Effect Of Granulocyte Colony-stimulating Factor Mobilization On The Distribution And Clonality Of T Cell Repertoire And The Functional Subsets Of γδ T Cells

Background and ObjectivesAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary means for treatment of hematologic and non-hematologic malignancies, aplastic anemia and other diseases, and even considered to be the only way to cure some malignancies. Graft-versus-host disease (GVHD) is the major complication after allo-HSCT, and one of the most important factors that constrain the efficacy of allo-HSCT. Recently, the peripheral blood stem cells obtained from recombinant human granulocyte colony-stimulating factor (rhG-CSF) mobilized donors have been used more frequently than bone marrow stem cells as the source of stem cells in allo-HSCT. In G-CSF mobilized peripheral blood stem cell transplantation (PBSCT), despite the presence of a more than10-fold higher number of mature T cells in the graft, the incidence or severity of GVHD, especially acute GVHD (aGVHD) is not elevated compared with bone marrow transplantation. Clinical and experimental data suggested that the protective effects of G-CSF against GVHD might result from the immune modulatory effect of G-CSF on T cells, including that G-CSF directly affected T cells or indirectly modulated T cell immune responses via effector cells and cytokines. However, these mechanisms could not fully explain all the mechanisms for G-CSF inducing immune tolerance.The proliferation and activation of T cells is activated mainly through the T cell receptor/CD3complex (TCR/CD3). T cells recognize specific ligands by specific TCR, which are heterodimers comprising either α/β or γ/δ chains. T cells are comprised of two major subpopulations, identified by their expression of either the Ωα or γδ TCR heterodimer. αβ+T cells, which are the predominant circulating population and can be subdivided into cells that express CD4+or CD8+antigens, are the major antigen-presenting cells and recognize foreign peptides in context of major histocompatibility complex (MHC) molecules. γδ+T cells, which represent approximately1-10%of peripheral CD3+T cells and are predominantly CD3+CD4-CD8-T cells, recognize specific antigen without MHC-restriction and are considered as linkage between innate and adaptive immune response. It is reported that γδ+T cells play an important role in anti-infection, anti-cancer effect and immune survelliance. Due to the lack of intracellular extended area, TCR requires CD3molecules to transduce activated signals into cells, leading to activation of cytoplasmic signaling mechanisms, thereby regulating the subsequent cellular response. CD3molecules contain multiple subunits, named CD3γ, CD3δ, CD3ε, CD3ζ and CD3η, and form three distinct dimers:CD3γε, CD3δε and CD3ζζ. CD3ζ chain is considered to play a critical role in TCR signal transduction.Some specific T-cell subsets that display clonal expansion might contribute to the pathology of GVHD. Previous studies found that αβ+T cells were the primary effector cells for GVHD; skewing distribution and oligoclonal expansion of TCRVβ subfamily T cells could be found in GVHD cases. Recently, some studies have shown that γδ+T cells have immunoregulatory function, and might also participate in the pathogenesis of GVHD. However, it is still unclear whether G-CSF mobilization could influence TCR repertoire of αβ+T cells and γδ+T cells as well as T cell signal transduction molecules. To explore the effect of G-CSF on TCR repertoire and T cell signal transduction molecules, we analyzed the distribution and clonality of TCRVa, Vβ, Vγ and Vδ subfamilies and the expression levels of CD3genes in peripheral blood mononuclear cells (PBMCs) from healthy donors before and after mobilization.Recent studies have demonstrated that regulatory T cells with suppressive function are not confined to CD4+T cells; CD8+T cells, CD4-CD8-T cells and γδ+T cells also have cell subset with suppressive function. In2009, Kang et al. initiatively found regulatory y8T cells (y8Tregs) with immunosuppressive function in normal mice. Moreover, they demonstrated that y8Tregs belonged to γδ+T cells, expressed Foxp3and CD25, and had suppressive effect on the proliferation of autologous naive CD4+T cells. Meanwhile, they found y8Tregs in tumor patients, including patients with lung cancer, hypopharyngeal carcinoma, renal cell carcinoma and gastric cancer. Subsequently, the research group further showed that y8Treg cell population in peripheral blood was significantly decreased in systemic lupus erythematosus patients compared with healthy controls, and the decrease of y8Tregs was correlated with the development of disease. Therefore, they proposed that y8Tregs might be employed as a potential therapeutic target in autoimmune disease immunotherapy. Moreover, human PBMCs could be induced to generate γδ Tregs in vitro by stimulating with anti-TCRyS and transforming growth factor-β (TGF-β). However, it remains unclear whether G-CSF could influence y8Tregs. In this study, we investigated the effect of G-CSF mobilization on the functional subsets of γδ+T cells, and explored the possibility of G-CSF inducing y8Tregs in vitro.The mechanism of G-CSF-mobilized PBSCT reducing the incidence of GVHD is still unclear. Different from most studies which analyzed from angle of direct and indirect pathway, we studied from the perspective of TCR and y8Tregs. So far there are no related reports. Therefore, the purpose of this study is to indentify the effect of G-CSF on TCR repertoire, T cell signal transduction molecules and functional subsets of γδ+T cells, and to explore the possibility of G-CSF inducing γδ Tregs in vitro. This finding will not only further reveal the immunomodulatory effects of G-CSF on T cells, provide new ideas to explain the mechanism of G-CSF-mobilized PBSCT reducing the incidence of GVHD, but it will also provide new immunotherapeutic strategies to further reduce GVHD and improve survival.Methods1. The distribution and clonality of TCRVa, V(3, Vy and V8subfamilies were analyzed in PBMCs from20donors before and after G-CSF mobilization, using reverse transcription polymerase chain reaction (RT-PCR) and genescan technique. The expression levels of TCRVy Ⅰ-Ⅲ and CD3genes before and after mobilization were detected in PBMCs from20donors by real-time RT-PCR. Combined with clinical data, the association between GVHD in recipients and the alteration of clonality of TCRVa, Vα, Vγ and Vδ subfamilies after G-CSF mobilization was analyzed.2. The percentages of functional subsets of γδ+T cells were analyzed in peripheral blood from15donors before and after G-CSF mobilization by flow cytometry. Combined with clinical data, the correlation between the percentages of functional subsets of donor γδ+T cells after mobilization and GVHD, relapse as well as survival in recipients was analyzed.3. The expression levels of immunoregulatory-associated molecules before and after G-CSF mobilization were detected in PBMCs from15donors by real-time RT-PCR. The concentration of cytokines in plasma before and after mobilization was detected in15donors by Liquichip technology. 4. PBMCs of healthy donors were cultured in complete RPMI-1640medium supplemented with10%fetal calf serum (FCS) and200IU/mL interleukin-2(IL-2) in6-well culture plates precoated with immobilized anti-human TCRγδ for9-12days. In some groups, G-CSF and/or TGF-β were added. The culture system was grouped based on the difference of added cytokines, including:①G-CSF group;②TGF-β group;③G-CSF+TGF-β group;④blank control group (①-③induction experimental groups).5. The percentages of functional subsets of γδ T cells in the three induction and control groups were analyzed by flow cytometry. The expression levels of immunoregulatory-associated molecules and G-CSFR gene of anti-TCRγδ-stimulated γδT cells were detected by real-time RT-PCR. The suppressive effect of anti-TCRγδ-stimulated γδT cells on the proliferation of autologous naive CD4+T cells was detected by two methods-carboxylfluorescein diacetate succinimidyl ester (CFSE) assays combined with flow cytometry and cell counting kit-8(CCK-8) method.6. The distribution and clonality of TCRVy and V8subfamilies were analyzed in anti-TCRγδ-stimulated y8T cells, using RT-PCR and genescan technique. The expression levels of TCRVy Ⅰ-Ⅲ genes in anti-γδ-stimulated y8T cells were detected by real-time RT-PCR.7. The concentration of cytokines in cultural supernatant of anti-γδ-stimulated γδ T cells was detected by Liquichip technology. The cytotoxic effect of anti-TCRγδ-stimulated y8T cells was detected by CCK-8method. The expression levels of Perf and Grmb genes in anti-TCRγδ-stimulated y8T cells were detected by real-time RT-PCR.8. Statistical analysis:all data was analyzed using SPSS13.0. Paired-samples t test was used to compare the numbers of expressed TCRVa, Vβ, Vγ and Vδ subfamilies, the expression levels of TCRVy Ⅰ-Ⅲ and CD3genes, the percentages of functional subsets of γδ T cells, the expression levels of immunoregulatory-associated molecules and G-CSFR gene, as well as the concentration of cytokines between pre-G-CSF-mobilized and G-CSF-mobilized group. Chi-squared or Fisher exact test was used for comparison of the expression frequencies of TCRVa, Vβ, Vγ and V8subfamilies before and after mobilization. Binary logistic regression analysis was used to estimate the association between GVHD in recipients and the alteration of clonality of TCRVa, Vβ, Vγ and Vδ subfamilies after G-CSF mobilization. As the expression levels of four CD3genes did not meet normal distribution, Spearman’s correlation analysis was used to estimate the correlation between different gene expression levels from four CD3genes, and the correlation between donor age and the expression levels of CD3genes before and after G-CSF mobilization. One-way ANOVA was used for comparisons of the percentages of functional subsets of anti-TCRy8-stimulated y8T cells, the expression levels of immunoregulatory-associated molecules, G-CSFR and TCRVy Ⅰ-Ⅲ genes as well as the concentration of cytokines in anti-TCRγδ-stimulated γδ T cells, and the cytotoxic effect of anti-TCRγδ-stimulated γδ T cells. When variance was homogeneous, LSD method was used for further multiple comparisons. When variance was heterogeneous, the Welch method was corrected, and Dunnett T3method was used for further multiple comparisons. Spearman’s correlation analysis was used to estimate the correlation between the percentages of donor γδ T cells after mobilization and GVHD, relapse as well as survival in recipients. P<0.05was considered as statistically significant.Results1. The expression frequencies of TCRVa, Vβ, Vγ and V8subfamilies changed at different levels after G-CSF mobilization. Most TCRVa, Vβ, Vγ and V8subfamilies revealed polyclonality before and after mobilization. Clonal expanded T cells were identified in two cases before mobilization, distributed in TCRVa5, Vβ2, Vβ4, Vβ7and Vβ16, respectively. The clonal expanded T cells were predominantly distributed in Vβ16after mobilization, which were identified in5of20cases. Oligoclonality was detected in TCRVγ and Vδ subfamilies in3donors before mobilization and in another4donors after G-CSF mobilization, distributed in TCRVγⅡ, Vδ1, Vδ3and Vδ6, respectively. The expression levels of TCRVγⅠ, VγⅡ and VγⅢ genes after G-CSF mobilization were significantly lower than that before mobilization (t=2.758, t=3.072,t=3.388; P=0.013, P=0.006, P=0.003). The expression pattern of TCRVy Ⅰ-Ⅲ subfamilies before mobilization revealed as VγⅡ>VγⅠ>VγⅢ, and changed to VγⅠ>VγⅡ>VγⅢ after mobilization.The alteration of clonality of TCRVβ22gene repertoire after mobilization was associated with low incidence of GVHD (P=0.042, OR=12.500). Significant positive association was observed between the invariable clonality of TCRV81gene repertoire after mobilization and low incidence of GVHD in recipients (P=0.015, OR=0.047).2. The expression levels of CD3ζ, CD3γ and CD3s genes after mobilization were significantly lower than that before mobilization (t=2.724, t=2.130,t=3.303; P=0.013, P=0.046, P=0.004); while the expression level of CD38gene was similar before and after mobilization (t=1.930, P=0.069). The expression patterns of four CD3genes before and after mobilization both were CD3ζ>CD3ε>CD3δ>CD3γ.3. Compared with that before mobilization, the V81proportion was significantly increased (t=-3.939, P=0.001), whereas the proportions of total y8T cells and V82subsets were significantly decreased after G-CSF mobilization (t=2.998, t=3.240; P=0.011, P=0.006). The proportions of CD25+and CD27+subsets were also significantly increased after mobilization (t=-3.342,t=-3.077; P=0.006, P=0.015), and the proportion of Foxp3+subset was similar between the two groups (t=0.302, P=0.768). In addition, there was a significant increase in the proportions of CD27+V81, CD25+Foxp3+, CD25+CD27+and CD27+Foxp3+Vδ1subsets,t=-4.050,t=-3.027, t=-3.132, t=-3.942; P=0.017, P=0.002, P=0.011, P=0.014, P=0.003), and a significant decrease in the proportions of CD27+V82and CD25+V82subsets after mobilization (t=3.312, t=3.590; P=0.008, P=0.003). There was a negative correlation between the incidence of aGVHD in recipients and the percentages of donor CD25+CD27+Vδ1and CD27+Foxp3+V81subsets after mobilization (P=0.032, rs=-0.645; P=0.032, rs=-0.645), as well as between the incidence of chronic GVHD in recipients and the percentages of donor CD27+Vδ1, CD25+Vδ1and CD25+CD27+V81subsets after mobilization (P=0.014,rs=-0.710; P=0.021,rs=-0.589; P=0.014, rs=0.710).4. After G-CSF mobilization, the expression levels of TLR8and G-CSFR genes were significantly increased (t=-3.596, P=0.003; t=-2.224, P=0.043), the expression levels of GITR and T-bet genes were significantly decreased (t=3.035, P=0.009; t=2.644, P=0.019), and the expression levels of Foxp3, CD25, CTLA-4and GATA-3genes did not change significantly (t=0.210,t=0.992, t=1.291,t=2.009; P=0.837, P=0.338, P=0.217, P=0.064). The concentration of interleukin-10(IL-10) in plasma after mobilization was significantly higher than that before mobilization (t=-2.484, P=0.026). The concentration of interleukin-4(IL-4), interleukin-17(IL-17), interferon-y (IFN-y) and TGF-P in plasma was similar before and after mobilization (t=1.206,t=0.574,t=0.278,t=0.772; P=0.248, P=0.575, P=0.785, P=0.453).5. PBMCs of healthy donors were cultured in complete RPMI-1640medium supplemented with10%FCS and200IU/mL IL-2in6-well culture plates precoated with immobilized anti-human TCRγδ for9-12days. In some groups, G-CSF and/or TGF-β were added. After9days’ culture, γδT cells in the control group accounted for>70%of the cultured cells, most of which were Vδ2subsets. In the presence of G-CSF and/or TGF-β, the yield of y8T cells decreased, manifesting that the proportion of V82subsets decreased, whereas the proportion of Vδ1subsets increased. There were significant differences in the proportions of Foxp3+γδ T cells and Foxp3+Vδ1subsets in y8T cells in the three induction and control groups (F=7.283, F=5.512; P=0.011, P=0.024). Compared with that in the control group, the proportions of Foxp3+γδ T cells and Foxp3+Vδ1subsets in the G-CSF, TGF-β and G-CSF+TGF-P group were significantly increased (P=0.015, P=0.006, P=0.003; P=0.044, P=0.014, P=0.005). The proportions of Foxp3+γδ T cells and Foxp3+Vδ1subsets in the G-CSF group were similar to that in the TGF-β and G-CSF+TGF-β group (P=0.557, P=0.253; P=0.495, P=0.190). The proportion of Foxp3+Vδ2subsets was similar in γδ T cells in the three induction and control groups (F=1.393, P=0.314).6. There were significant differences in the proportions of CD25+Foxp3+γδ T and CD27+Foxp3+γδ T subsets in y8T cells in the three induction and control groups (F=24.092, F=25.779; P<0.001, P<0.001). Compared with that in the control group, the proportions of CD25+Foxp3+γδ T and CD27+Foxp3+γδ T subsets in the G-CSF, TGF-β and G-CSF+TGF-β group were significantly increased (P=0.001, P<0.001, P<0.001; P<0.001, P<0.001, P<0.001). The proportions of CD25+Foxp3+γδ T and CD27+Foxp3+γδ T subsets in the G-CSF group were similar to that in the TGF-β and G-CSF+TGF-β group (P=0.102, P=0.122; P=0.377, P=0.552). The difference was especially seen in Vδ1subsets. There were significant differences in the proportions of CD25+Foxp3+Vδ1and CD27+Foxp3+Vδ1subsets in y8T cells in the three induction and control groups (F=37.465, F=167.294; P<0.001, P<0.001), whereas there were no significant differences in the proportions of CD25+Foxp3+Vδ2and CD27+Foxp3+Vδ2subsets in γδ T cells in the three induction and control groups (F=3.385, F=3.245; P=0.075, P=0.081). The main proportion of Foxp3+γδ T cells possessed the CD27+CD25+phenotype. 7. There were significant differences in the expression levels of Foxp3, TLR8and G-CSFR genes in γδT cells in the three induction and control groups (F=35.184, P<0.001; F=73.769, P<0.001; F=444.383, P<0.001). The expression levels of Foxp3, TLR8and G-CSFR genes in the control group were significantly lower than that in the G-CSF, TGF-β and G-CSF+TGF-β group (P<0.001, P=0.001, P<0.001; P<0.001, P<0.001, P<0.001; P=0.030,P<0.001, P=0.012). The expression level of Foxp3gene in the G-CSF+TGF-β group was significantly higher than that in the G-CSF and TGF-β group (P=0.002, P=0.001). The expression level of Foxp3gene in the G-CSF group was similar to than that in the TGF-β group (P=0.686). The expression level of TLR8gene in the G-CSF group was significantly higher than that in the TGF-P and G-CSF+TGF-β group (P<0.001, P<0.001). The expression levels of CD25, CTLA-4, GITR, GATA-3and T-bet genes were similar in γδ T cells in the three induction and control groups (F=5.285, F=0.888, F=0.206, F=0.131, F=0.355; P=0.080, P=0.488, P=0.890, P=0.939, P=0.787).8. There was significant difference in the effect of cell proliferation suppression in γδT cells in the three induction and control groups (F=2030.991, P<0.001). γδT cells in the control group manifested a lower suppressive effect on the proliferation of CD4+T cells than that in the G-CSF, TGF-β and G-CSF+TGF-β group (P<0.001, P<0.001, P<0.001). There was no significant difference in the effect of cell proliferation suppression between G-CSF and TGF-P group, G-CSF and G-CSF+TGF-β group (P=0.178, P=0.271). We used TGF-β receptor inhibitor to pretreat CD4+T cells, and found that the inhibition effect could be reversed to some extent in the TGF-β and G-CSF+TGF-β group; whereas G-CSF combined with TGF-β receptor inhibitor could also have a certain inhibition effect.9. The distribution of TCRVy and V8subfamilies of y8T cells in the control group was consistent with that of magnetic-activated y8T cells. After the addition of G-CSF and/or TGF-β, the distribution of some TCRVy and V8subfamilies might change. TCRVy and V8subfamilies of magnetic-activated y8T cells and y8T cells in the control group in three volunteers all revealed polyclonality. Most TCRVy and V8subfamilies of y8T cells in the three induction groups also revealed polyclonality. y8T cells in the G-CSF and TGF-P group revealed oligoclonality in TCRV86in two of the three volunteers. y8T cells in the G-CSF+TGF-β group revealed oligoclonal trend in TCRV88in one volunteer. The expression levels of TCRVγⅠ, VγⅡ and VγⅢ genes of y8T cells in the control group were similar to that of y8T cells in the three induction groups (F=2.000, F=3.130, F=2.056; P=0.193, P=0.087, P=0.268). The expression pattern of TCRVγⅠ-Ⅲ subfamilies in y8T cells in the three induction and control groups all revealed as VγⅡ>VγⅠ>VγⅢ.10. There were significant differences in the amount of IFN-y secreted by γδ T cells in the three induction and control groups (F=5.626,.P=0.023). y8T cells in the control group produced higher amount of IFN-y than y8T cells in the TGF-P and G-CSF+TGF-β group (P=0.007, P=0.012). y8T cells in the three induction and control groups produced similar amount of IL-10, TGF-β, IL-4and IL-17(F=0.515, F=2.252, F=1.333, F=0.304; P=0.683, P=0.159, P=0.330, P=0.822).11. The expression levels of Perf and Grmb genes were similar in y8T cells in the three induction and control groups (F=0.017, F=0.147; P=0.997, P=0.929). The cytotoxic effect of anti-TCRγδ-stimulated y8T cells on Molt4and Raji cells did not differ significantly among the three induction and control groups (F=2.913, F=0.346; P=0.101,P=0.793). Conclusions1. G-CSF mobilization could not only influence the distribution and expression levels of TCRVa, Vβ, Vγ and V8subfamilies, but also change the clonality of αβ+T cells and γδ+T cells. This alteration of TCRVa, Vβ, Vγ and V8 subfamilies might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT. Significant positive association was observed between the alteration of clonality of TCRVβ22gene repertoire after mobilization and low incidence of GVHD, as well as between the invariable clonality of TCRVδ1gene repertoire after mobilization and low incidence of GVHD in recipients.2. G-CSF mobilization might mainly influence the expression levels of CD3ζ, CD3γ and CD3ε genes in TCR signal pathway, while the expression pattern of four CD3genes was unchanged after mobilization.3. After G-CSF mobilization, the proportions of total γδT cells and Vδ2subsets in peripheral blood were significantly decreased, the Vδ1proportion was significantly increased, including that the proportions of Foxp3+Vδ1, CD27+Vδ1and CD27+Foxp3+Vδ1subsets were significantly increased. There was a negative correlation between the incidence of GVHD in recipients and the percentages of donor CD27+Vδ1and CD27+Foxp3+Vδ1subsets after mobilization.4. PBMCs of healthy donors could be induced to generate γδ Tregs in vitro by stimulating with anti-TCRγδ and G-CSF. G-CSF-induced γδ Tregs primarily belonged to Vδ1subset with Foxp3+CD25+CD27+phenotype. G-CSF-induced γδ Tregs were manifested as significant up-regulation of Foxp3and other immunoregulatory-associated molecules as well as remarkable proliferation inhibition function, compared with γδ T cells in the control group. The immune phenotype, proliferation inhibition function and cytokine secretion of G-CSF-induced γδ Tregs were similar to that of TGF-β-induced γδ Tregs. However, G-CSF and TGF-β might induce γδ Treg by means of different ways.