White matter lesions in multiple sclerosis are enriched for CD20 ^dim CD8 ⁺ tissue‐resident memory T cells

Brain CD8+CD69+ tissue-resident memory T (TRM) cells comprise a CD20dim subset, which is proportionally larger in CD103-negative TRM cells. In multiple sclerosis (MS) lesions, CD20dim TRM-cell proportions are increased. CD20-expression is associated with higher levels of CXCR6, Ki-67, and granzyme B, supporting CD20dim TRM cells as a relevant subset in MS. The cluster of differentiation (CD)20 antigen is most well known as a component of the B-cell receptor complex [1]. As such, it is since decades used to identify B lymphocytes with flow cytometry and immunohistochemistry. In relapsing–remitting and progressive multiple sclerosis (MS), B-cell depleting anti-CD20 immunoglobulin (Ig)G1 antibodies are efficacious in suppressing inflammatory disease activity and delaying disability progression [2]. A 1993 study identified in healthy controls a circulating subpopulation of T lymphocytes with intermediate expression of CD20 [3]. Increased frequencies of these circulating CD20dim T cells were noted in MS, and these cells were also depleted by anti-CD20 IgG1 antibodies [4]. Notably, a recent study by Von Essen et al. reported that an enrichment of CD20dim T cells in the cerebrospinal fluid (CSF) of MS patients positively correlates with disease severity, implying that CD20-expressing T cells may also exist in the human brain [5]. Our group recently revealed the human brain to be populated by small numbers of perivascular predominantly CD8+ tissue-resident memory T (TRM) cells [6, 7]. In brain donors with advanced MS, we observed recruitment of CD8+ TRM cells from the perivascular space into MS lesions [8]. We now investigated whether rapid postmortem autopsy-acquired viable brain CD8+ TRM cells in control and MS donors constitutively express intermediate levels of CD20. First, we stained with immunohistochemistry tissue sections of human MS white matter lesions for T and B cells (Fig. 1A). As described by us and others previously, these lymphocytes were mostly found in the perivascular space [7]. We encountered CD20bright CD3− B cells, CD20dim CD3+ T cells, and CD20- CD3+ T cells in the perivascular space, surrounding an active white matter lesion. Our findings are in line with former reports of CD20+ CD4+ and CD8+ T cells in MS white matter lesions [9], and support the presence of CD20dim T cell in the brain. We earlier identified the normal postmortem human brain to be populated by predominantly CD8+ TRM cells [6, 7]. To analyze CD20 expression by these cells, we isolated and analyzed viable CD8+ T cells from postmortem human brain white matter and stained them for the TRM-cell markers CD69, CD103, CD49a, PD1, CD44, and CXCR6 (Fig. 1B, donor and sample information in Supporting Information Table S1). For comparison, CD8+ T cells from peripheral blood were analyzed. In the circulation, we encountered a small proportion of CD8+ T cells with intermediate CD20 expression, in accordance with earlier studies (Fig. 1B) [4, 5]. Contrastingly, brain CD8+CD69+ TRM cells showed a higher proportion of CD20dim cells. Of note, CD20 expression was higher in the CD103− when compared to the CD103+ CD8+CD69+ TRM-cell fraction (Fig. 1C). To exclude the possibility of B- and T-cell duplicates as substrate for the CD20 expression by T cells, despite singlet gating, we performed imaging flow cytometry and confirmed the presence of both CD20dim and CD20− brain CD8+CD69+ TRM cells (Supporting Information Fig. S1 and Fig. 1D). Lastly, since autofluorescence and multiple fluorochromes imply technical challenges, we confirmed with quantitative reverse transcription-polymerase chain reaction (RT-PCR) that brain CD8+ TRM cells express mRNA encoding CD20 (MS4A1) and not CD19 (Fig. 1E). We conclude that brain CD8+ TRM cells constitutively contain a CD20dim subpopulation corroborating the recent report of von Essen et al., showing an enrichment of CD20dim CD4+ and CD8+ T cells in the CSF compared to peripheral blood [5]. We recently showed postmortem MS white matter lesions to be enriched for CD8+ TRM cells [8]. To analyze a possible association of brain CD20dim CD8+ TRM cells with MS lesions, we stained control white matter and MS white matter lesions for CD3 and CD20 (Fig. 2A). An increased proportion of T cells displayed a CD20dim phenotype in MS lesions (Fig. 2B), coinciding with an increased T-cell number (Fig. 2C). In addition, we compared with flow cytometry CD20expression between CD8+ T cells isolated from control white matter, MS normal-appearing white matter (NAWM), and MS active lesions. All tissue blocks with lesions contained either active or mixed active/inactive lesions, as determined with human leukocyte antigen (HLA)-DR and myelin proteolipid protein immunohistochemistry (Fig. 2D). Both in the CD103− and CD103+ fractions of CD8+CD69+ TRM cells, an increased proportion of CD20dim T cells was observed in MS lesions when compared to control white matter (Fig. 2E). Notably, the higher proportions of CD20dim T cells observed with flow cytometry may reflect a higher sensitivity for this weakly expressed antigen when compared to immunohistochemistry. These findings confirm the association of brain CD20dim CD8+ TRM cells with active lesions in MS. Lastly, to explore a possible functional relevance of CD20 expression, we compared with flow cytometry expression of markers of tissue homing (CXCR6), activation (Ki-67), and cytotoxic potential (granzyme B and GPR56) between CD20− and CD20dim brain CD8+ TRM cells. Distribution patterns were overlapping between cells isolated from control white matter, MS NAWM, and MS lesions (Fig. 2F). Expression levels of CXCR6, Ki-67, and granzyme B were higher on CD20dim when compared to CD20− CD8+ TRM cells regardless of CD103 expression (all p = 0.021 to p < 0.001). This notion is in line with earlier studies, attributing to CD20dim T cells a higher expression of chemokine receptors and a higher cytotoxic potential [5]. We here demonstrate the presence of CD20dim CD8+ TRM cells among the physiological T-cell residents in the perivascular space and show the association of these cells with active MS lesions. The enrichment of CD20dim T cells in the circulation and CSF of people with MS suggests that this is a trait of T cells with a greater propensity to migrate into the perivascular space and into MS lesions. These data draw attention to CD20dim T-cell depletion as potential mechanism underlying the efficacy of anti-CD20 therapies. This research is funded by MS Research (MS 14-888), the Vriendenloterij, the German Research Foundation (FOR 2149), and the National MS Fonds (OZ2018-003). CCH, NFL, AMRB, KIMB, IH, and JH declare to have no commercial or financial conflict of interest. JS received consultancy and/or lecture fee from Biogen, Merck, Novartis, and Sanofi-Genzyme. The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202048665. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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