TY - JOUR
AU - Gilmour, K C
AB - Summary Assessment of thymic output by measurement of naive T cells is carried out routinely in clinical diagnostic laboratories, predominantly using flow cytometry with a suitable panel of antibodies. Naive T cell measurements can also be made using molecular analyses to quantify T cell receptor excision circle (TRECs) levels in sorted cells from peripheral blood. In this study we have compared TRECs levels retrospectively with CD45RA+CD27+ T cells and also with CD45RA+CD31+ T cells in 134 patient samples at diagnosis or during follow-up. Both panels provide naive T cell measurements that have a strongly positive correlation with TRECs numbers and are suitable for use with enumerating naive T cell levels in a clinical laboratory. Graphical Abstract Open in new tabDownload slide Naïve T cell measurement is essential in assisting diagnosis of primary immunodeficiency. This can be carried out using a combination of both flow cytometry and TRECs measurement. We show a strong positive correlation between TRECs levels and either CD45RA+CD27+ T cells or CD45+CD31+ T cells but not with CD3+ T cells. Graphical Abstract Open in new tabDownload slide Naïve T cell measurement is essential in assisting diagnosis of primary immunodeficiency. This can be carried out using a combination of both flow cytometry and TRECs measurement. We show a strong positive correlation between TRECs levels and either CD45RA+CD27+ T cells or CD45+CD31+ T cells but not with CD3+ T cells. CD31, CD45RA, naive T cells, TRECs Introduction Diagnosis of primary immunodeficiencies (PID), particularly severe combined immunodeficiency (SCID), relies upon laboratory assessment of basic lymphocyte measurements, lymphocyte subsets and increasingly naive T cell assessment by flow cytometry. The European Society for Immunodeficiencies (ESID) have published online guidelines (https://esid.org/Working-Parties/Clinical/Resources/Diagnostic-criteria-for-PID2#Q12) for diagnosis of the most common PIDs, and typically this requires the laboratory assessment of lymphocyte subset analysis as a minimum. In the United States, many states now carry out newborn screening for SCID routinely, utilizing the measurement of T cell receptor excision circles (TRECs) as a screening tool [1]. Thus, laboratory testing to measure thymic output accurately is now a prerequisite for both screening and for diagnosis of PID. Additionally, monitoring TRECs levels has proved to be an essential tool to monitor T cell immune reconstitution in haematopoietic stem cell transplant (HSCT), gene therapy and thymus transplant patients following treatment [2–5]. Quantification of peripheral naive and memory CD4+ and CD8+ T cells is carried out routinely in clinical diagnostic immunology laboratories, typically using combinations of cell surface markers such as CD4, CD8, CD45RA, CD45RO and CD27 antibodies with flow cytometric assessment [2,6]. However, it has been well documented that, although useful in assessing T cell reconstitution, immunophenotyping using these markers may not be able to measure thymic output accurately [7]. An alternative marker, CD31 [platelet endothelial cell adhesion molecule-1 (PECAM-1)], has been proposed as a more suitable target to quantify recent thymic emigrants (RTE) when used alongside CD45RA [7,8]. The PECAM-1 protein was first cloned, named and characterized as a cell adhesion molecule belonging to the immunoglobulin gene superfamily [9–11]. Further studies have shown that CD31 is a differentiation antigen whose expression is lost after subsequent T cell receptor (TCR) engagement and during CD4 T cell maturation into T helper type 1 (Th1) or Th2 effector cells [12,13]. However, despite the advantages of using the combined expression of CD45RA and CD31 to measure RTE, it has been demonstrated that not every naive T cell expressing CD31 is a newly formed T cell [14]. Thus, using a second tool, such as quantification of TRECs, to measure the level of RTE can be useful [15]. Most TCRs are comprised of α and β chains, with a small minority being formed of γ and δ chains. TRECs are formed during the ligation of the recombination signal sequences flanking the δ rec locus and the Ψ-Jα, leading to the deletion of the TCRD locus from within the TCRA locus on the α-chain during the normal process of variable, diversity and joining (VDJ) recombination (Fig. 1). The resulting excised piece of DNA contains a unique signal joint (sj) sequence, and thus is termed the sjTRECs [15]. This recombination event is identical in approximately 70% of αβ T cells, despite the enormous diversity generated during VDJ recombination [16]. The excised DNA subsequently forms an episomal circle from which TRECs takes its name. TRECs have proved useful in determining thymic output, as they are stable and not degraded easily [17,18]. In addition, TRECs are not replicated during mitosis, are subsequently diluted during cell proliferation and can therefore be used as a measure of RTE [15]. TRECs can be measured using a real-time polymerase chain reaction (PCR) approach [19] and are reported typically as TRECs per 106 cells [20]. Fig. 1 Open in new tabDownload slide Following deletion of the T cell receptor (TCR)-δ locus from the TCR-α locus, a signal joint (sj) T cell receptor excision circle (TREC) is formed. Real-time polymerase chain reaction (PCR) can then be used to quantify the sjTRECs as a measure of thymic output. Fig. 1 Open in new tabDownload slide Following deletion of the T cell receptor (TCR)-δ locus from the TCR-α locus, a signal joint (sj) T cell receptor excision circle (TREC) is formed. Real-time polymerase chain reaction (PCR) can then be used to quantify the sjTRECs as a measure of thymic output. In this study we compare results of RTE quantification between flow cytometric measurement (using CD45RA in combination with either CD27 or CD31 expression on both CD4+ and CD8+ T cells) and real-time PCR based TREC quantification. Materials and methods Patient samples Blood [ethylenediamine tetraacetic acid (EDTA)] was taken from patients for either routine diagnostic assessment or for routine follow-up assessment following treatment and sent to the clinical laboratories for naive T cell measurement as part of their standard care. Samples were collected between 2010 to the present and were analysed by flow cytometry within 48 h of collection. These patient samples were also cell-sorted on the same day as collection. Flow cytometry Enumeration of lymphocyte populations was carried out by flow cytometric analysis. Whole blood was labelled with combinations of monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), peridinin chlorophyll protein (PerCP) or fluorochrome combinations with cyanines (PerCP-Cy5.5, APC-Cy7 and PE-Cy7) (BD Biosciences, Hatfield, UK). Lymphocyte subsets were detected using a six-colour multi-test reagent containing CD3 FITC, CD16+56 PE, CD45 PerCP-Cy5.5, CD19 APC, CD4 PE-Cy7 and CD8–APC-Cy7, to which CD45RA V450 and CD27 V500 were added. Naive, effector and memory T cell populations were also detected using CD45RA FITC, CD31 PE, CD45 PerCP and CD4 or CD8 APC. Post-staining, red cells were lysed (FACsLyse), samples washed (Cell Wash) and fixed (Cell Fix); 10 000 lymphocyte events were acquired on a FACsCanto II and analysed using FACs diva software. Magnetic bead cell sorting CD3+ T cells were isolated using magnetic bead cell sorting with human whole blood CD3 MicroBeads on the autoMACs Pro Separator following the manufacturer's instructions (Miltenyi Biotec, Surrey, UK). DNA extraction DNA was extracted directly from the cell sorted CD3+ T cells using the QIAamp DNA Blood Mini kit following the manufacturer's instructions (Qiagen, Manchester, UK). Eluted DNA was quantified using the Nanodrop 1000 spectrophotometer (Labtech International Ltd, Heathfield, UK). Real-time quantitative qPCR TRECs were measured using a real-time quantitative assay, as described previously [21]. Briefly, 5 µl patient DNA was amplified in a 25 µl total volume PCR solution containing primers and probes for the TRECs, B cell kappa chain excision circles (KRECS) and T cell receptor alpha constant (TRACs) sequences with the Taqman Universal Mastermix (Life Technologies, Paisley, UK) in a 96-well plate on the Taqman 7500 Fast Real Time PCR System (Life Technologies). Standards for TRECs, KRECs and TRACs were prepared from a plasmid kindly provided by Sottini et al. [21], and this was also run in the assay to generate a standard curve. All patient DNA samples were run in triplicate alongside no-template controls. TREC levels for all patient samples were calculated subsequently per 106 CD3+ T cells. Statistical analyses To present the data accurately, a logarithmic adjustment of the TRECs counts was performed. This allows an accurate representation of the spread of TREC values. In order to provide an accurate representation of the relationship between TREC counts and CD3/CD45RA/CD27 and CD3/CD45RA/CD31 percentages, patients with TREC counts of zero were not included in the graphs (Figs 2–4). These 16 patients with TREC values of 0 had a CD3/CD45RA/27 or CD3/CD45RA/CD31 percentage of less than 10%. All these would be classified as SCID babies by either TRECs or immunophenotyping with markers of naive T cells. Fig. 2 Open in new tabDownload slide T cell receptor excision circles (TRECs) (per 106 CD3+ T cells) plotted against CD3+CD45RA+CD31+ naive T cells (percentage of overall CD3+ T cells). [Colour figure can be viewed at wileyonlinelibrary.com] Fig. 2 Open in new tabDownload slide T cell receptor excision circles (TRECs) (per 106 CD3+ T cells) plotted against CD3+CD45RA+CD31+ naive T cells (percentage of overall CD3+ T cells). [Colour figure can be viewed at wileyonlinelibrary.com] Fig. 3 Open in new tabDownload slide T cell receptor excision circles (TRECs) (per 106 CD3+ T cells) plotted against CD3+CD45RA+CD27+ naive T cells (percentage of overall CD3+ T cells). [Colour figure can be viewed at wileyonlinelibrary.com] Fig. 3 Open in new tabDownload slide T cell receptor excision circles (TRECs) (per 106 CD3+ T cells) plotted against CD3+CD45RA+CD27+ naive T cells (percentage of overall CD3+ T cells). [Colour figure can be viewed at wileyonlinelibrary.com] Fig. 4 Open in new tabDownload slide T cell receptor excision circles (TRECs) (per 106 CD3+ T cells) plotted against CD3+ T cells (percentage of overall lymphocytes). [Colour figure can be viewed at wileyonlinelibrary.com] Fig. 4 Open in new tabDownload slide T cell receptor excision circles (TRECs) (per 106 CD3+ T cells) plotted against CD3+ T cells (percentage of overall lymphocytes). [Colour figure can be viewed at wileyonlinelibrary.com] Results A total of 134 patient samples were analysed using CD4+CD45RA+CD31+ and CD8+CD45RA+CD31+ panels to assess naive T cell numbers. These samples also had absolute CD3+, CD4+ and CD8+ T cell counts measured. This enabled us to calculate the percentage of CD3+CD45RA+CD31+ T cells in each sample. These same samples also had CD4+CD45RA+CD27+ and CD8+CD45RA+CD27+ cells quantified using flow cytometry at the same time. Again, this permitted us to calculate the percentage CD3+CD45+CD27+ T cells in each sample. The remaining blood samples were then sorted into CD3+ T cells which were used subsequently to measure TREC levels. Sorting for CD4+ and CD8+ cells was not undertaken, as there were insufficient numbers of these cells in many samples. Thus, the CD3+ T cells were isolated instead to maximize the potential for obtaining enough cells for TREC analysis. The median CD3+CD45RA+CD31+ naive T cell level detected was 13% (range 0–77%), with a mean level of 21% naive T cells. The median CD3+CD45RA+CD27+ naive T cell level detected was 18% (range 0–91%), with a mean level of 25% naive T cells. The median TREC level detected was 3107 per million CD3+ T cells (range 0–66 073) with a mean TREC level of 6642 per million CD3+ T cells. The TRECs levels were plotted against CD3+CD45RA+CD31+ naive T cell levels to assess the overall correlation between the two (Fig. 2, correlation coefficient 0·76). Similarly, the TREC levels were plotted against CD3+CD45RA+CD27+ naive T cell levels (Fig. 3, correlation coefficient 0·75). There is a very minor difference between their correlation coefficients of less than 0·015. This is insignificant (a 2% difference). This is in contrast with the calculated correlation coefficient of 0·40 between TREC and CD3+ T cell levels (Fig. 4). This suggests that it is not possible to estimate/predict the TREC count from a total CD3 percentage. Discussion As most of the samples received in the diagnostic laboratory were from children with primary immunodeficiencies, or from children who had recently received a haematopoietic stem cell transplant, gene therapy or thymus transplant, it was expected that most would have low naive T cell numbers and TREC levels. This was borne out by the results obtained. As expected, the relationship between TREC levels and flow cytometric measurement of naive T cells using either panel of antibodies (CD3+CD45RA+CD27+ or CD3+CD45RA+CD31+) was very linear, with high levels of correlation as assessed using Spearman's rank order correlation. There was no significant difference between the use of either panels when comparing to TREC levels. Thus, there is no evidence to suggest that CD31 is a more appropriate cell surface marker of naive T cells than CD27 when used with CD45RA. Unsurprisingly, the use of either naive T cell markers showed a much stronger correlation with TRECs than by using CD3+ T cells alone. For highly accurate assessment of thymic output other measurements are still required, as the use of CD45RA+CD31+ (or CD45RA+CD27+) alone is still partially flawed. Krenger et al. [14] have shown that not every naive T cell expressing CD31 is a newly formed T cell. Other studies have shown that the nuclear protein, Ki67, a proliferation marker expressed from late stage G1 through to the end of mitosis [22], can be used in conjunction with TREC measurement to form a highly comprehensive model calculating thymic output [23,24]. However, although modelling thymic output with Ki67 and TRECs is more accurate, there are logistical problems with incorporating this into the routine diagnostic setting. Most routine clinical laboratories operate with a high throughput of samples, often having to perform a number of different tests on small blood samples from young infants. To incorporate non-cell surface nuclear protein markers into routine working practice is prohibitive to workflow. Thus, the use of CD31 or CD27 aligned with CD45RA may prove to be the flow cytometric panel of choice, especially if allied with TRECs measurement. This retrospective study of naive T cell assessment has shown that the widely used flow cytometry panel of CD4+ or CD8RA+ with CD45+CD27+ correlates strongly with TREC levels in paediatric samples. We have also shown that there is little to be gained by using CD45RA+CD31+ instead of CD45RA+CD27+ to measure naive T cells. However, there is a considerable advantage in using at least one of these naive T cell panels, as the CD3+ marker alone is not suitable for quantifying naive T cell numbers accurately. It is therefore suggested that CD45RA+ should be used with either CD27+ or CD31+ for enumerating naive T cells in routine diagnostic clinical laboratories. For clinical laboratories, where TRECs measurements may not be routinely available, either panel is sufficient for detecting the presence of naive T cells post-therapy or the absence/low levels of them in patients with SCID or other primary immunodeficiencies. Acknowledgements The authors would like to thank Luisa Imberti and Alessandra Sottini for providing the TRECs plasmid construct used in this study and the staff of Immunology and Haematology for routine immunophenotyping. The authors have no financial support to disclose. Disclosure None disclosed by all authors. Author contributions The study was designed by K. G. and S. A. The TRECs assay was developed at GOSH by S. A. The TRECs assay runs were performed by S. A. and S. K. The flow cytometry assays were performed by E. R. under the supervision of K. G. 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TI - A comparison of TRECs and flow cytometry for naive T cell quantification
JF - Clinical & Experimental Immunology
DO - 10.1111/cei.13062
DA - 2018-01-08
UR - https://www.deepdyve.com/lp/oxford-university-press/a-comparison-of-trecs-and-flow-cytometry-for-naive-t-cell-aQOYLlcdq2
SP - 198
EP - 202
VL - 191
IS - 2
DP - DeepDyve
ER -