A0452

For H&E staining, 5‐micron‐thick FFPE slides 5‐micron were washed twice for 5 min each in xylene, 100% ethanol, and 95% ethanol, followed by a brief rinse in water and a 15‐min incubation in hematoxylin. After a brief water rinse post‐incubation, slides were dipped in 0.25% hydrochloric acid‐ethanol, followed by brief dip in 1% lithium carbonate, a water rinse, and a 5‐min incubation in eosin Y. Slides were then briefly rinsed twice each with 95% ethanol, 100% ethanol, and xylene and were cover‐slipped.
IHC for CD3 and CD20 was performed on 5‐micron‐thick formalin‐fixed paraffin‐embedded whole tissue sections using the following antibodies (clone, catalog number, dilution, vendor): CD3 (polyclonal, A0452, 1:400, Dako, Agilent Technologies; Santa Clara, California, USA) and CD20 (monoclonal/L26, M0755, 1:140, Dako, Agilent Technologies; Santa Clara, California, USA), each using DIVA retrieval in the decloaker (Biocare Medical; Pacheco, California, USA) and a polymer detection system (Dako, Agilent Technologies; Santa Clara, California, USA), with counterstains using dab and hematoxylin.

Mouse tumors were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned by the Research Histology Core Laboratory at The University of Texas MD Anderson Cancer Center. Patient tumors were isolated and processed according to the phase I trial protocol^8,10 and obtained from MD Anderson. Immunohistochemistry was performed with either rat anti-mouse CD31 mAb (SZ31, Dianova, Hamburg, Germany) or rabbit anti-human CD3 pAb (A0452, Dako [Agilent], Santa Clara, CA) followed by rabbit anti-rat HRP- or goat anti-rabbit HRP-conjugated secondary antibody, respectively (Bio-rad Laboratories, Hercules, CA) and the DAB peroxidase substrate kit (Vector Laboratories, Inc. Burlingame, CA). Sections were then counterstained with hematoxylin. Tissue sections were blindly quantified by manual counting. Representative areas^2,3 from each CD31 stained section (n = 12 in total) with most intensive microvessel density was captured under the light microscope at 200x (Leica). MVD per tumor section was calculated from the average count of CD31⁺ vessels per representative area, averaging total number from n = 3 tumors per treatment group. Total image length is 384 µm. Immunofluorescence was performed using rat mAb CD4 (GK1.5, Abcam, Cambridge, MA) and rabbit pAb CD8 antibodies (ab4055, Abcam). Sections were examined with a Nikon microscope and camera and processed in ImageJ.

Histochemical and immunohistochemical staining was performed on sections cut to 6 µm from paraffin-embedded tissue blocks. Randomly chosen sections of every patient were deparaffinated and rehydrated and subsequently stained with H&E. Immunohistochemistry for CD3 (polyclonal rabbit anti-human CD3, diluted 1:50, A0452; Dako) was performed on sections adjacent to H&E-stained sections with the Ventana Benchmark GX automated staining system using a CC1 (Roche) pretreatment and the iView DAB Detection Kit (Roche). Sections were counterstained with hematoxylin and coverslipped with Entellan (Merck) as mounting medium. Age of infarcts was estimated by two experienced and independent neuropathologists (T. Arzberger and P.T. Nelson) on H&E sections according to published criteria (Mena et al., 2004 (link)). CD3 stains were scanned with Zeiss Axio Scan Z1 using a 20× objective. The infarct area was demarcated, and the absolute numbers and coordinates of intra- and extralesional CD3⁺ T cells were assessed manually using Qupath (version 0.2.2). Cell density was calculated as number of cells per cm² of defined lesion area. The R index for quantification of cell clustering per area was calculated as described above in R (version 3.6.0). Intralesional cell density was correlated with time after stroke onset for each sample.