<scp>CD200</scp> expression in <scp>CD4</scp> ⁺ T‐cells: Diagnostic implications across T‐cell lymphomas

We read with great interest the manuscript titled “CD200 expression on Sézary cells: A valuable tool for flow cytometric assessment of peripheral blood T-cell neoplasms”, published in Cytometry Part B: Clinical Cytometry, Volume 102, Issue 4, December 2021, authored by Shameli and Roshan (2022). In this manuscript, CD200 expression is described as an additional diagnostic marker for Sézary syndrome (SS), aiding in its differentiation from other T-cell neoplasms. Although CD200 expression has been well established in B-cell neoplasms, it has rarely been studied in T-cell neoplasms, particularly by flow cytometry (FCM). CD200 expression has previously been reported in nodular T follicular helper lymphoma (nTFHL) and in a subset of peripheral T-cell lymphomas (PTCL) using immunohistochemistry (IHC) (Dorfman & Shahsafaei, 2011). In this study, we evaluated its expression on circulating T-cells from various mature T-cell neoplasms using FCM. FCM was performed using 12-color analysis on a BD FACSLyric™ cytometer and analyzed with BD FACSuite™ software version 6.1 (Becton Dickinson, San Jose, CA, USA), as used in our routine diagnostic workflow. Daily instrument quality control (QC) was performed using cytometer setup and tracking (CST) beads. Fresh whole peripheral blood (PB) samples were stained using our lymphoid screening panel, which includes a combination of 16 monoclonal antibodies (Abs), notably anti-CD200 BV510 [clone MRC OK-104*], anti-CD8 FITC [clone SK1*], anti-CD56 PE [clone NCAM*], Kappa FITC/Lambda PE** [Agilent (Dako), Santa Clara, USA], anti-CD3 PerCP-Cy5.5 [clone SK7*], anti-CD19 PE-Cy7 [clone SJ2SC1*], anti-CD5 APC [clone L17F12*], anti-CD10 APC-R700 [clone H10a*], anti-CD2 APC-H7 [clone RPA-10*], anti-CD20 V450 [clone L27*], anti-CD4 V450 [clone RPA-T4*], anti-HLA-DR BV605 [clone G46-6*], anti-CD7 BV711 [clone M-701*], anti-CD23 BV711 [clone L233*], and anti-CD45 BV786 [clone HI30*] (*Becton Dickinson). Lymphocytes were gated based on CD3+CD4+ expression. All cases in our series retained detectable CD3 expression. Nevertheless, to account for the possibility of CD3 loss described in TFHL, the gating strategy was adjusted to include CD4+CD5+ lymphocytes, ensuring that potentially aberrant neoplastic populations would not be excluded from the analysis. In all samples, the mean fluorescence intensity (MFI) values of CD200 expression were measured in CD4+-gated CD3+ T-cells and CD8+-gated CD3+ T-cells. These MFI values, expressed in relative linear units on an arbitrary bi-exponential scale (ranging approximately from 10−2 to 105), were extracted for each subset. Since CD200 expression on CD8+ T-cells was consistently negative and stable, the ratio of CD200 MFI in CD4+ T-cells to CD8+ T-cells was calculated to assess relative expression levels. In all lymphoma cases, CD200 expression was specifically assessed on the neoplastic T-cell population, excluding non-neoplastic background CD4+ T-cells. Statistical analyses were conducted using the Kruskal–Wallis test, with multiple comparisons corrected using the Benjamini–Krieger–Yekutieli (BKY) procedure, which controls the false discovery rate. A q-value of less than 0.05 was considered statistically significant. FCM data were generated from a retrospective series (January 2022–January 2025) of mature T-cell neoplasms with PB involvement. The cohort included cases of adult T-cell leukemia/lymphoma (ATLL, n = 3), nodal T follicular helper lymphoma (nTFHL, n = 13), T-prolymphocytic leukemia (T-PLL, n = 4), peripheral T-cell lymphoma, NOS (PTCL, NOS, n = 5), Sézary syndrome (SS, n = 30), and non-tumoral PB samples (n = 15). All diagnoses were established according to the 2022 WHO-HAEM5 classification criteria for hematologic malignancies, as well as the International Consensus Classification of Mature Lymphoid Neoplasms. The present data are consistent with those of Shameli et al., showing higher CD200 expression on circulating Sézary cells compared to T-prolymphocytic leukemia (T-PLL) and non-tumoral CD4+ T-cells (Shameli & Roshan, 2022). Additionally, we report that CD200 expression, measured by the MFI ratio of neoplastic CD4+ T-cells to non-tumoral CD8+ T-cells, was significantly higher in nTFHL than in all other mature T-cell neoplasms, especially PTCL, NOS, and non-tumoral T-cells (Figure 1). The evaluation of the CD200 ratio is particularly useful for detecting circulating T-cells in TFHL and for the differential diagnosis of CD4+ T-cell neoplasms. This is especially relevant as TFHL represents a group of mature T-cell neoplasms with the phenotypic features and gene expression signature of TFH cells, which are crucial for germinal center formation, affinity maturation, and B-cell memory. Tumor progression in TFHL is influenced by the crosstalk between tumoral TFH cells, follicular dendritic cells (FDCs), and B-cells, as demonstrated in mouse models (Chtanova et al., 2004). Given that CD200, expressed by these cells, is an immune-suppressive molecule, targeting CD200 with immunotherapy could offer a promising approach to affect neoplastic CD200+ T-cells in TFHL, similar to the use of anti-PD-1 in this context (Guenova et al., 2014). Finally, CD200 expression is a valuable marker in the differential diagnosis of B-cell neoplasms with PB involvement, particularly in distinguishing chronic lymphocytic leukemia from mantle cell lymphoma, and hairy cell leukemia from splenic diffuse red pulp small B-cell lymphoma, as well as in T-cell neoplasms, with the highest expression in TFHL. Including this marker in a screening panel for lymphoid populations is therefore particularly useful for differential diagnosis. Moreover, our unpublished data indicate that CD200 is consistently expressed on TFHL in lymph node samples, paralleling the pattern observed in peripheral blood. This stability across tissue compartments supports its reliability as a diagnostic marker and reinforces its value in distinguishing TFHL from other CD4+ T-cell neoplasms. In conclusion, this study highlights the utility of CD200 evaluation to screen for PB involvement by mature T-cell neoplasms, provide an initial diagnosis, and guide further analyses. Larger cohort studies are needed to confirm these findings and assess CD200 expression as a potential therapeutic and prognostic marker in TFHL. The authors have no acknowledgments to declare. The authors have no conflict of interest. This manuscript respects the ethic policy of CHU Lyon for the treatment of human research participants. All procedures were conducted in accordance with the Helsinki Declaration and the Biological Resource Centre policy of the Hospices Civils de Lyon. Furthermore, the research protocol was approved by the institutional review board of the Hospices Civils de Lyon (25-5164). The authors declare no use of third-party material in this study for which formal permission is required. Data available upon reasonable request to the corresponding author