Fig 2: The Majority of CD4-MBL CAR/CXCR5 Transduced Cells are T central memory (TCM)(A) Representative flow cytometry plots of the starting cell population, day 9 mock, day 9 total transduced, and CAR/CXCR5 transduced subset of rhesus PBMCs. For panels 1–3, gates were set on live, CD3+, and CD8+ cells. For the transduced subset, gates were set on live, CD3+, MBL+, and CD8+ cells. Central memory was defined as CD28+CD95+. Effector memory was defined as CD28-, CD95+. (B) The percentages of central memory cells on day 9 in mock and total transduced cells and the CAR/CXCR5 subset of transduced cells from six individual animals. Mean and 95% confidence intervals are shown with bars. (C) Representative flow plot of CCR7 expression in cells identified as CD28+/CD95+. (D) CCR7 expression levels in the central memory subset of mock, total transduced cells and the CAR/CXCR5 subset of transduced cells from six individual animals. Mean and 95% confidence intervals are shown with bars.

Fig 3: 9-Day Transduction and Expansion Protocol Yields Sufficient Cells for Infusion, Which Are Viable and Maintain Co-expression of Two Transduced Genes(A) Total number of cells in culture on days 5 and 9 of the expansion protocol and (B) viability of the transduced cells using PBMCs from seven different animals. Cell number and viability was monitored by trypan blue exclusion counting on the countess cell counter. (C) Representative flow plots showing gating strategy and expression of both the CD4-MBL CAR and CXCR5 in transduced cells on day 9. (D) Expression of CAR, CXCR5, and co-expression of CAR and CXCR5 on days 5 and 9 in cells from seven different animals. The bars represent the mean value and 95% confidence interval.

Fig 4: Virus Production in PBMCs Was Reduced in Cells Transduced with CAR/CXCR5 Compared to Mock Transduced CellsPBMCs isolated from rhesus macaques infected with SIV mac 251 were transduced with CAR/CXCR5 or CXCR5 only using the 9-day protocol. Mock samples were treated identically without exposure to the transducing retrovirus. At the conclusion of the transduction and expansion procedure, equal numbers of mock and transduced cells were evaluated for the presence of viral RNA. Cell pellets were analyzed for the presence of gag mRNA relative to the housekeeping gene beta-actin by RT-PCR. Colors denote matched transduced or mock transduced cells from seven individual animals. A Wilcoxon matched pairs signed rank test was used to determine significance of the difference between mock and CAR/CXCR5 samples. The bar represents the median value.

Fig 5: Transduced Cells Demonstrated Specific Migration to the CXCR5 Ligand, CXCL13, In VitroTransduced Rhesus macaque PBMCs (one million cells in 100 μL X-Vivo-15 containing 0.1% BSA) were placed in the upper chamber of a transwell plate with a 5.0 μm membrane. The lower chamber contained X-Vivo/1% BSA only or X-Vivo/1% BSA and the chemokine CXCL13 (2.5 μg/mL). After incubating for 4 h at 37°C, cells were collected and counted on a CytoFLEX flow cytometer (Beckman). All samples were normalized with the addition of AccuCheck counting beads. Specific migration was determined by subtracting the number of cells that migrated to X-Vivo/BSA alone from the number that migrated to the chemokine and then dividing by the number of input cells. Colors denote cells from seven individual animals. A Wilcoxon matched pairs signed rank test was used to determine significance. The bar represents the median value.