Patient characteristics is summarized in Table 2. The median time from HCT was 17,4 (range 12 to 25) years. Twelve male and 8 female recipients received allo-HCT due to a variety of hematological disorders (Table 2). Eight (40%) recipients had a history of chronic GvHD. None of these recipients required active immunosuppressive treatment at the time of study enrollment. Infectious status was low in 12 recipients whereas the rest had high risk (8) infectious status according to our infectious risk stratification model (Table 1).
Table 2
Patients’ characteristics
Patient no.
|
Diagnosis
|
Sex (R/D)
|
Time since allo-HCT (years)
|
Age at allo-HCT (years)
R/D
|
Conditioning regimen
|
Chronic GvHD *
|
Infection risk status (low, high) **
|
1
|
CML
|
M/F
|
25
|
33/27
|
BuCy
|
-
|
Low
|
2
|
ALL
|
F/M
|
18
|
20/15
|
TBI
|
-
|
High
|
3
|
AML
|
M/M
|
15
|
23/25
|
BuCy
|
-
|
Low
|
4
|
AML
|
F/M
|
20
|
36/46
|
BuCy
|
Yes
|
Low
|
5
|
HES
|
M/F
|
19
|
32/33
|
BuCy
|
-
|
Low
|
6
|
CML
|
M/M
|
18
|
46/43
|
BuCy
|
-
|
Low
|
7
|
CML
|
F/F
|
17
|
22/10
|
BuCy
|
Yes
|
Low
|
8
|
PNH
|
M/M
|
18
|
27/20
|
BuCy
|
-
|
Low
|
9
|
CML
|
M/F
|
23
|
39/41
|
BuCy
|
Yes
|
High
|
10
|
AML
|
M/F
|
14
|
43/39
|
BuCy
|
Yes
|
High
|
11
|
AML
|
F/F
|
17
|
47/43
|
BuCy
|
Yes
|
High
|
12
|
CML
|
M/M
|
19
|
36/18
|
BuCy
|
-
|
High
|
13
|
ALL
|
F/M
|
24
|
28/24
|
BuCy
|
-
|
Low
|
14
|
AML
|
M/F
|
15
|
31/28
|
BuCy
|
-
|
Low
|
15
|
CML
|
M/M
|
20
|
44/43
|
BuCy
|
-
|
Low
|
16
|
MDS
|
F/F
|
12
|
42/43
|
BuCy
|
Yes
|
High
|
17
|
CML
|
F/M
|
17
|
38/43
|
BuCy
|
-
|
High
|
18
|
AML
|
F/M
|
12
|
38/38
|
BuCy
|
Yes
|
Low
|
19
|
CML
|
M/M
|
13
|
33/22
|
BuCy
|
Yes
|
High
|
20
|
AML
|
M/F
|
12
|
41/54
|
BuCy
|
-
|
Low
|
* History of chronic cGvHD |
** Status assessment according to Table 1. |
(CML – chronic myelogenous leukemia, ALL – acute lymphoblastic leukemia, AML – acute myelogenous leukemia, HES – hypereosinophilic syndrome, PNH – paroxysmal nocturnal hemoglobinuria, MDS – myelodysplastic syndrome, R – recipient, D – donor, MRD – matched related donor, MUD – matched unrelated donor, BuCy – busulfan & cyclophosphamide, TBI – total body irradiation) |
Results
Pairwise (recipients vs donors) comparison of telomeric length
Median of telomeric length, expressed in kb per chromosome end) in CD8+ lymphocytes was significantly greater in D (2,1kb [95%CI 1,8;2,7]) compared to R (1,7kb [95%CI 1,4;1,9]) (p = 0,02; n = 40). There were also similar tendencies in CD4+ and CD19+ lymphocyte subpopulations, respectively D – 2,2kb [95%CI 1,8;3,8], R – 1,6kb [95%CI 1,4;2,4] (p = 0,1; n = 40) and D – 2,3kb [95%CI 2,1;2,9], R- 2,1kb [95%CI 1,7;2,4] (p = 0,076; n = 40), although they have not reach statistical significance. We have not found differences in the CD56+ population (D – 2kb [95%CI 1,8;2,3], R – 2kb [95%CI 1,5;2,3] (p = 0,53) (n = 40)) (Fig. 1.).
Also, we have not found any statistically significant differences nor any obvious trends in TL between recipients grouped according to infection status and GvHD status (Supplementary materials – Table 1. – Table 8.).
Proinflammatory cytokine concentrations
There were no statistically significant differences in proinflammatory cytokine concentrations i.e. Il-1β, IL-2, IL-4, IL-6, IL-10, TNF-α and IL-17 in any recipient-donor pair measured at a single time point after allo-HCT (Supplementary materials – Table 9.). Additionally, we have found no statistically significant differences in proinflammatory cytokine concentrations between recipients of allo-HCT grouped according to chronic GvHD history (Supplementary materials – Table 10.).
The analysis of proinflammatory cytokine concentrations in recipients of allo-HCT, grouped according to the infection status (low risk versus high risk) (see Table 1.) has shown statistically significant differences in concentrations of IL-4 and TNF-α. The median IL-4 concentration was higher in low-risk recipients – 0,07 pg/ml (95%CI 0,03;0,15) than in high-risk recipients – 0,00 pg/ml (95% CI 0,00; 0,02) (p = 0,003). Similarly, the median TNF α concentration was also higher in. low risk recipients – 0,36 pg/ml (95%CI -0,15;2,19) than in high risk recipients – 0,22 pg/ml (95%CI 0,03;0,39) (p = 0,0449) (Table 3.).
Table 3
Proinflammatory cytokine concentrations comparison between recipients of allo-HCT grouped according to infection risk status.
|
Low risk
(n = 12)
|
High risk
(n = 8)
|
P-value
U Mann-Whitney
|
IL-2 (pg/ml)
|
|
|
0,1770
|
mean (SD)
|
0,22 (0,51)
|
0,03 (0,04)
|
|
range
|
0,00–1,83
|
0,00–0,10
|
|
median
|
0,09
|
0,01
|
|
95%CI
|
[-0,10;0,55]
|
[0,00;0,06]
|
|
IL-4 (pg/ml)
|
|
|
0,0030
|
mean (SD)
|
0,09 (0,09)
|
0,01 (0,01)
|
|
range
|
0,00–0,27
|
0,00–0,03
|
|
median
|
0,07
|
0,00
|
|
95%CI
|
[0,03;0,15]
|
[0,00;0,02]
|
|
IL-6 (pg/ml)
|
|
|
0,3348
|
mean (SD)
|
1,19 (1,50)
|
0,69 (0,20)
|
|
range
|
0,38 − 5,42
|
0,48 − 1,10
|
|
median
|
0,51
|
0,61
|
|
95%CI
|
[0,23;2,14]
|
[0,52;0,86]
|
|
IL-10 (pg/ml)
|
|
|
0,8471
|
mean (SD)
|
0,62 (0,86)
|
0,45 (0,27)
|
|
range
|
0,00–3,20
|
0,11 − 0,87
|
|
median
|
0,45
|
0,39
|
|
95%CI
|
[0,08;1,17]
|
[0,23;0,67]
|
|
TNF α (pg/ml)
|
|
|
0,0449
|
mean (SD)
|
1,02 (1,85)
|
0,21 (0,21)
|
|
range
|
0,09 − 6,78
|
0,00–0,62
|
|
median
|
0,36
|
0,22
|
|
95%CI
|
[-0,15;2,19]
|
[0,03;0,39]
|
|
IL-1B (pg/ml)
|
|
|
0,9692
|
mean (SD)
|
0,21 (0,52)
|
0,04 (0,08)
|
|
range
|
0,00–1,73
|
0,00–0,22
|
|
median
|
0,00
|
0,00
|
|
95%CI
|
[-0,12;0,54]
|
[-0,03;0,11]
|
|
IL-17F (pg/ml)
|
|
|
0,93851
|
mean (SD)
|
0,08 (0,28)
|
0,08 (0,24)
|
|
range
|
0,00–0,98
|
0,00–0,67
|
|
median
|
0,00
|
0,00
|
|
95%CI
|
[-0,10;0,26]
|
[-0,11;0,28]
|
|
Immunophenotype analysis
Median percentage of T CD4 + was significantly greater in D than in R: 44,3% (95%CI 37,2;48,3) and 40,1% (95%CI 31,9;40,8) respectively (p = 0,05; n = 34). In contrast CD19+ percentage was greater in R than in D: mean 11,3% (95%CI 9,8;13,5) and 8,5% (95%CI 7,8;11,9) respectively (p = 0,03; n = 34). (Table 4) Moreover we observed difference trends in few others lymphocyte subpopulations (p value approaching 0.05, Table 4). Among the population of CD4+ there was greater percentage of effector memory (CD4+ EM) cells in R than D: 28,8% (95%CI 23,4;37,5) and 19,8% (95%CI 16,5;27,8) (p = 0,07; n = 34) respectively and lower percentage of CD4+ naïve cells in R than D: 24,5% (95%CI 16,9;33,8) and 38% (95%CI 28,8;43,5) (p = 0,06 n = 34) respectively. Among the CD8+ subpopulation there was greater percentage of CD8+ expressing eomesodermin (CD8+ Eomes) in R – 39,4% (95%CI 29,7;47,7) than D – 31,5% (95%CI 24,2;36,7) (p = 0,07; n = 34). Among the CD19 + population there was greater percentage of B1 lymphocytes in D – 21,7% (95%CI 17;27,5) than R – 17,2% (95%CI 12,8;24,3) (p = 0,08; n = 34) and greater percentage of B2 lymphocytes in R – 81,6% (95%CI 74,4;86,4) than D – 77% (95%CI 71,1;82) ( p = 0,07; n = 34) ) (Table 4.).
Table 4
Immunophenotypic differences between recipients and donors of allo-HCT
|
R
(n = 17)
|
D
(n = 17)
|
P-value
|
CD4+
|
|
|
0.05
|
mean (SD)
|
36,4 (8,4)
|
42,8 (10,4)
|
|
range
|
19,9–49,5
|
16,8–58,4
|
|
median
|
40,1
|
44,3
|
|
95%CI
|
[31,9;40,8]
|
[37,2;48,3]
|
|
CD4+ Effector Memory
|
|
|
0.07
|
mean (SD)
|
30,4 (13,2)
|
22,2 (10,7)
|
|
range
|
13,0–59,0
|
9,0–54,1
|
|
median
|
28,8
|
19,8
|
|
95%CI
|
[23,4;37,5]
|
[16,5;27,8]
|
|
CD4+ Naive
|
|
|
0.06
|
mean (SD)
|
25,3 (15,9)
|
36,1 (13,8)
|
|
range
|
4,6–55,3
|
2,4–52,9
|
|
median
|
24,5
|
38,0
|
|
95%CI
|
[16,9;33,8]
|
[28,8;43,5]
|
|
CD8+ Eomes
|
|
|
0.07
|
mean (SD)
|
38,7 (16,3)
|
30,4 (11,2)
|
|
range
|
1,3–66,9
|
11,1–49,5
|
|
median
|
39,4
|
31,5
|
|
95%CI
|
[29,7;47,7]
|
[24,2;36,7]
|
|
CD19+
|
|
|
0.03
|
mean (SD)
|
11,7 (3,4)
|
9,8 (3,9)
|
|
range
|
7,4–20,0
|
5,9–19,5
|
|
median
|
11,3
|
8,5
|
|
95%CI
|
[9,8;13,5]
|
[7,8;11,9]
|
|
B1
|
|
|
0.08
|
mean (SD)
|
18,5 (10,8)
|
22,2 (9,8)
|
|
range
|
2,6–49,8
|
5,7–47,4
|
|
median
|
17,2
|
21,7
|
|
95%CI
|
[12,8;24,3]
|
[17,0;27,5]
|
|
B2
|
|
|
0.07
|
mean (SD)
|
80,4 (11,2)
|
76,5 (10,2)
|
|
range
|
48,1–97,2
|
50,4–93,7
|
|
median
|
81,6
|
77,0
|
|
95%CI
|
[74,4;86,4]
|
[71,1;82,0]
|
|
CD4+/CD8+ ratio
Median CD4+/CD8+ ratio was higher in donors than in recipients of allo-HCT – 2,1 (95%CI 1,3;2,1) and 1,5 (95%CI 1,8;2,6) respectively (p = 0,0396) (n = 38) (Table 5.).
Table 5
CD4+/CD8+ ratio in recipients of allo-HCT and their donors.
|
R
(n = 19)
|
D
(n = 19)
|
P-value (U Mann-Whitney)
|
CD4+ to CD8+
|
|
|
0,0396
|
mean (SD)
|
1,7 (0,9)
|
2,2 (0,9)
|
|
range
|
0,7 − 4,6
|
1,0–4,6
|
|
median
|
1,5
|
2,1
|
|
95%CI
|
[1,3;2,1]
|
[1,8;2,6]
|
|
1U Mann-Whitney |
Analysis of the recipients of allo-HCT depending on the GvHD status
Immunophenotype analysis according to GvHD and infectious status
Differences in immunophenotype were tested in recipients divided into two groups: with and without history of cGvHD history. We have found significant differences in the percentage of T Helios− with the expression of eomesodermin (T Helios− Eomes+), B1 with the expression of PD1 and B2 with the expression of PD1 – in all aforementioned the higher percentage was found in cGvHD group p-value was 0,0,227, 0,0147 and 0,0448 respectively (n = 34). In contrast, a higher percentage in the group without cGvHD was found in the population of CD19+ cells with the expression of PD1. (Table 6.).
Table 6
Immunophenotype comparison between recipients of allo-HCT grouped according to chronic GvH disease history.
Parameter
|
cGvHD
|
Without cGvHD
|
P-value
|
Treg Helios- Eomes
|
|
|
0,0227
|
mean (SD)
|
4.1 (1.3)
|
8.7 (4.8)
|
|
range
|
2.4–5.4
|
4.2–19.1
|
|
median
|
4.6
|
7.2
|
|
95%CI
|
[2.7;5.5]
|
[5.2;12.1]
|
|
B1 PD1
|
|
|
0,0147
|
mean (SD)
|
4.0 (2.7)
|
10.4 (5.5)
|
|
range
|
0.2–8.7
|
3.6–18.7
|
|
median
|
3.7
|
9.7
|
|
95%CI
|
[1.2;6.9]
|
[6.4;14.3]
|
|
B2 PD1
|
|
|
0,0448
|
mean (SD)
|
0.7 (0.7)
|
1.8 (1.8)
|
|
range
|
0.1–2.1
|
0.6–6.2
|
|
median
|
0.5
|
1.1
|
|
CD19 PD1
|
|
|
0,0147
|
mean (SD)
|
1.2 (0.9)
|
3.3 (2.3)
|
|
range
|
0.2–2.9
|
1.2–8.9
|
|
median
|
0.9
|
3.0
|
|
95%CI
|
[0.2;2.2]
|
[1.6;4.9]
|
|
Analysis of the recipients of allo-HCT depending on the infection status
Immunophenotype analysis
Differences in immunophenotype were also tested in recipients divided again into two groups: low risk and high risk of infection. We have found significant differences in the percentage of NK cells (CD56+), which was higher in low risk recipients’ group (p = 0,0344). Furthermore, among the NK cells population we have found differences in the NK cells with the expression of perforin (NK Perforin) and CD28. NK Perforin percentage was higher in low risk recipients group (p = 0,0079) and NK CD28+ percentage was higher in high risk patients group. There was also a difference in the percentage of NK dim cells – it was higher in low risk recipients group (p = 0,0344) (Table 7.).
Table 7
Immunophenotype comparison between recipients of allo-HCT grouped according to infection risk status.
Parameter
|
Low risk
|
High Risk
|
P-value
|
%NK Perforin+
|
|
|
0,0079
|
mean (SD)
|
86.4 (29.8)
|
57.9 (44.0)
|
|
range
|
2.2–99.8
|
1.4–92.3
|
|
median
|
95.2
|
82.0
|
|
95%CI
|
[65.1;107.7]
|
[11.7;104.1]
|
|
%NK CD28+
|
|
|
0,0344
|
mean (SD)
|
6.5 (8.7)
|
14.3 (9.5)
|
|
range
|
1.8–30.8
|
3.6–27.5
|
|
median
|
3.8
|
11.1
|
|
95%CI
|
[0.3;12.7]
|
[4.4;24.3]
|
|
%NK CD56dim
|
|
|
0,0433
|
mean (SD)
|
18.5 (12.6)
|
6.5 (5.8)
|
|
range
|
0.1–45.9
|
0.1–15.4
|
|
median
|
20.0
|
6.9
|
|
95%CI
|
[9.5;27.5]
|
[0.4;12.6]
|
|
%NK
|
|
|
0,0448
|
mean (SD)
|
22.1 (13.0)
|
10.5 (3.1)
|
|
range
|
9.3–52.0
|
7.1–15.3
|
|
median
|
22.3
|
9.6
|
|
95%CI
|
[12.8;31.4]
|
[6.6;14.3]
|
|