Fig 2: Ectopic lymphoid structures in progressive MS are characterized by infiltration of lymphocytes, FDCs and plasma cells. (A) Parenchyma of FFPE sections of brain and spinal cord of progressive MS patients were screened for infiltrated regions by H&E staining. (B) IF staining for CD3+ T cells and CD20+ B cells on serial sections were used to determine the infiltration score. Score 0, no or <5 lymphocytes; score 1, at least five but <30 lymphocytes; score 2, 31 to 60 lymphocytes; score 3, more than 60 lymphocytes. (C) Meninges and sulci of FFPE sections of brain and spinal cord of progressive MS patients were screened for infiltrated regions by H&E staining. (D) IF staining for CD3+ T cells and CD20+ B cells on serial sections were used to determine the infiltration score. Score 0, no or <5 lymphocytes; score 1, at least five, but <30 lymphocytes; score 2, 31 to 60 lymphocytes; score 3, more than 60 lymphocytes. (E) Whole slides were screened for infiltration on H&E, representative infiltration area depicted in the box (F), and serial sections were stained, depicted in the box (G–O). eLFs are characterized by (G) CD3+ T cells and CD20+ B cells, (H) CD4+ T cells and CD138+ plasma cells, (I) CD3+ T and CD3+CD4+ T helper cells (J) Ki67+ proliferating cells, (K) CD35+ and, (L) CD21+ FDCs, (M) CD68+ macrophages as well as (N) BCL-6+ and (O) CXCR5+ GC-like lymphocytes. (P) CD3+CD8+ cytotoxic T cells as well as some CD3+CD27+ memory T cells were also present in eLFs. Scale bars (A–D), (F) indicate 100 µm; (E) indicates 2,000 µm; (G–P) indicate 50 µm.

Fig 3: Follicle-like structures of SPMS brains are devoid of FOXP3 expression, but exhibit NFATc1+ cells. (A) Representative meningeal follicle-like structure of SPMS spinal cord, which was screened based on H&E staining and characterized by >60 lymphocytes (score 3), detection of Ki67+, CD35+/CD21+, and CD138+ cells on serial sections, termed F+. F–, if no or not all criteria were fulfilled. (B) Follicle-like structures could be characterized by CXCR5+, (C) but not by FOXP3+ cells. (D) NFATc1+ cells were present in follicle-like structures. (E) IHC stainings of Ki67, CD35, CD21, BCL-6, CXCR5, and NFATc1 were quantified as frequency of total cells in follicle-like structures (F+) and less defined infiltrates (F-). Ki67: F-, M = 1.52, SD = 3.73, n = 45; F+, M = 1.89, SD = 3.55, n = 38; Mann Whitney test, U = 734.0, p = 0.161. CD35: F-, M = 15.41, SD = 22.95, n = 22; F+, M = 19.05, SD = 14.75, n = 21; Mann Whitney test, U = 159.0, p = 0.081. CD21: F-, M = 5.68, SD = 10.63, n = 21; F+, M = 3.51, SD = 5.60, n = 20; Mann Whitney test, U = 183.5, p = 0.488. BCL-6: F-, M = 0.32, SD = 1.08, n = 44; F+, M = 0.49, SD = 1.62, n = 38; Mann Whitney test, U = 803.0, p = 0.642. CXCR5: F-, M = 14.72, SD = 17.24, n = 30; F+, M = 20.11, SD = 15.73, n = 29; Mann Whitney test, U = 319.0, p = 0.080. NFATc1: F-, M = 7.15, SD = 11.68, n = 28; F+, M = 16.36, SD = 21.46, n = 31; Mann Whitney test, U = 307.0, p = 0.043. Scale bars A-D indicate 100 µm. *, p < 0.05.

Fig 4: Detection of CD19 on myeloma cells using flow cytometry and dSTORM. a Flow cytometric analysis of CD19 expression on primary myeloma cells purified from bone marrow aspirates. Gating strategy for dot plots shown: FSC/SSC plasma cell gate ? 7-AAD- ? CD138+/CD38+. Plots show expression of CD19 or isotype control (Ctrl, x-axis) vs. CD138 (y-axis). Exemplary patients where myeloma cells comprised a CD19-positive fraction by FC (patient M012 & patient M016), were CD19-negative (patient M019) or showed an ambiguous expression (patient M022). Data for all patients are shown in Table 1 and Supplementary Fig. 1. b Detection of CD19 on multiple myeloma cells using dSTORM. Myeloma cells were identified by transmitted light microscopy and expression of CD138 and CD38 as detected by conventional wide-field fluorescence microscopy. CD19 was detected on primary myeloma cells using conventional wide-field fluorescence and dSTORM. Images depict CD19 molecules in the bottom plasma membrane (attached to glass surface) of a CD19-positive (top row) and a CD19-negative myeloma cell (bottom row). Small panels display magnification of boxed regions revealing the markedly enhanced sensitivity of dSTORM. c Exemplary images of CD19-positive cells as detected by dSTORM. Myeloma cells were identified as stated in b and CD19 was detected on primary myeloma cells using dSTORM for patients M012 (top image: 3.5 CD19/µm2; bottom image: 4.9 CD19/µm2), M016 (top image: 3.3 CD19/µm2; bottom image: 1.8 CD19/µm2, M019 (top image: 0.16 CD19/µm2; bottom image: 0.21 CD19/µm2 and M022 (top image: 3.6 CD19/µm2; bottom image: 5.2 CD19/µm2. The representative images depict CD19 molecules in the basal plasma membrane. Scale bars, 3 µm and 0.4 µm (magnification) (b), 5 µm (c)