Characteristic <scp>CD45RA</scp>/<scp>CD45RO</scp> maturation pattern by flow cytometry associated with the <scp>CD45 C77G</scp> polymorphism

Abstract

Extended T-cell immunophenotyping is used in the evaluation for both primary and secondary immune disorders. The distinction between naïve and memory T-cells includes the expression patterns of CD45RO and CD45RA as well as other markers. CD45 isoform expression depends on the stage of T-cell maturation, activation, and differentiation. Naïve T-cells express the isoform CD45RA and the isoform CD45RO is primarily found on primed/memory T-cells. By flow cytometric assays using fluorochrome conjugated antibodies against CD45RA and CD45RO, there is a typical staining pattern seen within the CD4+ T lymphocyte and CD8+ T lymphocyte compartments (see reference cases in Figure 1). A well-studied polymorphism in the extracellular domain of CD45 is the C77G point mutation in a splice silencer region of exon 4, resulting in memory/effector lymphocytes of C77G carriers expressing both CD45RA and CD45RO. A characteristic CD45RA/CD45RO staining pattern of lymphocytes from C77G individuals has been reported (Tchilian et al., 2006). In a retrospective study approved by our institutional review board (IRB #13310), we identified six patients with the characteristic CD45RA/CD45RO staining pattern out of 309 expanded lymphocyte immunophenotyping panels from 273 patients (M:F ratio of 0.9:1, average age 18 years, range 0–83) performed from January 2017 through August 2020. The patients were identified by natural language search and/or visual inspection of flow cytometry plots. Pertinent information was obtained from review of the electronic medical record. Five of the 6 patients had genotyping performed by Invitae (San Francisco, CA) as part of their clinical work-up. The presence or absence of the CD45 C77G polymorphism was included in the supplemental variant call file and denoted as NM_002838.4: PTPRC c.177C>G silent variant. Flow cytometric analysis was performed using a 6-color FACSCanto II (Becton Dickinson Biosciences, BD, San Jose, CA) analyzer using the following antibodies (all from BD Biosciences): CD45RO (FITC), CD45RA (PE), CD8 (PerCP-Cy5.5), CD4 (PE-Cy7), CD3 (APC), and CD45 (APC-H7). The peripheral blood samples were processed by a stain-lyse method using a Beckman Coulter Whole Blood Lyse Kit (Immuno-Lyse followed by fixative). Data was analyzed in FACSDiva software (Becton Dickinson). Doublets were excluded by a forward scatter area by height (FSC-A by FSC-H) plot and debris were excluded by a forward by side-scatter plot. CD45 by side-scatter (SSC-A) was used to establish a lymphocyte gate. CD3 positive CD4 positive lymphocytes or CD3 positive CD8 positive lymphocytes were used as the input gate for CD45RA by CD45RO plots. Normalized mean fluorescence intensity (MFI) values were calculated for the CD45RO populations relative to non-T lymphocytes. Absolute values for lymphocyte subsets were calculated from the separately reported white blood cell differential count. A subset of the patients had the same samples re-run with additional wash steps with nearly identical results and all of the patients had repeat flow cytometry studies from different draw dates between 1 month and 15 months apart showing a similar CD45RA by CD45RO maturation pattern (data not shown). Patient details for the six individuals with the characteristic CD45RA/CD45RO staining pattern are presented in Table 1. Five of the patients were under the age of 10 (average age for this subgroup 4 years old, range 6 months to 9 years), and one was an adult. Five of the patients presented with an infectious history, prompting evaluation in the Allergy/Immunology Clinic and laboratory evaluation with immunoglobulins, flow cytometry and genetic testing. Three had a history of allergic diseases, and one (the adult) had a history of autoimmune disease. Work-up led to formal diagnosis of primary immunodeficiency in only one patient (Case 4—specific antibody deficiency). All five patients tested were heterozygous for the CD45R C77G polymorphism. Flow cytometry scattergrams showing the characteristic CD45RA by CD45RO maturation pattern are shown in Figure 1. There is acquisition of CD45RO expression with retained CD45RA expression of variable intensity, resulting in a “double-positive” population with a curved, or upside-down “U,” shape. In the six patient cohort, the CD45RO MFI relative to non-T lymphocytes ranged from 19 to 65 (average 51) in the CD4+ T cells and 11 to 47 (average 33) in the CD8+ T cells. The corresponding relative MFIs for the reference cases were 48 and 66 (for the CD4+ T cells) and 35 and 59 (for the CD8+ T cells). When quantitative assessment of the absolute value for the CD4 + CD45RO+ T-cell population was performed using the sum of the single positive CD45RA-CD45RO+ and the double positive CD45RA + CD45RO+ populations (using the right upper quadrant values in the plots shown), four patients fell within normal limits for age and two had slightly low levels. Regarding the patients with slightly low levels, the absolute value for the sum of the single positive CD45RA-CD45RO+ population and the double positive population in Case 4 was 172 cells per microliter (reference range for age 230–630) and in Case 5 was 179 (reference range for age 220–660). The characteristic CD45RA/CD45RO curved pattern seen with the C77G polymorphism is different than either the CD45RA/CD45RO dual positive population expressing both isoforms in low density and falling along the linear negative slope line from CD45RA positive to CD45RO positive and the distinct CD45RA/CD45RO dual positive population expressing both isoforms with high density (“Ddull” and “Dbright” populations respectively) as described in the 1996 paper by Hamann, Baars, Hooibrink, and van Lier (1996). Recognized since the mid-1990s, the exon 4 C77G polymorphism has a low mutation frequency, between 0% and 3.5% in studies of healthy control populations from Europe and the United States (Tchilian et al., 2006). All patients in our cohort identified as White/Caucasian; additional information on ancestry was not available to us. An increased frequency of the polymorphism has been reported in certain disease states including systemic sclerosis, hepatitis C, and autoimmune hepatitis. In our cohort, the characteristic CD45RA by CD45RO maturation pattern was seen in 2% of patients (6/273), which is concordant with the reported mutation frequency for the polymorphism in a healthy control population. The aim of this letter to the editor is to bring the pattern of CD45RA/CD45RO staining associated with the C77G polymorphism to the awareness of the general flow cytometry community. Such an awareness could prevent unnecessary repeat flow cytometric studies which may be performed to confirm the immunophenotypic pattern. We recommend a description of the observed immunophenotypic pattern within the flow report with a reference to the association of the phenotypic pattern with the C77G polymorphism. In our opinion, for the semi-quantitative evaluation of memory/primed CD45RO T-lymphocytes in patients with this maturation pattern, the double positive CD45RA + CD45RO+ population should be used to compare to published reference ranges for the CD45RA-CD45RO+ population. Dr Roshini Abraham for making us aware of the 2006 Tchilian paper. Dr Jeffrey Wilson and Dr Larry Borish for contributing clinical information. Barbara Murphy for expert help creating the figure. Nothing to report.

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