Fig 1.
Distribution of CTV-labeled infused T cell product in different tissues at 2 days post-treatment.
Location of cell trace violet (CTV)-labeled infused T cells (pseudo-colored magenta) was determined in a chronically SIV-infected ART-naïve rhesus macaque, animal R14025 at two days post-treatment (DPT). CTV-labeled infused T cell product was detected in both the follicular and extrafollicular areas of lymphoid tissues. Representative images from spleen, lymph node (LN), bone marrow, ileum, lung, liver, brain, and rectum. Tissues were stained with anti-IgM or anti-CD20 (green) to label B cells and delineate B cell follicles (green). Arrowheads point to CTV-labeled cells. Scale bars = 100 μm.
Fig 2.
CAR/CXCR5-T cells home to lymphoid follicles and recognize SIV-infected cells in vivo.
Location of CAR/CXCR5-T cells (red) within lymphoid tissues was determined in a chronically SIV-infected ART-naïve rhesus macaque, animal R14025 at 2 days post-treatment (DPT). Representative image of spleen tissue section showing duplex detection of CAR/CXCR5 construct (red) and SIV (pseudo-colored white) using RNAscope ISH combined with immunofluorescence using a custom-made probe for detection of CAR/CXCR5 construct and a probe specific for SIV. The white haze within the B cell follicle represents SIV virions trapped by the follicular dendritic cells (FDC) network. Scale bar = 100 μm. The right panels are enlargements showing an interaction between two CAR/CXCR5-transduced T cells and an SIV-infected cell. The tissue was stained with DAPI (blue), and anti-CD20 (green) to label B cells and delineate B cell follicles. Scale bar = 10 μm. Confocal images were collected using a 20× objective. The curves tool in Photoshop was used to increase the contrast of each image in a similar manner.
Table 1.
Animal information.
Fig 3.
A timeline of the Rhesus macaque pilot studies.
Animals were infected with SIV mac251 and ART suppressed. Cells were collected for transduction either post-infection (T1) or pre-infection (T2). ART was interrupted at the time of CAR/CXCR5 cell infusion. Blood and tissue samples were collected at indicated intervals post-infusion.
Table 2.
Cell infusions.
Fig 4.
Viral loads over time in (A) untreated control animals, (B) pilot study T1 animals and (C) pilot study T2 animals. Viral loads were determined by measurement of gag mRNA in plasma using reverse transcription (RT) polymerase chain reaction (PCR). The asterisk in panel B (T1) indicates the increase in viral load after infusion of the SIV containing T cell product. (D) Viral loads for the three animal groups one month post-infusion (27–30 DPI). The bar represents the median. (E) Total viral burden represented by area under the curve from day 0 to day 56 post-infusion. The bar represents the median for each of the groups.
Fig 5.
CAR/CXCR5-T cells continue to expand in vivo.
CAR/CXCR5-T cells expanded at the edges of B cell follicles in vivo at 2 DPT and accumulated within B cell follicles at 6 DPT. (A) Representative image from LN tissue from Rh2850 stained 2 DPT showing CTV-labeled cell proliferation in the extrafollicular (EF) area near the follicular (F) zone. Tissues were stained with anti-CD3 (blue) to label T cells and anti-CD20 (green) to label B cells and delineate B cell follicles (F). The infused T cell product labeled with CTV is pseudo-colored magenta. Cells within F showed lower fluorescence intensity indicating division. Scale bar = 50 μm. (B) Representative image of LN tissue section, from Rh2858 visualized using RNAScope ISH combined with IF, showing expansion of CAR/CXCR5-T cells at the edge of follicles at 2 DPT. The right panel is an enlargement from the left panel showing a cluster of CAR/CXCR5-T cells (red) that appear to be expanding at the edge of the follicle. Tissues were stained with DAPI (blue) and anti-IgM (green) to label B cells and delineate B cell follicles (F), with the more brightly stained germinal center in the center of the F. (C) Representative image of LN tissue from Rh2850, showing CAR/CXCR5-T cell (red) proliferation in F and EF at 6 DPT detected by RNAScope ISH combined with IF. Tissues were stained with anti-IgM (Blue) and anti-Ki67 (green) to mark activation and proliferation. B cell follicles are delineated with white lines. Scale bars = 100 μm. Confocal images were collected using a 20× objective. (D) Percentage of Ki67+ CAR/CXCR5 T cells in the F (green) and EF (blue) areas for each of the T2 animals.
Fig 6.
CAR/CXCR5-T cells localize to over 90% of the follicles and persist for up to 28 days.
CAR/CXCR5-T cells successfully homed to over 90% of the B cell follicles at 6 DPT and persisted for up to 28 DPT in SIV-infected ART-suppressed/released animals. Representative images of LN tissue section from Rh2858, showing CAR/CXCR5-T cells (red) detected using RNAScope ISH combined with IF using a custom-made probe for detection of the CAR/CXCR5 construct. (A) Confocal image showing a whole LN tissue section. (B) Enlargement of the delineated area in (6A) showing that CAR/CXCR5-transduced T cells (red) successfully homed to a B cell follicle (green). The tissue was stained with DAPI (blue), and anti-CD20 (green) to label B cells and delineate B cell follicles. The confocal image is collected with a 20× objective. Scale bar = 100 μm. (C) Levels of CAR/CXCR5-T cells over time after infusion in F (green) and EF areas (blue) of LN. The animal that lost control of the virus infection is marked with a red star. Samples not determined are marked ND. (D) Percentage of follicles that contained CAR/CXCR5-T cells over time post-infusion. (E) Frequency of CD4-MBL+ cells in CD3+ PBMCs as determined by flow cytometry and (F) copies of CAR in the total cell population in PBMCs as determined by quantitative real-time PCR at the indicated time points post-infusion.
Table 3.
T2 animals in vivo CAR/CXCR5+: vRNA+ E:T at 6 days post-infusion.
Fig 7.
Infusion of CAR/CXCR5-T cells into SIV-infected rhesus macaques results in lower viral loads post-ART interruption.
At 28 DPT, treated animals showed a reduction in viral (v)RNA compared to untreated control animals. (A) A representative image from a LN tissue section showing the abundance of SIV vRNA+ cells and free virions trapped by follicular dendritic cells network (pseudo-colored white) detected using RNAscope ISH combined with IF in treated from Rh2850 (left panels) versus untreated control from R11002 (right panels). The tissue was stained with DAPI (blue), and anti-CD20 (green) to label B cells and delineate B cell follicles. The white haze within the B cell follicle represents SIV virions trapped by the follicular dendritic cells (FDC) network. Confocal images were collected with a 20× objective. Scale bar = 100 μm. (B) Levels of viral RNA in F and EF areas. The animal that lost viral control of the virus infection is marked with a red star. (C) The percentage of follicles with free virions bound by FDC.