Monitoring of molecular profiling of regulatory T cell biomarkers by using non- invasive strategies to predict outcome in renal transplantation

doi:10.21203/rs.3.rs-267696/v1

Background. T-cell mediated acute rejection (aTCMR) is still an issue in kidney transplantation for it is associated with chronic rejection, graft loss, and overall worse outcomes. For these reasons, a standard non-invasive molecular tool to detect is desirable to offer a simpler monitoring of kidney transplant recipients (KTRs). The purpose of our study was to examine, in peripheral blood before and after transplantation, the expression patterns of regulatory T cell (Treg)-related genes: the forkhead box P3 (FOXP3) and the two CTLA-4 isoforms (full-length and soluble) to predict aTCMR onset, de novo donor-specific antibodies (DSA) development and renal dysfunction one year after transplantation.

Methods. We profiled by using a relative quantification analysis (qRT-PCR 2-∆∆CT) the circulating mRNA levels of these biomarkers in peripheral blood of 89 KTRs within the first post-transplant year (at baseline and 15, 60, 365 days after transplantation and when possible at acute rejection) and compared the results with 24 healthy controls.

Results. The three mRNA levels drastically reduced 15 days after transplantation and gradually recovered at one year in comparison with baseline, with very low levels by the time of aTCMR for FOXP3 (RQ=0.445, IQR=0.086-1.264, p=0.040). Healthy controls exhibited higher FOXP3 levels than patients at baseline (median RQ: 2.132, IQR=1.664-2.895 vs. p=0.005). Noteworthy, solCTLA-4 displayed a dual profile: on the one hand, at multivariate analysis solCTLA-4 transcripts at 15 days were associated with an increased risk of aTCMR over time (HR=3.905, 95%CI: 0.958-15.916, p=0.050), and graft dysfunction at one year (AOR=3.683, 95%CI=1.165-12.079, p=0.027); on the other hand, pre-transplant levels showed a protective association with de novo DSAs development (HR=0.189, 95%CI=0.078-0.459, p<0.001).

Conclusions. Peripheral blood mRNA levels of Treg-associated molecules might help shape immunosuppression, tailor monitoring and achieve a better long-term clinical course of kidney transplantation in the wake of “precision medicine”. While the potential of these molecules as a therapeutic target needs further investigation, we found out preliminary evidence that solCTLA-4 can qualify as a candidate non-invasive biomarker of cellular and humoral alloreactivity in clinical transplantation.

Urology & Nephrology

CTLA-4

FOXP3

RT-PCR

kidney transplantation

cellular acute rejection

DSA development

graft dysfunction.

Table 1: Demographic and clinical characteristics of the transplant study groups

Variables	Frequencies N (%)
Recipient gender: - males - females	65 (73%) 24 (27%)
Recipient age (years, mean±SD)	52.5±11.5
Donor age (years, mean±SD)	50.9±15.9
Donor gender: - males - females	59 (66%) 30 (34%)
Donor type: - brain-dead donors - living donors	84 (94.3%) 5 (5.7%)
Time on RRT (months, mean±SD)	54.3±35.0
RRT type: - haemodialysis - peritoneal dialysis	74 (83.1%) 15 (16.9%)
No of HLA mismatches (median and IQR)	3MM (1-5)
Class I PRA (%, mean±SD)	4.4±11.1
Class II PRA (%, mean±SD)	1.8±7.8
CIT (minutes, mean±SD)	630±265
WIT (minutes, mean±SD)	44±13
Induction: - basiliximab - anti-thymocyte globulins (rATG)	82 (92%) 7 (8%)
Calcineurin inhibitor: - cyclosporine - tacrolimus	14 (16%) 75 (84%)
Proliferation signal inhibitor: - everolimus - mycophenolic acid	12 (13%) 77 (87%)
CMV-positive donor/CMV-negative recipient	11 (12.4%)
Previous transplantation	6 (6.7%)
Delayed graft function	32 (36%)

Abbreviations: CIT, cold ischaemia time; CMV, cytomegalovirus; HLA, human leukocyte antigen; IQR, interquartile range; PRA, panel-reactive antibodies, RRT, renal replacement therapy; SD, standard deviation; WIT, warm ischaemia time.

Table 2 - Comparison of immunological biomarkers (log 2^-DCT), demographic and clinical parameters in the two groups of patients with aTCMR within 1 year (n=11) or stable transplant (n=78).

VARIABLE	aTCMR (n=11)	aTCMR-free (n=78)	P value	OR=
^aFOXP3, baseline	1.580 (1.410-1.670)	1.585 (1.370-1.760)	0.909
^aFOXP3, 15d	1.150 (0.718-1.675)	1.260 (0.950-1,510)	0.999
^aFOXP3, 60d	1.345 (1.003-1.648)	1.180 (0,940-1,390)	0.284
^aFOXP3, 1y	1.320 (1.030-1.420)	1.350 (1,095-1,423)	0.776
^aflCTLA-4 baseline	0.910 (0.730-0.980)	0.810(0.600-1.008)	0.586
^aflCTLA-4 15d	0.655 (0.010-1.093)	0.570 (0.340-0.740)	0.691
^aflCTLA-4 60d	0.585 (0.320-1.020)	0.630 (0.440-0.870)	0.999
^a flCTLA-4 1y	0.870 (0.740-1.260)	0.730 (0.508-0.863)	0.210
^asolCTLA-4 baseline	0.320 (0.060-0.590)	0.235 (0.035-0.443)	0.376
^asolCTLA-4 15d	0.365 (-0.073-0.648)	-0.070 (-0.440-0.280)	0.043
^asolCTLA-4 60d	0.120 (-0.170-0.230)	0.155 (-0.190-0.363)	0.539
^asolCTLA4 1y	0.070 (-0.530-0.670)	0.110 (0.060-0.500)	0.999
Recipient gender (%): -males -females	8/11 (72.7) 3/11 (27.3)	57/78 (73.1) 21/78 (26.9)	1.000
Donor gender (%): -males -females	6/11 (54.5) 5/11 (45.5)	53/78 (67.9) 25/78 (32.1)	0.498
^bRecipient age	54.7±14.3	52.2±11.1	0.372
^b Donor age	51.8±20.4	50.8±15.3	0.747
Donor type (%): -brain-dead donors -living donors	11/11 (100) 0/11 (0)	73/78 (93.6) 5/78 (6.4)	1.000
Delayed graft function (%)	4/11 (36.4)	28/78 (35.9)	1.000
Induction (%) : -Basiliximab -Anti-thymocyte globulin	8/11 (72.7) 3/17 (27.3)	74/78 (94.9) 4/78 (5.1)	0.038	0.144
Proliferation signal inhibitor (%): -Everolimus -Mycophenolic acid	5/17 (29.4) 12/17 (70.6)	7/72 (9.7) 65/72 (90.3)	0.048	3.869
Calcineurin inhibitor (%): -Cyclosporine -Tacrolimus	2/11 (18.2) 9/11 (81.8)	12/78 (15.4) 66/78 (84.6)	0.682
^b HLA Mismatches (MM)	3.8 ± 1.4	3.0±1.2	0.050
PRA n= (%) -Class I -Class II	2/11 (18.2) 2/11 (18.2)	34/78 (43.6) 25/78 (32.1)	0.188 0.493
^b Time on RRT (months)	51.5±26.6	54.7±36.1	0.963
^b CIT (min)	717.0±218.6	617.9 ±269.4	0.212
^b WIT (min)	48.0±16.5	43.4±12.1	0.416
Previous transplants (%)	1/11 (9.1)	5/78 (6.4)	0.558
Maintenance therapy change %	6/11 (54.5)	13/78 (16.7)	0.010	6.000
CMV reactivation (%)	5/11 (45.5)	17/78 (21.8)	0.131

a(median±interquartiles), b (mean±SD)

Table 2A - Univariate and multivariate Cox regression analyses for the risk of aTCMR after kidney transplantation.

VARIABLE	Univariate analysis			Multivariate analysis^a -2ln likelihood: 46.938
	HR	95% CI	P	HR	95% CI	P
Baseline
Membrane CTLA4	1.178	0.366—3.788	0.784	—	—	—
Soluble CTLA4	2.382	0.744—7.624	0.144
FOXP3	0.691	0.196—2.433	0.565	—	—	—
At 15 days
Membrane CTLA4	1.053	0.321—3.457	0.932	—	—	—
Soluble CTLA4	3.164	0.836—11.981	0.090	3.905	0.958-15.916	0.057
FOXP3	0.698	0.202—2.408	0.569	—	—	—
At 60 days
Membrane CTLA4	2.537	0.480—13.404	0.273	—	—	—
Soluble CTLA4	1.431	0.362—5.661	0.609	—	—	—
FOXP3	4.343	0.868—21.715	0.074	—	—	—
At 365 days
Membrane CTLA4	1.898	0.160—22.480	0.611	—	—	—
Soluble CTLA4	0.711	0.056—8.955	0.792	—	—	—
FOXP3	0.749	0.095—5.895	0.783	—	—	—
Recipient age	1.007	0.963—1.053	0.758	—	—	—
Donor age	0.999	0.969—1.031	0.972	—	—	—
Recipient gender	1.128	0.363—3.506	0.835	—	—	—
Donor gender	0.930	0.337—2.567	0.889	—	—	—
Type of dialysis	0.575	0.185—1.790	0.340	—	—	—
Dialysis duration	0.752	0.398—1.424	0.382	—	—	—
CMV reactivation	2.033	0.738—5.604	0.170	—	—	—
Previous transplant	1.466	0.973—2.210	0.067	—	—	—
HLA mismatch	1.229	0.885—1.707	0.219	—	—	—
CIT (min)	0.799	0.251—2.544	0.704	—	—	—
WIT (min)	1.000	0.998—1.002	0.859	—	—	—
Type of donor	1.030	0.992—1.069	0.120	—	—	—
Type of induction	0.259	0.082—0.814	0.021	0.148	0.024-0.923	0.041
Chronic use of steroids	0.667	0.213—2.093	0.488	—	—	—
CNI (tacrolimus vs. cyclosporine)	23.310	0.011-51229.762	0.423	—	—	—
PSI (mTOR vs. mycophenolate)	3.926	1.425—10.813	0.008	7.017	1.714-28.725	0.007
Immunosuppression change	9.175	3.178—26.488	0.000	—	—	—
Infratherapeutic CNI level at 15 days	0.821	0.291—2.311	0.708	—	—	—
Infratherapeutic CNI level at 60 days	1.303	0.483—3.514	0.601	—	—	—
DGF	0.729	0.252—2.114	0.561	—	—	—
^a Model summary: χ²(1)=15.901, p<0.001. Covariates initially introduced in the multivariable model and then elided were: soluble CTLA4 at 15 days, FOXP3 at 60 days. Abbreviations: CI, confidence intervals; CIT, cold ischemia time; CMV, cytomegalovirus; CNI, calcineurin inhibitor; DGF, delayed graft function; HLA, human leukocytes antigens; HR, hazard ratio; PRA, panel-reactive antibodies; mTORi, mammalian target of rapamycin inhibitor; PSI, proliferation signal inhibitor; WIT, warm ischemia time.

Table 3 - Univariate and multivariate Cox regression analyses for the risk of development of de novo donor-specific antibodies after kidney transplantation (p<0.05).

VARIABLE	Univariate analysis			Multivariate analysis^a -2ln likelihood: 103.840
	HR	95% CI	P	HR	95% CI	P
Baseline
Membrane CTLA4	0.479	0.181—1.263	0.137	—	—	—
Soluble CTLA4	0.296	0.126—0.694	0.005	0.189	0.078-0.459	<0.001
FOXP3	1.603	0.467—5.501	0.453	—	—	—
At 15 days
Membrane CTLA4	0.810	0.352—1.862	0.619	—	—	—
Soluble CTLA4	0.399	0.196—0.810	0.011	—	—	—
FOXP3	1.494	0.579—3.851	0.406	—	—	—
At 60 days
Membrane CTLA4	0.413	0.120—1.427	0.162	—	—	—
Soluble CTLA4	0.205	0.074—0.563	0.002	—	—	—
FOXP3	1.194	0.370—3.854	0.767	—	—	—
At 365 days
Membrane CTLA4	0.969	0.216—4.356	0.967	—	—	—
Soluble CTLA4	1.570	0.350—7.045	0.556	—	—	—
FOXP3	1.826	0.304—10.972	0.510	—	—	—
Recipient age	0.975	0.943—1.008	0.132	—	—	—
Donor age	1.003	0.977—1.029	0.847	—	—	—
Recipient gender	1.240	0.460—3.341	0.671	—	—	—
Donor gender	0.507	0.224—1.151	0.104	—	—	—
Type of dialysis	1.378	0.408—4.659	0.606	—	—	—
Dialysis duration	1.005	0.994—1.016	0.383	—	—	—
Don CMV IgG+/Rec CMV IgG-	1.201	0.355—4.060	0.768	—	—	—
Previous transplant	0.676	0.091—5.025	0.702	—	—	—
HLA mismatch	0.956	0.694—1.318	0.785	—	—	—
CIT (min)	0.999	0.998—1.001	0.411	—	—	—
WIT (min)	0.999	0.969—1.031	0.960	—	—	—
Type of donor	3.235	0.949—11.025	0.061	—	—	—
Type of induction	1.321	0.308—5.659	0.708	—	—	—
Chronic use of steroids	1.366	0.183—10.183	0.761	—	—	—
CNI (tacrolimus vs. cyclosporine)	0.743	0.218—2.529	0.635	—	—	—
PSI (mycophenolate vs. mTOR)	1.550	0.527—4.562	0.426	—	—	—
Immunosuppression change	1.330	0.524—3.376	0.549	—	—	—
Infratherapeutic CNI level at 15 days	1.020	0.430—2.424	0.964	—	—	—
Infratherapeutic CNI level at 60 days	1.002	0.442—2.275	0.996	—	—	—
DGF	0.958	0.406—2.263	0.922	—	—	—
CMV reactivation	0.605	0.206—1.780	0.361	—	—	—
^a Model summary: χ²(1)=14.217, p<0.01. Covariates initially introduced in the multivariable model and then elided were: soluble CTLA4 at 15 days, soluble CTLA4 at 60 days. Abbreviations: CI, confidence intervals; CIT, cold ischemia time; CMV, cytomegalovirus; CNI, calcineurin inhibitor; DGF, delayed graft function; HLA, human leukocytes antigens; HR, hazard ratio; PRA, panel-reactive antibodies; mTORi, mammalian target of rapamycin inhibitor; PSI, proliferation signal inhibitor; WIT, warm ischemia time.

Table 4 - Immunological, demographic and clinical parameters in the two groups of patients with or without graft dysfunction (eGFR<60ml/min/1.73m² ) one year after transplantation.

VARIABLE	Graft dysfunction (N=50)	No dysfunction (N=36)	P value	OR=
FOXP3
Baseline^a	1.590 (1.408-1.823)	1.520 (1.295-1.678)	0.033
15 days^a	1.270 (0.999-1.520)	1.125 (0.510-1.435)	0.156
60 days^a	1.270 (0.970-1.398)	1.160 (0.940-1.430)	0.952
One year^a	1.420 (1.280-1.660)	1.215 (1.015-1.363)	0.055
flCTLA-4
Baseline^a	0.850 (0.623-1.060)	0.825(0.580-0.980)	0.713
15 days^a	0.610 (0.365-0.845)	0.550 (-0.020-0.740)	0.072
60 days^a	0.620 (0.443-0.870)	0.645 (0.285-0.913)	0.763
One year^a	0.890 (0.2800-1.290)	0.740 (0.640-0.800)	0.790
solCTLA-4
Baseline^a	0.240 (0.105-0.443)	0.280 (-0.125-0.508)	0.457
15 days^a	-0.050 (-0.225-0.340)	-0.190 (-0.700-0.260)	0.039
60 days^a	0.145 (-0.008-0.370)	0.090 (-0.230-0.330)	0.559
One year^a	0.500 (-0.06-0.633)	0.100 (0.005--0.318)	0.402

Recipient gender (M/F)	37/50	25/36	0.825
Donor gender (M/F)	29/50	27/36	0.161
Recipient age^b	55.7±10.4	47.4±11.3	0.0002
Donor age^b	57.9±12.6	40.6± 15.1	<0.0001
Induction: - Basiliximab (%) - Anti-thymocyte globulins (%)	48/50 (96.0) 2/50 (4.0)	31/36 (86.1) 5/36 (13.9)	0.124
CNI (cyclosporine vs. tacrolimus)	10/50 (20.0)	4/36 (11.1)	0.378
mTORi (%)	8/50 (16.0)	4/36 (11.1)	0.754
HLA mismatches^b	3.1±1.1	3.0±1.5	0.785
Class I PRA (%)	14/50 (28.0)	19/36 (52.8)	0.035
Class II PRA (%)	10/50 (20.0)	14/36 (38.9)	0.092
Time on dialysis (months)^b	58.4±36.9	46.4±26.4	0.160
Cold ischemia time (min)^b	632.4±294.1	620.2±227.4	0.836
Warm ischemia time (min)^b	43.1±14.0	44.8±11.1	0.175
Previous transplants (%)	5/50 (10.0)	1/36 (2.8)	0.394
Maintenance therapy change (%)	13/50 (26.0 )	6/36 (16.7)	0.443
Proteinuria at one year (mg/l)^b	159.9±180.4	148.2±156.8	0.564
DSA development (%)	12/50 ( 24.0)	7/36 (19.4)	0.811
DGF (%)	21/50 (42.0)	10/36 (27.8)	0.260
CMV reactivation (%)	13/50 (26.0)	8/36 (22.2)	0.457

^a (median±interquartiles), ^b(mean±SD), ^c Mann Whitney U test. Real-time biomarkers values expressed as log 2^∆CT. Graft dysfunction is defined as an eGFR<60ml/min/1.73m² one year after transplantation.

Table 4A - Univariable and multivariable logistic regression for the risk of graft dysfunction at one year from kidney transplantation.

Variables	Univariable analysis			Multivariable analysis^a -2log likelihood: 69.886
	OR	95% CI	P	AOR	95% CI	P
Baseline
Full-length CTLA4	2.033	0.643-6.428	0.227	—	—	—
Soluble CTLA4	2.610	0.882-7.729	0.083	—	—	—
FOXP3	3.111	0.869-11.133	0.081	—	—	—
At 15 days
Full-length CTLA4	2.462	0.897-6.752	0.080	—	—	—
*Soluble CTLA4*	2.628	1.015-6.800	0.046	3.683	1.145-11.845	0.029
FOXP3	2.301	0.836-6.331	0.107	—	—	—
At 60 days
Full-length CTLA4	1.962	0.519-7.412	0.320	—	—	—
Soluble CTLA4	1.726	0.559-5.328	0.343	—	—	—
FOXP3	1.132	0.376-3.402	0.826	—	—	—
At one year
Full-length CTLA4	1.356	0.163-11.318	0.778	—	—	—
Soluble CTLA4	2.574	0.302-21.913	0.387	—	—	—
FOXP3	4.381	0.261-73.437	0.304	—	—	—
Recipient age	1.072	1.026-1.121	0.002	—	—	—
Recipient gender	1.095	0.417-2.873	0.854	—	—	—
Donor age	1.094	1.049-1.141	0.000	1.084	1.033-1.137	0.001
Donor gender	0.395	0.150-1.036	0.059	—	—	—
Type of donor	1.085	0.172-6.852	0.931	—	—	—
Previous transplantation	3.043	0.326-28.445	0.329	—	—	—
HLA mismatch	1.106	0.787-1.554	0.563	—	—	—
cRF (first class)	0.421	0.174-1.019	0.055	—	—	—
cRF (second class)	0.552	0.220-1.387	0.206	—	—	—
CIT	1.000	0.999-1.002	0.547	—	—	—
WIT	1.004	0.971-1.038	0.820	—	—	—
Type of renal replacement therapy	1.796	0.586-5.502	0.305	—	—	—
Dialysis time	1.001	0.989-1.001	0.889	—	—	—
CMV reactivation	1.230	0.449-3.371	0.688	—	—	—
Type of induction^b	0.258	0.047-1.415	0.119	—	—	—
CNI Tacrolimus vs. Cyclosporine	0.500	0.143-1.744	0.277	—	—	—
Use of everolimus	1.302	0.351-4.831	0.693	—	—	—
Immunosuppression change	3.111	0.929-10.422	0.066	—	—	—
DGF	2.534	0.965-6.658	0.059	—	—	—
Development of DSA	1.579	0.531-4.699	0.412	—	—	—
Proteinuria at one year (mg/l)	1.002	0.998-1.005	0.344
^aModel summary: χ²(3)= 25.088, p=0.000; Nagelkerke R²=0.406; Hosmer and Lemeshow χ² test=3.715, p=0.882. Covariates initially introduced in the multivariable model and then elided were: recipient age, cRF class I and DGF; ^b use of anti-thymocyte globulins vs. use of anti-IL2 receptor-α monoclonal antibodies; ^c vs. tacrolimus. Abbreviations: OR, odds ratio; CI, confidence intervals; AOR, adjusted OR; BMI, body mass index; HLA, human leukocyte antigens; CIT, cold ischemia time; WIT, warm ischemia time.

No competing interests reported.

Monitoring of molecular profiling of regulatory T cell biomarkers by using non- invasive strategies to predict outcome in renal transplantation

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