Characterization and Comparison of Cord Blood (CB) vs Adult Peripheral Blood (APB) CD4+CD25+ NAÏVE and Antigen Presentation Cell (APC) Induced T Regulatory (TREG): Significant Increase in APC Induced CB vs APB CD4+/CD25+/CTLA-4+ TREG.
Authors: Chang Chih-Chao , van de Ven Carmella , Satwani Prakash , Oberfeld Nora , Simpson Lynn , Cairo Mitchell S. 
Published: April 2020

Abstract
The mechanism(s) responsible for the significant decrease in the incidence of a+cGVHD following UCBT remain to be determined (Cairo et al, 90:4665 Blood 1997). Treg (CD4+CD25+) cells have been demonstrated to be critically important in maintaining self-tolerance, transplantation tolerance, controlling autoimmunity and tumor immunity (Sakaguchi et al, Immuno Rev 2001) and suppressing aGVHD in a rodent model (Hoffman et al, J Exp Med2002; Taylor et al, Blood2002). We have previously reported that naïve CB vs APB CD4+CD25+ vs CD4+CD25− selectively express amplified genes including CTLA, GITR, and Foxp3 among others (Oberfield/Cairo et al, Blood697a, 2003). In this study, we compared naïve and APC induced CB vs APB CD4+CD25− and CD4+CD25+ T cells with respect to gene profiles, immunophenotype, suppressive activity and mechanisms of suppression. CD4+CD25+ and CD4+CD25− cells were purified by negative selection for CD4 and positive selection for CD25 (Miltenyi Biotech), respectively. APC were generated from APB monocyte (M-iDC) or MUTZ-3 stimulated with GM-CSF (100ng/ml), IL-4 (10ng/ml) and TNF-a (2.5ng/ml) for 7d (MUTZ-iDC). Suppression assays were preformed with autologous CD4+CD25− (Th), allo-M-iDC or MUTZ-iDC (stimulators) and CD4+CD25+ cells (effectors) pulsed with 3H-thymidine. Gene expression of for Foxp3, CTLA-4, CD25, GITR, Tnfrsf-1B, IFRF4, SOCS2, Rarg, IL-10, IFN-γ, TGFß-1 and GAPDH were quantified by RT-PCR. MUTZ-iDC expressed significantly increased levels of CD1a, Cd11c, CD83, CD86, CMRF44 and induced similar primary but significantly reduced secondary MLR in naïve CB CD4+ T cells (p&lt;0.02). Naïve CB and APB CD4+CD25+ vs CD4+CD25− expressed similar increased mRNA levels of genes including CTLA-4, GITR, Foxp3, Rarg, Tnfrsf-1B, IFR4 and CD25 and increased protein expression of CTLA4 (p&lt;0.001) and GITR (p&lt;0.001). However, APB but not CB CD4+CD25+ T cells significantly suppressed MUTZ-iDC stimulated Th responses (CB 8±3 % vs APB 52±5%, p&lt;0.02). MUTZ-iDC stimulated CB and APB CD4+ T cells induced similar amplification of Foxp3 and CTLA-4 in CD25+ vs CD25− T cells but no significant difference in GITR, Rarg and Tnfrsf-1B. However, CD4+/CD25+/CTLA-4+ subset was significantly induced following CB vs APB MUTZ-iDC stimulation (CB 27±5% vs APB 10±2%, p&lt;0.0023, n=8) and was CD62L positive. Most importantly, both CB and APB CD4+CD25+ T cells significantly suppress CD4+Th responses with MUTZ-iDC vs Allo-M-iDC stimulation (CB, p&lt;0.001, vs APB, p=0.0012). However, there was no significant difference in either CB or APB IL10 or TGFß1 at both mRNA or protein levels. Taken together, these data suggest that activated CB CD4+CD25+ T cells induced similar primary but decreased secondary MLR and have similar Foxp3, GITR, CTLA-4 gene expression compared to APB but that MUTZ-iDC vs allo-M-iDC induced CB CD4+CD25+ T cells are associated with a significant intrinsic suppression activity with increased CTLA-4+/CD25+ expansion in CB vs APB, suggesting the importance of alloantigen specificity. This suppression does not appear to be mediated by IL10 or TGFß1. Our results provide insight into the mechanism(s) in alloantigen recognition and activation of CB vs APB CD4+Treg and these findings may in part responsible for the decrease in a+cGVHD seen post UCBT.