IL 2 rg Cytokines Enhance Umbilical Cord Blood CD 34 + Cells Differentiation to T Cells

nozadh@tbzmed.ac.ir, Tayefih@yahoo.com 2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(Suppl 1), 615-619 doi: 10.15171/apb.2015.083 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Hematopoietic stem cell (HSC) have been used in clinical transplantation for curative treatment of malignant and non-malignant diseases. 1,2Umbilical cord blood (UCB) is an alternative source of HSCs transplantation in treatment of leukemia.For a long time CD34 positive has been accepted as a pan marker of HSC in bone marrow and UCB.However, CD34+ fraction is a heterogeneous population of immature stem and progenitor cells.3][4][5] Developing T cells from a common lymphoid progenitor cells pass through a series of distinct phases that are marked by changes in the expression of the T-cell receptor and cell-surface proteins such as CD3 complex during functional maturation. 6revious studies showed that the IL2rg cytokines(common cytokine receptor γ chain) are essential for the function of several cytokines, including IL-2, IL-7, IL-15 and some others. 7IL-2 plays an essential role in regulating the size of the peripheral T cell pool 8 and is reported to influence NK cell differentiation and function. 9IL-7 is the major IL2rg cytokine which involved in B and T cell development. 10- 12It has been shown that IL-7 knockout mice have a 10fold reduction in B cells. 12,13However it is unclear the role of these cytokines on T cell development from cord blood cells.In adults, setting of CB transplantation influences apoptosis in T cells following stem cell transplantation more than adult peripheral blood T cells and these cells are also more susceptible to apoptosis after being allopriming in vitro. 14ord blood is one of stem cell sources for transplantation in leukemia patients.However the number of getting cell per each baby delivery is limited.In order to get more effective CB transplantation, many laboratories do ex vivo expansion of HSCs/HPCs.Therefore, they apply some extrinsic regulators such as stem cell factor (SCF) which improve homing and proliferation of UCB cells in preclinical models 15,16 and Fetal liver tyrosine kinase-3ligand (Flt3) to enhance short-term expansion, proliferation and differentiation of HSCs. 17As it is known, IL-7 has a mutual role in B cell development as well as in induced NK cell differentiation. 18,19][24] Therefore, it is important to understand the effect of these cytokines on the T cell expansion in cord blood context, since T cell is important player in immunity.In this study, we evaluated the potential of CD34+ cord blood cells differentiation to T cells.We also established the best cytokine condition for development of T cells derived from cord blood mononuclear cells.

Cell isolation
Cord blood samples collected from full-term normal deliveries, were diluted 2:1 with phosphate-buffered saline (PBS) (SIGM).Subsequently, mononuclear cells were isolated by centrifugation on Ficoll-paque (GE healthcare, 1.078 g/ml) at 850 gm for 25 minutes.The mononuclear cells were collected, washed twice and resuspended in RPMI1640 (Gibco) supplemented with 10% FBS (Gibco) either for culture or for freezing.

Monoclonal antibodies and flow cytometry
Monoclonal antibodies (conjugated with different fluorochromes) used to stain cell-surface antigens were: CD34 (581; Abcam) and CD3 (UCHT1; R&D).We evaluated the cultured cells by flow cytometeric analysis every week.Propidium iodide (1.0 mg/mL; Invitrogen) were used to exclude dead cells from the analysis.Cells were analyzed by BD caliber (BD ebioscience), between10000 to 30000 events were collected and analyses were performed using flowing software (Perttu Terho, version: 2.5.1.).

Statistical analysis
All results are expressed as mean (SD).The statistical significances between groups were determined using the Student t test and one-way ANOVA.P < 0.05 was considered to be statistically significant.The analysis performed by GraphPad Prism software (version: 5.04).Experimental Ethical matters have been approved by Ethical committee of Tabriz University of medical Sciences.

Role of cytokines in generation of T cells from cord blood CD34+/-cells
Several cytokines are known to up regulate and control the generation of T cells.][24] We cultured 1x 10 5 cord blood mononuclear cells for 21 days in presence of different combination of SCF, FL, IL2, IL7, and IL15.Harvested cells evaluated by FACS at distinct time points gating on lymphoid mononuclear cells.We gated CD3+ cells on CD34+ and CD34fractions separately to evaluate the percentage of CD3+ T cells derived from cultured mononuclear cells (Figure 1).As shown in (Figure 1), CD34+ fraction significantly produce CD3+ cells about 60 t0 80 percent more than CD34-fraction.In presence of the combination of all cytokines, the percentage of T cells increased from day 7 to day 21 significantly from 10% to around 93% (Figure 2).IL-2 in compare to IL7 and IL15, produced higher CD34+T cells (92.4%) (Figure 2C).However with addition of IL7 and IL15 the percentages of CD34+T cells were 87.4% and 80.3% respectively (Figure 2D, E).Moreover in combination of all cytokines the percentage of T cells was (78.2%) (Figure 2F).CD3+CD34-cells increased in presence of different combination of mentioned cytokines from day 7 to day 21 as like as CD3+CD34+ cells.There is significant difference between expansion of CD3+CD34+ and CD3+CD34-in different time points (Figure 2A, B, C, D, and F).

Discussion
Stem cell transplantation is a standard treatment for hematological disorders.Umbilical cord blood is an alternative source of hematopoietic stem cell and it is currently used for transplantation. 25However, the limiting number of cells in cord blood is a challenging factor in transplantation especially in adult recipients. 26 this study, we evaluated the potential of UCB CD34+ cells to differentiate to T cells.We cultured mononuclear cord blood cells in presence of IL2, IL7 and IL15.We compared percentage of T cells derived from CD34+ and CD34-at different time points.Previous studies showed relationship between IL2 and IL7 with T cell differentiation, [22][23][24] It has been reported that knockout in IL7 gene reduces mature T cell dramatically and also cause decline in the size of thymus. 27,28IL2 deficiency cause reduction in the T cell progenitors and block the T cell maturation. 29It is very important to understand whether T cell derived cord blood cells are affected by IL2, IL7 and IL15.We tested T cell differentiation from cord blood mononuclear cells in presence of IL2 and IL7.We found that IL-2 in comparison to IL7 has more influence on T cell differentiation from CD34+ cord blood cells.We obtained same result in agreement with previous studies which have displayed that the percentage of T cells increased in presence of IL7 (Marcel R. M. and colleagues in 2001). 30However IL15 did not increase the T cell differentiation.This finding is in agreement with other studies which have shown that IL15 is a key cytokines for NK cell development.Therefore our data illustrated that this cytokines can be used in ex vivo expansion of UCB HSCs.These findings are worth for clinicians, who are working on cord blood transplantation.It is important to understand, whether they should use cytokines for the expansion of cord blood cells before transplantation or in vivo injection during transplantation.As shown IL2 and IL7 alone increase the T cell differentiation from cord blood cells more than other combinations.Although Donor T cell from bone marrow transplantation attack and kill the recipient cells and cause the relapse because of the graft versus host disease, however in cord blood transplantation these negative effect is not severe and clinicians can prevent the minor negative effect of derived T cell in cord blood transplantation by injection of T cell preventive drugs.These findings could be helpful to improve the cord blood transplantation for leukemia patients and probably it should be replaced with other types of transplantation.However, still the types of T cell derived from cord blood CD34 positive cells remain unknown.

Conclusion
In summary, we illustrated that either IL2 or IL7 versus other cytokine combinations, generate more T cell from cord blood CD34 cells, probably this cytokines can be useful for ex vivo and in vivo expansion of cord blood stem cell.However it is crucial to understand whether T cell derived cells are cytolytic or other types.

Figure 1 .
Figure 1.Representative FACS profile of 10 5 cultured cord blood mononuclear cells.A. Control.B. Sample