Enumerating regulatory T cells in cryopreserved umbilical cord blood samples using FOXP3 methylation specific quantitative PCR

Abstract

Background: Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples.

Study design and methods: To establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples.

Results: Epigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing.

Conclusion: Epigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.

Fig 1. Flow cytometry gating of CD3+ cells and Tregs with viability and total Treg events.

A; example gating strategy for CD3+, CD3+CD4+ cell and Tregs (CD127^lowFOXP3^hi) content in fresh CB and segments. Shown is an example from a fresh CB. Fresh CB and thawed segments were RBC lysed and stained for the surface markers CD45, CD3, CD4, CD127, and CD25, followed by the fixable live/dead stain eFluor 506. After washing the cells were fix/permeabilised and stained with FOXP3. Left to right top row; (i) gating CD45+ cells, (ii) excluding dead cells (eFluor506+), (iii) gating SSC^low/CD3+ cells and (iv) CD45^hiSSC^low lymphocytes (R5). Second row, (v) CD3+CD4+ cells are gated from R5 cells and then (vi) effectors (CD127^hi) and Tregs (CD127^low) CD25+ cells. (vii) FOXP3 expression on gated effectors (eff) and Tregs. (viii) gated Tregs from CD127^lowFOXP3^hi CD25+ cells. B; Proportion of eFluro506^- cells in the CD45⁺ and CD3⁺ gated populations fresh CB or frozen segments. C; number of live Tregs (as gated 1A) events acquired from fresh CB or frozen segments. Results shown are of non-parametric unpaired Mann-Whitney U tests *** = p ≤ 0.001, ** = p ≤ 0.01 and * = p ≤ 0.05.

Fig 2. CD3+ cell and Treg content in fresh CB and segments by flow cytometry and MS-qPCR.

Fresh CB and thawed segments were RBC-lysed and stained for the surface markers CD45, CD3, CD4, CD127, and CD25, followed by the fixable live/dead stain eFluor 506. After washing, the cells were fix/permeabilised and stained with FOXP3. DNA was extracted from paired samples. A; proportion of CD3+ cells by flow cytometry (live CD3+ of live CD45+ cells) and by MS-qPCR. B; proportion of Tregs by flow cytometry (live Treg of live CD45+ cells) and by MS-qPCR. C; Ratio of Treg to CD3+ by flow cytometry and by MS-qPCR. Results of non-parametric unpaired Mann-Whitney U tests or Wilcoxon tests for paired observations *** = p ≤ 0.001, ** = p ≤ 0.01 and * = p ≤ 0.05.

Fig 3. Comparison of CD3+ cell and Treg enumeration in male and female samples.

A; Comparison between male and female fresh samples. B; comparison between male and female samples from frozen segments. (i); Proportion of CD3+ cells measured by flow cytometry (ii); Proportion of CD3+ cells by TcSDR MS-qPCR enumeration. (iii); Proportion of FOXP3+ cells by flow cytometry and (iv); by TSDR MS-qPCR enumeration. Shown is the results of Mann-Whitney U tests *** = p ≤ 0.001, * = p ≤ 0.05.

Fig 4. Comparison of flow cytometric and MS-qPCR data for CD3+ cell, Treg content, and Treg/CD3+ cell ratio in fresh samples.

Cellular enumerations, from fresh CB samples, using flow cytometry were compared with epigenetic enumerations performed on the same samples. Male and female derived samples are as indicated. The results of a Pearson’s correlation coefficient are shown. A; Proportion of CD3+ cells by flow cytometric or with TcSDR MS-qPCR enumeration. B; Proportion of Treg by flow cytometry (as gated in Fig 1A) or with TSDR MS-qPCR enumeration C; Ratio of Treg/CD3+ cells using the two methods.

Fig 5. Comparison of flow cytometric and MS-qPCR data for CD3+ cell, Treg content, and Treg/CD3+ cell ratio in frozen samples.

Cellular enumerations, from frozen CB segments, using flow cytometry were compared with epigenetic enumerations performed on the same samples. Male and female derived samples are as indicated. The results of a Pearson’s correlation coefficient are shown. A; CD3+ cells by flow cytometric or with TcSDR MS-qPCR enumeration. B; Treg by flow cytometry or with TSDR MS-qPCR enumeration. C; Ratio of Treg/CD3+ cells using the two methods.