Ascorbic Acid Promotes Increased mRNA Expression of Rearranged TCR-Alpha and TCR-Beta Genes During In Vitro T Cell Development.
Authors: Manning Jared R. , Mitchell Birgitta , Appadurai Daniel Anand , Ornelas Loren , Spangrude Gerald J. 
Published: October 2019

Abstract
Abstract 2623

Introduction:
A complete understanding of factors that drive T cell differentiation from hematopoietic progenitors remains a fundamental goal of hematology. T cells are key regulators and effectors in defense against infections and malignancies. Aberrations in T cell regulation play causative roles in autoimmune and graft-versus-host disease. The OP9-DL1 stromal cell line enables investigation of lymphocyte development in vitro. Lymphocyte progenitors (KLS, Thy1.1 -) harvested from murine adult bone marrow and seeded onto the OP9-DL1 stromal line can be followed through stages of maturation by immunophenotyping. We previously demonstrated that addition of a stabilized form of vitamin C, phospho-ascobate (pAsc), to culture media promoted T lineage differentiation. To address the mechanism of this effect, we employed quantitative RT-PCR and spectratyping analysis to examine mRNA expression of rearranged complementarity-determining region 3 (CDR3) polymorphisms in T cell receptor (TCR) beta variable (BV) and alpha variable (AV) genes in the presence or absence of pASC.

Methods:
Lymphocyte progenitor cells (KLS, Thy1.1-) were sorted from adult mouse bone marrow and 1000–2000 progenitors were seeded per well in a 24 well plate coated with OP9-DL1 stromal cells. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/mL) and SCF (100 ng/mL) plus or minus pAsc (100 mcg/mL). Cells were passaged, counted and reseeded with fresh media and supplements twice a week over a 21 day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, TCR beta chain and TCR gamma-delta chains. Total RNA from cultured cells was isolated at day 21, reverse transcribed to cDNA, and analyzed by RT-PCR for differential expression of BV and AV genes using gene-specific primers for BV1, BV4, BV8.2, BV13, AV2, and AV8 with corresponding beta constant (BC) and alpha constant (AC) primers. For spectratyping, RT-PCR amplicons were generated using BV or AV gene-specific primers for BV1, BV4, BV8.2, BV13, AV1, AV2, AV5, AV8, AV10, AV13, AV16, AV18, and AV19 with corresponding BC and AC primers. These products were then re-amplified with the same gene-specific primers but with fluorochrome-labeled nested BC or AC primers. Spectratype analysis was performed on labeled amplicons by capillary electrophoresis.

Results:
T cell differentiation was markedly advanced by the addition of pAsc, with the majority of cells co-expressing CD4/CD8 and TCR beta/CD3. Transfection of a functionally rearranged TCR beta gene failed to rescue cells cultured without pAsc to the double positive stage; similar results were obtained with bone marrow cells derived from TCR alpha-beta transgenic donor mice. Cells cultured with pAsc demonstrated an average 5 fold increase (5.08 ± 0.40) in expression of BV genes and an average 13 fold increase (13.46 ± 2.18) of AV genes. pAsc did not induce alterations in the spectratype distributions of BV amplicons compared those generated under non-pAsc conditions, nor to distributions derived from thymic cDNA. However, spectratype distributions of AV amplicons generated under pAsc conditions more closely resembled those derived from thymic and lymph node cDNA than distributions generated from non-pAsc conditions.

Conclusions:
In our in vitro model, the addition of pAsc promotes robust differentiation of adult mouse bone marrow progenitors to T cells co-expressing CD4/CD8 and alpha-beta TCR. However, the mechanism by which pAsc exerts its effect remains elusive. We suspect that pAsc enhances an already pre-programmed process. The fact that transfection of a functional TCR beta gene fails to rescue differentiation, coupled with our observation that pAsc has no effect on BV spectratypes suggests that enhancement of beta selection is not involved. Rather, the AV spectratyping data suggest that pAsc exerts its effect temporally near alpha gene rearrangement, possibly via enhancement of the TCR signal transduction cascade. Further work will include PCR microarray analysis encompassing multiple signal transduction cascades involved in hematopoietic progenitor differentiation. These findings will help develop a model for future mechanistic studies and for ex vivo expansion of immune cells for therapeutic use.

Disclosures:
No relevant conflicts of interest to declare.