Hs00918595_m1, PRF1-Hs00169473_m1, SH2D1B-Hs01592483_m1, SLC8A1-Hs01062258_m1, TCL1A-Hs00951350_m1, TGFB1-Hs00998133_m1, Hs00963364_m1,

TLR5-Hs00152825_m1,

TMEM176B-Hs00962650_m1,

TNFRSF13B-

TNFRSF13C-Hs00606874_g1,

TNFRSF17-Hs03045080_m1,

TNFSF13B-

Hs00198106_m1, GAPDH-Hs99999905_m1, HPRT1-Hs01003267_m1, PGK1-Hs99999906_m1, 18SHs99999901_s1 ) in the whole blood RNA and in the biopsies RNA. Quantitative RT-PCR based on TLDA technology was carried out as described elsewhere (18). Real-time RT-qPCR data were quantified using the SDS 2.4 software package (Applied Biosystems) and relative gene expression 7

ACCEPTED MANUSCRIPT values were determined using the comparative 2-ΔΔCt method of the Relative quantification (RQ) Manager Software v 1.2.1 (Applied Biosystems) with normalization to endogenous control (GAPDH and PGK1) in the case of gene expression from the whole blood and to PGK1 in the case of biopsies gene expression. Endogenous controls were chosen from four candidate genes (PGK1Hs99999906_m1, HPRT1-Hs01003267_m1, GAPDH-Hs99999905_m1 and 18S-Hs99999901_s1) using NormFinder (www.mdL.dk) as the gene with the most stable expression. IGLL1 gene was not amplified with employed primers. All investigated mRNAs were measured in triplicates for each

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sample.

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2.1.5. Statistical Analyses

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Nonparametric tests were used to estimate statistical significance, as n˂10 in group comparisons and data distribution did not conform to a normal distribution. Normality of the data was tested using Kolmogorov-Smirnov normality test. Kruskal-Wallis test for continuous or the χ2 test

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for categorical variables was used to compare differences between patient groups’ characteristics. Data are presented as median [min, max] or as absolute numbers (n). Statistical significance of

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differences in gene expression and absolute numbers of lymphocyte subpopulations in particular time points was evaluated by Kruskal-Wallis test and differences within the study period were evaluated by paired Friedman test. Two-sided P values were considered as statistically significant

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when < 0.05. Statistical analyses were performed using SPSS v.20.0 (SPSS, Inc., Chicago, IL) and

Rejections and graft function

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3.1.

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3. RESULTS

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GraphPad InStat v. 3. 05 for Windows (GraphPad software, San Diego, CA).

Regarding kidney graft function, there were no differences in serum creatinine levels during 1-year follow-up (Table 1). Less patients on standard steroid immunosuppression developed borderline changes and 2 patients had acute TCMR at POD 6 and 278. One patient from steroid avoidance group and 1 from steroid withdrawal group developed acute TCMR at POD 6 and 98, respectively (Table 2). Chronic TCMR was diagnosed in 2 patients from steroid withdrawal group at 3months protocol biopsy and one of them finally lost graft at 13 months after transplantation. There were more polyomavirus BK nephropathies in the standard of care group (Table 2).

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ACCEPTED MANUSCRIPT One patient from steroid avoidance group had been treated by steroids due to thrombotic microangiopathy and borderline changes since POD 21. In 3 patients from steroid withdrawal group prednisone was added after 3 months due to acute TCMR, chronic TCMR and leukopenia, respectively. At the time of peripheral blood transcripts monitoring at POD 7 and 14 all patients were on scheduled immunosuppression. 3.2. Histological findings and gene transcripts in 3-months protocol biopsies

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The influence of early steroids withdrawal on allograft function at 3 months was evaluated by comparison of Banff scores in protocol biopsies of particular treatment groups (n=33) (Supplemental

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Table 1) and in a subset of available samples (n=19) gene expression of rejection and tolerance

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associated markers was analyzed by RT-qPCR.

At 3-months protocol biopsies there were 4 borderline changes (1 in steroid avoidance group

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and 3 in steroid withdrawal group), 2 chronic TCMR and 1 acute TCMR (all in steroid withdrawal group) (Table 2). Vascular fibrosis and arteriolar hyalinization Banff scores were not significantly

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higher in steroid withdrawal group (Supplemental Table 1).

There were no differences in measured genes between standard of care (n=5) and steroid withdrawal (n=8) and steroid avoidance (n=6) immunosuppression protocols with the exception of

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higher BAFF-R expression in steroid withdrawal group (p=0.043) (data not shown). According to

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histological evaluation from these 19 measured 3-months protocol biopsies, 13 biopsies had almost normal findings, 3 biopsies in steroid avoidance arm had borderline changes and in steroid 10-day withdrawal there were 1 acute TCMR and 2 chronic TCMR findings. These three biopsies with

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rejections findings had statistically significant higher expression of T cell related rejection markers FOXP3 and PRF1 (p<0.01) and GZMB and IFNG (p<0.01) and B-cell tolerance associated markers CD79B, FCRL2, SLC8A1, TCL1A, BAFF (p<0.05) and SH2D1B (p<0.01) which clearly reflects higher

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inflammatory burden during allograft rejection. 3.3. Lymphocytes subsets in peripheral blood Time-dependent dynamic changes of lymphocyte subpopulation in particular patients’ groups were calculated by paired Friedman test. The increase in absolute numbers of CD19+ B lymphocytes were observed in all 3 groups (Steroid avoidance group: p=0.008, Steroid withdrawal group: p=0.018 and Standard of care group: p=0.045) (Fig.1). The CD19+ B lymphocytes increase from POD 0 to 7 was more obvious in patients receiving steroids (1.97 x standard of care group, 1.75 x in steroid withdrawal group) compared to 1.5 x in steroid avoidance group). The absolute numbers of NK cells significantly decreased (almost to 57.1% at POD 7 and to 53.6% at POD 14 of their pretransplantation 9

ACCEPTED MANUSCRIPT value) only in patients receiving standard immunosuppression (standard of care group) (p=0.006). A declining trend in NK cells at POD 7 was observed also in steroid withdrawal group (p=0.084, Wilcoxon rank test with Bonferroni correction). There were no significant changes in CD3+ T cells and Tregs subpopulations.

3.4. Peripheral blood transcripts monitoring

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To address the influence of steroids on gene expression of peripheral T-cell and B-cell rejection and tolerance-associated markers, especially during first 14 days, we compared the

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expressions in patients with two steroid-free regimens and steroid-based immunosuppression.

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Both steroid-free regimens were associated with higher cytotoxic T lymphocytes and NK cells derived GZMB (granzyme B) expression at POD 14 (p=0.020) and PRF1 (perforin) at POD 7 (p=0.036). The higher proinflammatory cytokine IL-2 expression at POD 7 (p=0.034) was detected only in no-

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steroids group (Table 3). This reflects immediate negative effect of even low dose steroids on IL-2 transcripts. In later time-points (≥90 days) no significant differences were found among groups

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(Supplemental Figure 1). There were no differences in the expression of Th1 associated cytokine IFNG (interferon gamma) or Th2 associated TGFβ (transforming growth factor beta).

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When analyzing time-dependent gene expression changes in particular treatment groups, the most significant decrease in expression of GZMB and PRF1 was observed in standard of care group

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(p=0.0034 and 0.0064, respectively) (Table 3). Perioperative steroid bolus administration was associated with the decreased expression of

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NK cells derived SH2D1B (SH2 domain-containing protein 1B) at POD 7 in all 3 groups, and its expression returned to pretransplant levels just in steroids avoidance group at POD 14 (Table 3). This fact clearly demonstrates that steroid free period allowed faster reestablishment of

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pretransplant NK cell immunity. The expression of B-cell activating factor (BAFF), an essential factor for B cells growth, was not elevated by steroid treatment. Steroids given in the standard of care group increased the expression of BAFF-R (high affinity receptor of B-cell activating factor) at POD 14 (p=0.044) compared to both steroid avoidance and withdrawal regimens. No differences were found in the expression of the other two BAFF receptors (TACI, BCMA) and of previously described B cell associated tolerance markers MS4A1 (cell surface antigen of B lymphocytes), TCL1A, HS3ST1, PNOC, CD79B, FCRL1, FCRL2 and of an anti-inflammatory IL-10 cytokine among groups.

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ACCEPTED MANUSCRIPT 4. DISCUSSION In this study we showed the capability of low dose initial steroids to reduce peripheral some of Th1 genes transcripts at first posttransplant week. This observation has clinical relevance as steroid free protocols were shown to be associated with the increased risk of acute rejections in kidney transplantation (2, 6, 7). Our study aimed to monitor rejection and tolerance associated transcripts in a subset of low risk patient cohort enrolled into large prospective international study (9). This large two arms study clearly showed that even patients who received steroids for 10 days

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after transplantation had lower rejection rate than patients without steroids (8.7% vs 13.6%, p=0.006). Although rejection occurred rarely in our cohorts and small rejection number doesn’t allow

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further statistical evaluation, clearly, higher Th1 transcripts in steroid free regimens may represent

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underimmunosuppression and rejection risk that might be confirmed while using much larger patient cohort. The high rejection occurrence observed in steroid free immunosuppression in high risk

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groups (21) further supports this hypothesis.

Nevertheless, some clinical studies have shown a relative safety of early steroids withdrawal in low risk kidney transplant recipients (8). In our study, all patients were at low risk (defined as panel

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reactive antibodies lower than 20%) and received basiliximab induction and maintenance immunosuppressive therapy consisting of once daily tacrolimus and mycophenolate mofetil. Higher expressions of genes associated with rejection and inflammation (GZMB, PRF1 and IL-2) were

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observed in peripheral blood of patients in both steroid avoidance and steroid withdrawal arms

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within 14 days after transplantation. Within the first week after transplantation the magnitude of alloimmune response is clearly greater and steroid avoidance might be less beneficial for some patients. Our results also supports the findings of recent Cochrane metaanalysis showing significantly

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higher risk of acute rejection in patients treated with steroids for less than 14 days after transplantation (22). There were no differences in evaluated transcripts at later time (≥3 months).

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We cannot rule out lower alloimmune response or minimal effects of low dose steroids in the standard of care group (23). Our findings correspond to Gaber study (2) showing that patients randomized to early steroid withdrawal had statistically significantly more rejections during first 90 days after transplantation. In our cohort steroids significantly decreased the absolute numbers of NK cells early after transplantation suggesting their protective role. The increased expression of three cytotoxic effector molecules (granulysin, granzyme and perforin) in steroid free regimen was observed early after transplantation in another study (24), however patients in steroid free arm received additional extended daclizumab for 3 weeks while not in steroid-treated patients, therefore the effect of antiIL2R antagonist on Th1 transcripts cannot be ruled out.

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ACCEPTED MANUSCRIPT In our study, no influence of steroids withdrawal or avoidance on expression of IFNγ was found. IFNγ overexpression has been observed in acute allograft rejection in earlier studies (25, 26), however, direct association of IFNγ transcripts with rejection was not confirmed by microarray studies (27-29). In the present study no changes in IL-10 transcripts, a cytokine associated with regulation and Tregs function, in peripheral blood were shown. This finding is in line with the observation of others (30). Recently, B cell related markers of operational tolerance were described (16, 17) and their possible role was further validated in rejection free immunosuppression-treated

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patients (18, 31, 32). In our study the effects of steroids on B-cell related tolerance markers in kidney transplant recipients were evaluated for the first time. Steroids decreased the expression of NK cell

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derived SH2D1B (SH2 domain-containing protein 1B) early after transplantation. In later time points

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there were no differences among treatment groups, suggesting that initial high dose of steroids might prevent the expression of SH2D1B found in operationally tolerant patients later than 1 year after transplantation (20). SH2D1B promotes NK cell activation (33), however in cooperation with

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CD84 it might also participate in the activation of memory B cells (34). The decreased expression of SH2D1B in the peripheral blood of patients suffering from acute kidney allograft rejection (35, 36)

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and increased expression in operationally tolerant patients (17, 20) also suggest its effects in the regulation of alloimmune response. Steroids were shown in vitro to suppress B cell proliferation, differentiation and IgG production (37). Contrary to peripheral blood, intragraft SH2D1B expression

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was increased during rejection and this observation is in line with other reports (38, 39). Even though

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the sample size of our study is small, the decrease of NK cells and rejection associated markers in peripheral blood of steroids treated patients is obvious. Rigorous inclusion and exclusion criteria for ADVANCE study, also used for selection of control standard of care group, prevent the influence of

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potential confounding factors.

These results suggest that initial steroids enhance immunosuppression through inhibiting

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expression of T cell effector molecules despite basiliximab induction and tacrolimus/mycophenolate mofetil concomitant therapy. Interestingly, later steroid withdrawal might be advantageous as in both steroid free regimens some of operational tolerance B-cell associated markers were actually increased. Such observation however needs further clinical validation in a larger cohort.

ACKNOWLEDGEMENTS This study was supported by the Grant Agency of the Czech Republic (No. P301/11/1568), by Ministry of Health of the Czech Republic (MZO 00023001) and by the Grant Agency of the 12

ACCEPTED MANUSCRIPT Ministry of Health of the Czech Republic (No. 15-26865A). The authors thank Romana Polackova, Marie Kolarova, Katarina Barcikova and Eva Faberova for their technical assistance and coordination of the sample collection as well as to the patients and nurses for their cooperation and help. We also thank to Marek Cernoch for English correction.

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15. Muthukumar T, Dadhania D, Ding RC, Snopkowski C, Naqvi R, Lee JB, et al. Messenger RNA for FOXP3 in the urine of renal-allograft recipients. New Engl J Med. 2005;353(22):2342-51. 16. Sagoo P, Perucha E, Sawitzki B, Tomiuk S, Stephens DA, Miqueu P, et al. Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans. J Clin Invest. 2010;120(6):1848-61. 17. Newell KA, Asare A, Kirk AD, Gisler TD, Bourcier K, Suthanthiran M, et al. Identification of a B cell signature associated with renal transplant tolerance in humans. J Clin Invest. 2010;120(6):183647. 18. Viklicky O, Krystufkova E, Brabcova I, Sekerkova A, Wohlfahrt P, Hribova P, et al. B-cellrelated biomarkers of tolerance are up-regulated in rejection-free kidney transplant recipients. Transplantation. 2013;95(1):148-54. 19. Haas M, Sis B, Racusen LC, Solez K, Glotz D, Colvin RB, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant. 2014;14(2):272-83. 20. Sagoo P, Perucha E, Sawitzki B, Tomiuk S, Stephens DA, Miqueu P, et al. Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans. J Clin Invest. 2010;120(6):1848-61. 21. Kasiske BL, Chakkera HA, Louis TA, Ma JZ. A meta-analysis of immunosuppression withdrawal trials in renal transplantation. J Am Soc Nephrol. 2000;11(10):1910-7. 22. Haller MC, Royuela A, Nagler EV, Pascual J, Webster AC. Steroid avoidance or withdrawal for kidney transplant recipients. Cochrane Database Syst Rev. 2016(8):CD005632. 23. Vitalone MJ, O'Connell PJ, Wavamunno M, Fung CL, Chapman JR, Nankivell BJ. Transcriptome changes of chronic tubulointerstitial damage in early kidney transplantation. Transplantation. 2010;89(5):537-47. 24. Satterwhite T, Chua MS, Hsieh SC, Chang S, Scandling J, Salvatierra O, et al. Increased expression of cytotoxic effector molecules: different interpretations for steroid-based and steroidfree immunosuppression. Pediatr Transplant. 2003;7(1):53-8. 25. Kirk AD, Bollinger RR, Finn OJ. Rapid, Comprehensive Analysis of Human Cytokine MessengerRna and Its Application to the Study of Acute Renal-Allograft Rejection. Hum Immunol. 1995;43(2):113-28. 26. Nast CC, Zuo XJ, Prehn J, Danovitch GM, Wilkinson A, Jordan SC. Gamma-Interferon GeneExpression in Human Renal-Allograft Fine-Needle Aspirates. Transplantation. 1994;57(4):498-502. 27. Sarwal M, Chua MS, Kambham N, Hsieh SC, Satterwhite T, Masek M, et al. Molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling. New Engl J Med. 2003;349(2):125-38. 28. Flechner SM, Kurian SM, Head SR, Sharp SM, Whisenant TC, Zhang J, et al. Kidney transplant rejection and tissue injury by gene profiling of biopsies and peripheral blood lymphocytes. American Journal of Transplantation. 2004;4(9):1475-89. 29. Reeve J, Einecke G, Mengel M, Sis B, Kayser N, Kaplan B, et al. Diagnosing Rejection in Renal Transplants: A Comparison of Molecular- and Histopathology-Based Approaches. American Journal of Transplantation. 2009;9(8):1802-10. 30. Chakraborty A, Blum RA, Mis SM, Cutler DL, Jusko WJ. Pharmacokinetic and adrenal interactions of IL-10 and prednisone in healthy volunteers. J Clin Pharmacol. 1999;39(6):624-35. 31. Rebollo-Mesa I, Nova-Lamperti E, Mobillo P, Runglall M, Christakoudi S, Norris S, et al. Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment? Am J Transplant. 2016. 32. Moreso F, Torres IB, Martinez-Gallo M, Benlloch S, Cantarell C, Perello M, et al. Gene expression signature of tolerance and lymphocyte subsets in stable renal transplants: results of a cross-sectional study. Transpl Immunol. 2014;31(1):11-6. 33. Perez-Quintero LA, Roncagalli R, Guo H, Latour S, Davidson D, Veillette A. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cgamma, Ca++, and Erk, leading to granule polarization. J Exp Med. 2014;211(4):727-42. 14

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34. Tangye SG, van de Weerdt BC, Avery DT, Hodgkin PD. CD84 is up-regulated on a major population of human memory B cells and recruits the SH2 domain containing proteins SAP and EAT2. Eur J Immunol. 2002;32(6):1640-9. 35. Flechner SM, Kurian SM, Head SR, Sharp SM, Whisenant TC, Zhang J, et al. Kidney transplant rejection and tissue injury by gene profiling of biopsies and peripheral blood lymphocytes. Am J Transplant. 2004;4(9):1475-89. 36. Sarwal M, Chua MS, Kambham N, Hsieh SC, Satterwhite T, Masek M, et al. Molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling. N Engl J Med. 2003;349(2):125-38. 37. Haneda M, Owaki M, Kuzuya T, Iwasaki K, Miwa Y, Kobayashi T. Comparative analysis of drug action on B-cell proliferation and differentiation for mycophenolic acid, everolimus, and prednisolone. Transplantation. 2014;97(4):405-12. 38. Dominy KM, Roufosse C, de Kort H, Willicombe M, Brookes P, Behmoaras JV, et al. Use of Quantitative Real Time Polymerase Chain Reaction to Assess Gene Transcripts Associated With Antibody-Mediated Rejection of Kidney Transplants. Transplantation. 2015;99(9):1981-8. 39. Hidalgo LG, Sis B, Sellares J, Campbell PM, Mengel M, Einecke G, et al. NK Cell Transcripts and NK Cells in Kidney Biopsies from Patients with Donor-Specific Antibodies: Evidence for NK Cell Involvement in Antibody-Mediated Rejection. American Journal of Transplantation. 2010;10(8):181222.

Figure legends

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Figure 1 Significant time-dependent changes of CD19 and NK cells in particular treatment

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group. Differences were calculated by paired Friedman test.

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TABLE 1. Patient´s characteristics Standard of care (n=10)

p

44 [20; 65] 10/4

46 [18; 63] 6/3

56 [28; 67] 8/2

0.231 0.800

46.7 [36.6; 64.2] 12/2 27.5 [23.8; 36.7] 2.5 [1; 5]) 2 [0; 16]) 795.5 [156; 2746] 17.1 [14.7; 22.9]

53.2 [40.9; 68.2] 7/2 28.8 [21.1; 33.9] 2 [0; 5] 0 [0; 8] 760.5 [209; 1225] 16.1 [13.5; 24]

61.9 [42.4; 69.1] 7/3 27.9[20.7; 33.9] 3.5 [2; 5] 6 [0; 25] 572 [161; 1257] 19.0 [15.9; 25.6]

0.057 0.869 0.926 0.142 0.098 0.579 0.043

13/1

8/1

8/2

0.632

16.1 ±6.6 13.2 ±5.5 8.3 ± 2.6

11.3 ±4 14.7 ±5.8 10.2 ± 2.3

0.075 0.270 0.227

122.7 [79.4; 235.7] 125.2 [72.5; 311.5]

132.5 [90.6; 168.2] 112.2 [85.8; 204.3]

0.988 0.722

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16.5 ±5.9 13.6 ±6.4 9.15 ±2.8

PT

Steroid withdrawal (n=9)

143.7 [86.6; 289.2] 137,6 [72.3; 283.5]

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Donor´s characteristic Age, years* Gender (male/female), n Recipient´s characteristics Age, years* Gender (male/female) PreTx BMI* HLA mismatch* PRA, %* Dialysis vintage, days* Cold ischemia, hours* Hemodialysis/Peritoneal dialysis, n Tacrolimus blood level (µg/L) At POD 7 At POD 14 At POD 90 Serum creatinine (µmol/L) At 3 months* At 1 year*

Steroid avoidance (n=14)

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*Data are presented as medians [min; max] or as means ±SD

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Standard of care

Borderline changes

2

4

2

Acute TCMR

1

0

1

Thrombotic microangiopathy

1

0

Pyelonephritis

1

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1

0

0

Borderline changes

1

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For cause 3-12 months

Steroid withdrawal

3

0

Acute TCMR

0

1

0

Chronic TCMR

0

2

0

BKVN

0

1

2

Acute TCMR

0

0

1

0

0

0

0

0

1

Chronic TCMR

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BKVN

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3-months protocol

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For cause by 3 months

Histological finding

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Biopsy

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TABLE 2. Histological findings within first year

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Abbreviation: TCMR: T cell mediated rejection, BKVN: polyomavirus BK nephropathy (defined as positive biopsy for SV40 immunostaining)

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ACCEPTED MANUSCRIPT TABLE 3 Peripheral gene expressions of GZMB, PRF1, IL2 and SH2D1B (presented as medians of relative quantity) in particular study groups (steroid avoidance, steroid withdrawal and standard of care) at 0, 7 and 14 days after kidney transplantation. Differences among groups were calculated by Kruskal Wallis test* and differences among particular time-points in one group by paired Friedman test**.

P value **

PRF1

0 7 14

P value**

IL2

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0 7 14

P value**

SH2D1B

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P value**

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7 14

0.59 0.376 0.591 0.017

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1.1585 0.742 0.8955 0.03 1.314 0.6845 0.8745 0.03 1.752 2.8465 2.467 0.135

Standard of care

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0.826 0.197 0.277 0.005

P value *

1.5055 0.3405 0.4125 0.0034 1.6765 0.438 0.5305 0.0063 1.785 1.735 2.1 0.601

0.978 0.050 0.020

0.708 0.244 0.266 0.012

0.769 0.024 0.003

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0 7 14

Steroid withdrawal POD 10 1.119 0.451 0.775 0.029 1.198 0.68 0.814 0.154 1.888 1.845 1.537 0.569

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Steroid avoidance

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GZMB

Postoperative day

0.907 0.036 0.084 0.824 0.034 0.753

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Fig. 1

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ACCEPTED MANUSCRIPT Highlights 

Steroid avoidance in kidney transplantation is associated with higher early acute rejection rate.



Early upregulation of several Th1 associated transcripts found in the steroid avoidance

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group reflects higher susceptibility for early acute rejection.

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