3.1 PBMC Recovery and Viability Remained Stable after Long-term Cryopreservation
Fifty-seven peripheral blood samples were randomly obtained from the Xijing Hospital. After obtaining PBMCs by density gradient centrifugation, the cells of each patient were divided into five tubes, the fresh sample was used for subsequent experiments, and the remaining four samples were frozen for preservation to determine whether frozen time could affect the quality of PBMCs. The total cell numbers were detected using a Bio-Rad automatic cell counter, and the total number of frozen PBMCs was significantly reduced compared with that in fresh samples (P < 0.0001). However, there was no significant change after cryopreservation for 1, 3, or 6 months, and the statistical results showed 1 m vs. 3 m, P = 0.48; 1 m vs. 6 m, P = 0.84; 3 m vs. 6 m, P = 0.11 (Figure 2A). The viability of the PBMCs was also reduced significantly after cryopreservation (P < 0.0001) but remained stable during the different cryopreservation times, the statistical results showed that 1 m vs. 3 m, P = 0.99; 1 m vs. 6 m, P = 0.10; 3 m vs. 6 m, P = 0.05 (Figure 2B), indicating that although cryopreservation affects the cell recovery efficiency and viability of PBMCs, PBMCs still maintain a stable state during long-term cryopreservation.
3.2 T Cell Subtypes in the PBMCs were not Susceptible to Long-term Cryopreservation
Peripheral blood mononuclear cells contain two kinds of immune cell subtypes, namely, innate immune cells and adaptive immune cells. To further elucidate the changes in the PBMC subtypes after cryopreservation, the expression of the immune cell markers between freshly isolated and cryopreserved PBMCs were detected by flow cytometry. Firstly, the proportion of total leukocytes showed no significant difference during long-term cryopreservation. Then, the phenotypes of the innate immune cells were observed(Figure 3A). Compared with the freshly isolated PBMCs, the number of monocytes and ILC were not only reduced significantly after cryopreservation (P < 0.01 for monocytes; P < 0.01 for ILC) but also changed dynamically during long-term cryopreservation (1 m vs. 3 m, 3 m vs. 6 m, P < 0.05, P < 0.05 for monocytes; 1 m vs. 6 m, 3 m vs. 6 m, P < 0.05, P < 0.01 for ILC) (Figure 3B, G). Further analysis of the ILC subtypes showed that ILC1 and ILC3 were affected by long-term cryopreservation (1 m vs. 3 m, 1 m vs. 6 m, 3 m vs. 6 m, P < 0.05, P < 0.05, P < 0.01 for ILC1; 1 m vs. 3 m, 1 m vs. 6 m, 3 m vs. 6 m, P < 0.01, P < 0.01, P < 0.01 for ILC3), except for ILC2 (Figure 3G), and the proportions of these subtypes in ILC were changed either during long-term cryopreservation or in freshly isolated PBMCs(Figure 3H). Although the number of natural killer (NK) cells decreased significantly compared with that of the freshly isolated cells (Table 1), the NK cells remained stable during long-term cryopreservation (Figure 3C). The phenotypes of the adaptive immune cells were observed, and there was a significant change in the percentages of T cells and B cells between the fresh and cryopreserved adaptive immune cells (Figure 3D, Table 1). However, there was no difference in the percentages of T cells and natural killer T (NKT) cells between the PBMCs cryopreserved at different times (Figure 3E, F).
Table 1
The effect of fresh isolation and cryopreservation on innate and adaptive immune cells
|
Fresh(%)
|
1 m(%)
|
|
3 m(%)
|
|
6 m(%)
|
|
|
mean(range)
|
mean(range)
|
Fresh vs.1m
P
|
mean(range)
|
Fresh vs.3m
P
|
mean(range)
|
Fresh vs.6m
P
|
Lymphocytes
|
86.6(72.2-94.9)
|
87.8(80-94.6)
|
ns
|
87.5(79.3-94.1)
|
ns
|
89.3(81.9-95.7)
|
ns
|
T
|
73.9(59.4-80.7)
|
82.2(67.1-89.5)
|
<0.0001
|
81.3(68.2-87.6)
|
<0.0001
|
81.0(67.3-88.6)
|
<0.0001
|
B
|
5.77(2.5-7.3)
|
4.56(2.4-7.0)
|
0.0004
|
5.51(3.0-7.7)
|
ns
|
4.19(1.7-7.9)
|
0.0004
|
NK
|
12.3(6.8-24.6)
|
10.0(4.1-23.0)
|
0.0136
|
7.6(3.2-19.0)
|
<0.0001
|
8.9(3.9-18.7)
|
<0.0001
|
NKT
|
1.7(0.1-9.3)
|
1.8(0.1-10)
|
ns
|
1.3(0.1-10.3)
|
ns
|
1.6(0.1-12)
|
ns
|
Monocyte
|
3.9(0.5-12.4)
|
0.2(0.0-0.8)
|
0.0004
|
0.6(0.0-1.9)
|
0.0008
|
0.2(0.0-0.8)
|
0.0004
|
ILC
|
44.1(29.9-60.2)
|
19.1(11.8-28.6)
|
0.0002
|
16.6(11.6-30.1)
|
0.0004
|
31.1(13.6-55.6)
|
ns
|
ILC1
|
36.0(25.1-54.6)
|
14.4(9.4-21.4)
|
0.0013
|
10.3(6.2-21.1)
|
0.0012
|
28.0(11.1-53.0)
|
ns
|
ILC2
|
4.3(1.8-8.1)
|
1.7(0.7-3.7)
|
0.0176
|
1.7(0.9-2.9)
|
0.0148
|
1.6(0.8-2.1)
|
0.0308
|
ILC3
|
3.9(1.7-6.3)
|
3.0(1.4-5.6)
|
ns
|
4.5(2.1-6.1)
|
ns
|
1.5(1.0-2.2)
|
0.0088
|
3.3 T cell Proportion, Apoptosis, and Proliferation were not Affected by Long-term Cryopreservation
T cell response is an important part of cellular immunity and is involved in various types of biological functions against diseases and infections. Previous results have already confirmed the stability of T cell proportion after cryopreservation for 1, 3, and 6 months, respectively. T cells can be divided into CD4+ helper T cells and CD8+ cytotoxic T cells. The percentages of CD4+ T had no difference between the freshly isolated and cryopreserved PBMCs. Interestingly, the percentages of CD8+ T cells in the cryopreserved PBMCs were significantly reduced when compared with that in freshly isolated PBMCs (fresh vs. 1 m, 3 m, 6 m, P < 0.0001, P < 0.0001, P < 0.001; 1 m vs. 3 m, 6 m, P < 0.0001, P < 0.0001), indicating that the number of T cells decreased after cryopreservation which was mainly influenced by CD8+ T cells (Figure 4A).
Although the viability of PBMCs have been proven to remain stable during cryopreservation in previous studies, cryopreservation may disrupt the integrity of the cell membrane, change the mitochondrial membrane potential, and cause cell apoptosis. The results indicated that the apoptosis of CD4+ T and CD8+ T cells were not affected by cryopreservation (Figure 4B). On the other hand, proliferation of CFSE-labeled T cells was assessed, and the proliferation capacity between the freshly isolated and cryopreserved PBMCs changed significantly after stimulation with T cell activator and IL-2 (fresh vs. 1 m, 3 m, 6 m, P < 0.05, P < 0.05, P < 0.05), but no significant change was observed after prolonging the freezing time. This change in T cells was mainly caused by the CD4+ T cells, and the proliferation of CD8+ T cells was unchanged between the freshly isolated and cryopreserved PBMCs during cryopreservation (Figure 4C). Although the proliferation ratio of the T cells was not affected by the extension of freezing time, it seemed that the passages of the T cells were changed, and after 72 h of stimulation under the same conditions, the number of proliferating cells cryopreserved for 3 or 6 months was significantly less than that of the cells cryopreserved for 1 month (Figure 4D). In the experiment, we also found that sufficient cell numbers had a great influence on the results of proliferation (data not shown).
3.4 Proportions of the Activated T Cells, Naïve T Cells, Central Memory T Cells, Effector T Cells, and Effector Memory T Cells were Dynamically Changed after Long-term Cryopreservation
Further study is necessary as there are several T cell subtypes according to their status and activation. Proportion of activated CD3+ T cells decreased significantly not only between the freshly isolated and cryopreserved PBMCs but also in the different cryopreservation times (fresh vs. 6 m, P < 0.01; 1 m vs. 3 m, 3 m vs. 6 m, P < 0.01, P < 0.0001). This change was mainly caused by CD8+ T cells (Figure 5A, Table 2).
Table 2
Effect of long-term cryopreservation on different stage of T cells
|
Fresh(%)
|
1 m(%)
|
|
3 m(%)
|
|
|
6 m(%)
|
|
|
|
|
mean(range)
|
mean(range)
|
Fresh vs.1m
P
|
mean(range)
|
Fresh vs.3m
P
|
1m vs.3m
P
|
mean(range)
|
Fresh vs.6m
P
|
1m vs.6m
P
|
3m vs.6m
P
|
CD4
|
|
|
|
|
|
|
|
|
|
|
Naïve
|
31.9(4.9-66.5)
|
30.1(12.8-63.4)
|
ns
|
31.6(15.0-48.4)
|
ns
|
ns
|
14.8(5.4-40.5)
|
<0.0001
|
<0.0001
|
<0.0001
|
Central memory
|
41.0(22.5-54.5)
|
44.3(24.3-58.6)
|
ns
|
34.1(24.1-42.3)
|
0.0013
|
<0.0001
|
44.0(27.2-58.7)
|
ns
|
ns
|
<0.0001
|
Effector
|
4.8(2.5-11.4)
|
4.8(2.4-9.7)
|
ns
|
2.9(0.9-8.8)
|
<0.0001
|
<0.0001
|
3.8(0.8-14.8)
|
0.0264
|
ns
|
ns
|
Effector memory
|
22.4(7.2-38.5)
|
20.2(8.2-30.4)
|
ns
|
31.6(15.7-50.0)
|
<0.0001
|
<0.0001
|
72.4(14.1-58.3)
|
<0.0001
|
<0.0001
|
0.0014
|
Actived
|
1.5(0.3-3.9)
|
2.1(0.6-6.1)
|
ns
|
1.4(0.6-2.5)
|
ns
|
0.0467
|
1.8(1.0-3.7)
|
ns
|
ns
|
ns
|
CD8
|
|
|
|
|
|
|
|
|
|
|
Naïve
|
27.1(5.4-52.6)
|
31.3(13.1-50.9)
|
0.0449
|
26.7(7.1-47.9)
|
ns
|
0.0003
|
16.6(3.7-46.7)
|
<0.0001
|
<0.0001
|
<0.0001
|
Central memory
|
6.5(1.9-12.5)
|
8.1(2.3-13.6)
|
0.0178
|
4.7(1.3-8.0)
|
0.0073
|
0.0001
|
6.2(1.7-14.0)
|
ns
|
ns
|
0.0091
|
Effector
|
41.0(25.6-74.6)
|
37.9(21.6-63.2)
|
0.0065
|
39.1(22.8-70.1)
|
ns
|
ns
|
45.1(24.2-79.3)
|
0.0053
|
<0.0001
|
<0.0001
|
Effector memory
|
25.3(25.6-58.2)
|
23.2(21.6-63.2)
|
ns
|
29.5(22.8-70.1)
|
0.0024
|
<0.0001
|
32.1(24.4-79.3)
|
<0.0001
|
<0.0001
|
ns
|
Actived
|
2.0(0.5-4.7)
|
1.9(0.8-4.3)
|
ns
|
1.7(0.4-4.2)
|
ns
|
ns
|
0.5(0.1-2.2)
|
<0.0001
|
<0.0001
|
<0.0001
|
T cells, whether expressing C-C chemokine receptor type 7 (CCR7) and CD45RA or not, can be divided into naïve T cells, central memory T cells, effector T cells, and effector memory T cells, and can be further divided into CD4+ T or CD8+ T cells. Compared with that in freshly isolated cells, the proportions of naïve CD4+ T cells and naïve CD8+ T cells both decreased after cryopreservation for 6 months, and these changed dynamically at different freezing times. Although the proportion of CD4+ T central memory (Tcm) and CD8+ Tcm decreased significantly at 3 months, there was no significant change in 6 months as compared to that in freshly isolated cells. The proportions of effector memory cells (Tem) in both the CD4+ or CD8+ T cells, which decreased slightly after 1 month of cryopreservation, increased significantly with the extension of the freezing time (Figure 5B, C, Table 2). The effector CD4+ and CD8+ T cells also tended to increase after a decrease in the cryopreservation time (Figure 5B, C, Table 2). These results suggest that cryopreservation resulted in a significant reduction in the proportion of naïve T cells and that other subtype T cells increased after long-term cryopreservation but still differed from that in freshly isolated samples.
3.5 Functional T Cells Remained Stable after Long-term Cryopreservation, expect for Tregs
To determine whether functional T cells were affected by long-term cryopreservation, the Th cells were marked with IFN-γ, IL-4, and IL-17, the T follicular helper cells (Tfh) were marked with CD45RO and C-X-C motif chemokine receptor 5 (CXCR5), and the Tregs were marked with CD25, CD127, and FOXP3. As expected, the functional T cells remained stable after long-term cryopreservation, and compared with that in freshly isolated PBMCs, the percentage of inflammatory T cells Th17 remained stable both before and after cryopreservation, Th1 and Th2 increased after 1 month of cryopreservation, and then remained stable after longer cryopreservation (Figure 6A–C, Table 3). The number of Tfhs decreased slightly after cryopreservation for 1 month and remained unchanged after 3 months or longer (Figure 6D, Table 3). The CD4+ T cells, which express the surface marker CD25 together with intracellular FOXP3, are Tregs. Down-modulated IL-7 receptor CD127 is also used to identify Tregs. Percentages of the CD25+CD127low Tregs reduced significantly after cryopreservation, but there was no noticeable change after long-term cryopreservation. The results of CD25+FOXP3+Tregs led to the same conclusion (Figure 6E, Table 3). The percentages of naïve Tregs and memory Tregs in the CD4+ T cells that were identified with CD45RO decreased significantly after cryopreservation and remained unchanged with increasing freezing time, and the proportions of naïve Tregs and memory Tregs in the Tregs dramatically changed (Figure 6F, Table 3).
Table 3
Effect of long-term cryopreservation on functional T cells
|
Fresh(%)
|
1 m(%)
|
|
3 m(%)
|
|
|
6 m(%)
|
|
|
|
|
mean(range)
|
mean(range)
|
Fresh vs.1m
P
|
mean(range)
|
Fresh vs.3m
P
|
1m vs.3m
P
|
mean(range)
|
Fresh vs.6m
P
|
1m vs.6m
P
|
3m vs.6m
P
|
Th1
|
10.4(5.4-19.4)
|
18.1(3.4-31.9)
|
0.0003
|
11.1(1.7-26.0)
|
ns
|
<0.0001
|
13.7(5.1-22.7)
|
ns
|
0.0293
|
ns
|
Th2
|
2.2(0.7-4.1)
|
3.5(1.0-6.1)
|
0.0048
|
1.6(1.0-3.1)
|
ns
|
0.0057
|
1.3(0.7-2.2)
|
ns
|
0.0010
|
ns
|
Th17
|
1.3(0.5-2.2)
|
1.5(0.6-2.3)
|
ns
|
1.3(0.7-1.7)
|
ns
|
ns
|
1.0(0.7-1.7)
|
ns
|
ns
|
ns
|
Tfh
|
10.4(1.9-20.8)
|
7.1(2.2-13.0)
|
0.0491
|
9.0(5.0-15.6)
|
ns
|
ns
|
7.9(3.0-15.4)
|
ns
|
ns
|
ns
|
Tregs(CD25+CD127low)
|
5.1(2.7-9.2)
|
2.6(1.6-3.9)
|
<0.0001
|
2.3(0.7-4.7)
|
<0.0001
|
ns
|
2.1(1.0-3.3)
|
<0.0001
|
<0.0001
|
ns
|
Tregs(CD25+Foxp3+)
|
3.8(2.1-8.2)
|
1.8(0.9-2.9)
|
0.0006
|
1.1(0.6-1.8)
|
<0.0001
|
0.0006
|
1.0(0.4-2.0)
|
<0.0001
|
0.0006
|
ns
|
naïve Tregs/CD4
|
0.9(0.3-2.0)
|
1.0(0.3-1.6)
|
ns
|
0.6(0.3-1.1)
|
ns
|
0.0004
|
0.4(0.1-1.0)
|
0.0026
|
<0.0001
|
0.0003
|
memory Tregs/CD4
|
3.1(1.7-7.0)
|
0.9(0.3-1.4)
|
<0.0001
|
0.4(0.2-0.7)
|
<0.0001
|
<0.0001
|
0.7(0.2-1.8)
|
<0.0001
|
ns
|
0.0099
|