T-cell allorecognition of donor glutathione S-transferase T1 in plasma cell-rich rejection

Table 1 Patient characteristics

Group	Patient	Gender	LT date	Original disease	Baseline IS	PBMC extraction date	Years after Tx	Treatment at PBMC extraction
1	1	M	06-05-99	Alcoholic cirrhosis	CYA (N), MMF, ST	12-04-12	13	CYA (N), MMF, ST
	2	F	07-05-07	Cirrhosis probably autoimmune	CYA, MMF, ST	16-04-12	5	CYA (N), MMF, ST
	3	F	02-07-00	HCV cirrhosis	CYA (N), ST, BASILISIMAB	13-03-12	12	TAC, AZA, ST
	4	F	18-09-03	Alcoholic cirrhosis	CYA, MMF, ST	09-05-12	9	TAC, MMF, ST
	5	M	02-11-01	HCV + alcoholic cirrhosis	CYA (N), ST, BASILISIMAB	14-06-12	11	MMF, ST
	6	F	27-03-09	Primary biliary cirrhosis	CYA, MMF	12-04-12	3	MMF, ST
	7	F	18-11-06	Secondary biliary cirrhosis	CYA (N), MMF, ST	08-05-12	6	CYA, MMF
2	8	M	23-11-96	HBV cirrhosis	CYA (N), MMF, ST	19-06-12	16	CYA (N), MMF
	9	F	03-06-96	Agenesis of the bile ducts	CYA, ST	21-05-12	16	TAC
3	10	M	12-7-06	Alcoholic cirrhosis + hepatocarcinoma	TAC, MMF, ST	16-04-12	6	MMF, SIR
	11	M	12-02-09	HBV cirrhosis	TAC, MMF, ST	16-04-12	3	MMF, SIR
	12	M	06-07-10	Non-alcoholic steatohepatitis	TAC (10 d) CYA, RAPA	17-04-12	2	MMF, SIR
	13	M	19-04-11	HCV cirrhosis+ hepatocarcinoma	CYA, ST	18-04-12	1	CYA, ST
	14	M	18-01-09	HCV cirrhosis	TAC, MMF, ST	02-05-12	3	TAC
	15	F	18-06-04	Alcoholic cirrhosis	TAC	07-05-12	8	MMF, EVE
	16	M	30-07-08	HCV cirrhosis	TAC, MMF, ST	08-05-12	4	MMF, EVE
	17	M	20-12-04	HCV cirrhosis	TAC, MMF, ST	22-05-12	7	TAC, MMF
	18	F	20-09-99	HCV cirrhosis	CYA, ST	18-06-12	13	CYA
4	E	F	09-03-09	Alcoholic cirrhosis	CYA, ST	21-03-12	3	CYA, MMF
	G	M	16-11-08	HCV cirrhosis	TAC, DACLIZUMAB	30-04-12	3	CYA
	J	F	22-05-10	Hepatocarcinoma	CYA, ST	02-05-12	2	CYA
	L	M	28-07-11	Primary biliary cirrhosis	TAC, MMF, ST	19-06-12	1	TAC

HBV: Hepatitis C virus; HCV: Hepatitis C virus.

Table 2 Glutathione S-transferase T1 peptides and amino acid position and sequence

Pool	Amino acid #	Amino acid sequence
1	1-15	MGLELYLDLLSQPCR
	7-21	LDLLSQPCRAVYIFA
	13-27	PCRAVYIFAKKNDIP
	19-33	IFAKKNDIPFELRIV
	25-39	DIPFELRIVDLIKGQ
2	31-45	RIVDLIKGQHLSDAF
	37-51	KGQHLSDAFAQVNPL
	43-57	DAFAQVNPLKKVPAL
	49-63	NPLKKVPALKDGDFT
	55-69	PALKDGDFTLTESVA
3	61-75	DFTLTESVAILLYLT
	67-81	SVAILLYLTRKYKVP
	73-87	YLTRKYKVPDYWYPQ
	79-93	KVPDYWYPQDLQARA
	85-99	YPQDLQARARVDEYL
4	91-105	ARARVDEYLAWQHTT
	97-111	EYLAWQHTTLRRSCL
	103-117	HTTLRRSCLRALWHK
	109-123	SCLRALWHKVMFPVF
	115-129	WHKVMFPVFLGEPVS
5	119-133	MFPVFLGEPVSPQTL
	125-139	GEPVSPQTLAATLAE
	131-145	QTLAATLAELDVTLQ
	137-151	LAELDVTLQLLEDKF
	143-157	TLQLLEDKFLQNKAF
6	149-163	DKFLQNKAFLTGPHI
	155-169	KAFLTGPHISLADLV
	161-175	PHISLADLVAITELM
	167-181	DLVAITELMHPVGAG
	173-187	ELMHPVGAGCQVFEG
7	179-193	GAGCQVFEGRPKLAT
	185-199	FEGRPKLATWRQRVE
	191-205	LATWRQRVEAAVGED
	197-211	RVEAAVGEDLFQEAH
	203-217	GEDLFQEAHEVILKA
8	209-223	EAHEVILKAKDFPPA
	215-229	LKAKDFPPADPTIKQ
	221-235	PPADPTIKQKLMPWV
	226-240	TIKQKLMPWVLAMIR

Table 3 Specific immune response after stimulation of T lymphocytes in culture with glutathione S-transferase T1 peptides¹

Group	Pat #	CD8+			CD4+			CD4+CD8low
		IL-4	IFNγ	IL-4/IFNγ	IL-4	IFNγ	IL-4/IFNγ	IL-4	IFNγ	IL-4/IFNγ
1	1	-	-	-	1.24%	1.41%	-	Δ	Δ	Δ
	2	1.7%	-	-	2.04%	-	-	8.23%	-	3.44%
	3	4.92%	-	3.54%	4.93%	-	31.34%	10.77%	2.25%	78.95%
	4	-	-	-	-	-	-	-	-	9.54%
	5	-	-	-	-	-	-	-	-	4.56%
	6	2.36%	2.03%	1.15%	3.71%	2.45%	2.14%	Δ	Δ	Δ
	7	-	-	-	3.19%	2.35%	5.63%	Δ	Δ	Δ
2	8	-	-	-	-	-	-	Δ	Δ	Δ
	9	-	-	-	-	-	-	Δ	Δ	Δ
3	10	-	-	-	-	-	-	-	-	6.63%
	11	-	-	-	-	-	-	-	-	-
	12	-	-	-	-	-	-	-	-	-
	13	-	-	-	-	-	-	Δ	Δ	Δ
	14	-	-	-	2.65%	-	4.71%	Δ	Δ	Δ
	15	-	-	-	1.45%	-	5.18%	Δ	Δ	Δ
	16	-	-	-	-	-	-	Δ	Δ	Δ
	17	-	-	-	-	-	-	1.68%	4.6%	29.58%
	18	-	-	-	-	-	-	2.51%	4.13%	43.05%
Control	E	-	-	-	-	-	-	Δ	Δ	Δ
	G	-	-	-	-	-	-	Δ	Δ	Δ
	J	-	-	-	-	-	-	Δ	Δ	Δ
	L	-	-	-	-	-	-	Δ	Δ	Δ

¹Pathways of activation are defined by the production of IL-4, IFNγ or both cytokines simultaneously. “-“: No activation; “Δ”: Analysis was not possible due to the low number of gated cells. Group 1: don+/rec-, with Abs, with PC-rich rejection; Group 2: don+/rec-, with Abs, without PC-rich rejection; Group 3: don+/rec-, without Abs, without PC-rich rejection; Control group: don+/rec+.

Table 4 Class I and class II human leukocyte antigen genotypes of the patients

Pat #	HLA-A*		HLA-B*		DRB1*
1	01	03	07	57	11	15
2	01	66	08	41	03	13
3	11	29	07	35	07	13
4	02	11	51	60	04	13
5	30	-	13	18	03	07
6	02	11	35	44	07	08
7	26	29	38	44	01	03
8	23	24	14	52	01	11
9	11	68	35	44	01	14
10	02	33	14	35	01	07
11	01	29	57	61	01	04
12	01	33	44	64	01	07
13	01	-	08	18	04	07
14	29	-	44	-	07	-
15	01	30	08	51	03	07
16	02	29	39	44	07	11
17	03	32	37	44	03	12
18	03	11	14	49	07	-

Table 5 Identification of several regions of the glutathione S-transferase T1 protein, whose peptides would be suitable to be presented by human leukocyte antigen class I and II alleles of the patients based on in silico predictions

Recipient’s HLA	Peptide sequence	Aa position	Length	Percentile rank
Class I
A*01:01	FTLTESVAILLY1	62-73	12	0.1
A*02:01	YIFAKKNDIPFEL	18-30	13	0.1
	CLRALWHKVMFPV	110-122	13	0.1
	IKQKLMPWVLAMI	227-239	13	0.1
A*03:01	SVAILLYLTRKYK1	67-79	13	0.2
A*11:01	ESVAILLYLTRK1	66-77	12	0.1
A*29:02	FLTESVAILLY1	62-73	12	0.2
B*07:02	SPQTLAATLAEL	129-140	12	0.1
	RPKLATWRQRVEAA	188-201	14	0.2
B*08:01	FAQVNPLKKVPAL	45-57	13	0.2
	LAWQHTTLRRSCL	99-111	13	0.2
	DPTIKQKLMPWVL	224-236	13	0.2
B*35:01	YPQDLQARARVDEY	85-98	14	0.1
B*44:02	TESVAILLY1	65-73	9	0.15
	AELDVTLQL	138-146	9	0.15
Class II
DRB1*01:03	GDFTLTESVAILLYL1	60-74	15	0.6
DRB1*07:01	ALKDGDFTLTESVAI1	56-70	15	0.4
DRB1*11:01	AILLYLTRKYKVPDY1	69-83	15	0.5
DRB1*12:01	SVAILLYLTRKYKVP1	67-81	15	0.72
DRB1*13:01	LTESVAILLYLTRKY1	64-78	15	0.17
DRB1*14:01	SVAILLYLTRKYKVP1	67-81	15	0.48
DRB1*15:01	ESVAILLYLTRKYKV1	66-80	15	0.41

¹Highly antigenic areas of the GSTT1 protein whose peptides are shared by HLA class I and II alleles. GSTT1: Glutathione S-transferase T1; HLA: Human leukocyte antigen.