PD1-Targeted Transgene Delivery to Treg Cells

Abstract

Achieving the precise targeting of lentiviral vectors (LVs) to specific cell populations is crucial for effective gene therapy, particularly in cancer treatment where the modulation of the tumor microenvironment can enhance anti-tumor immunity. Programmed cell death protein 1 (PD-1) is overexpressed on activated tumor-infiltrating T lymphocytes, including regulatory T cells that suppress immune responses via FOXP3 expression. We developed PD1-targeted LVs by incorporating the anti-PD1 nanobody nb102c3 into receptor-blinded measles virus H and VSV-G_mut glycoproteins. We assessed the retargeting potential of nb102c3 and evaluated transduction efficiency in activated T lymphocytes. FOXP3 expression was suppressed using shRNA delivered by these LVs. Our results demonstrate that PD1-targeted LVs exerted pronounced tropism towards PD1⁺ cells, enabling the selective transduction of activated T lymphocytes while sparing naive T cells. The suppression of FOXP3 in Tregs reduced their suppressive activity. PD1-targeted glycoprotein H provided greater specificity, whereas the VSV-G_mut, together with the anti-PD1 pseudoreceptor, achieved higher viral titers but was less selective. Our study demonstrates that PD1-targeted LVs may offer a novel strategy to modulate immune responses within the tumor microenvironment with the potential for developing new therapeutic strategies aimed at enhancing anti-tumor immunity.

Keywords: FOXP3; PD1; Treg; lentivector; nanobody; retargeting.

Figure 1

(A) Schematic representations of retargeted measles (Hc∆18AA-102c3, 4AHc∆24AA-102c3), Nipah (Nip∆34Gm4-102c3), and VSV (VSVG_mut+102c3R) glycoproteins tested for lentivector retargeting. (B) Predicted structure of the homodimer of the measles H-protein head fused with nb102c3. (C) Microphotographs of the syncytia formation test performed on HEK-293T and HEK-293T-PD1 cells transfected with Hc∆18AA-102c3, 4AHc∆24AA-102c3, and Nip∆34Gm4-102c3 with corresponding F-protein-encoding plasmids and the lentivector plasmid encoding tagRFP (red channel). (D) Infectious titers of all retargeted LVs (i.u.) and selectivity (fold). (E) FACS plots of HEK-293T and HEK-293T-PD1 cells transduced with a corresponding lentivector carrying the tagRFP sequence (1 mL of non-concentrated lentivirus sample per 35 mm well).

Figure 2

(A) qPCR quantitation of PDCD1 levels in PBMCs cultured with IL-2 (ctrl), IL-2 + PHA-M (1), PMA + ionomycin + PHA-M (2), IL-2 + PHA-M + PMA + ionomycin (3), and IL-2 + PHA-M + PMA + ionomycin + IFNγ + IL-4 + IL-12 (4), **—p = 0.007. (B) nanoLuc luminescence levels normalized to cell count after transduction with lentiviral vectors of CD4⁺ T cells with or without transduction enhancers (Treatment 3, as in (A), and Treatment 3-PB-F108), and in the presence of 50 µg/mL azidothymidine (Treatment 3 + AZT). (C) nanoLuc luminescence levels normalized to cell count after transduction with control (4AHc∆24) and targeted (4AHc∆24AA-102c3, VSVGmut+102c3R) lentivectors, **—p < 0.0075. (D) nanoLuc luminescence levels in transduced CD4⁺ T cells, **—p = 0.0018, ***—p = 0.0005.

Figure 3

shRNA knockdown of FOXP3 with a non-targeted lentivector. (A) Schematic representation of shRNA-expressing lentivector and its cassette integrated in the genome. (B) qPCR quantitation of FOXP3 expression in stimulated CD4⁺ T cells and Tregs transduced with shFOXP3 and control shRNA, **—p = 0.0065. (C) qPCR quantitation of FOXP3 expression in PD1⁺ CD4⁺ T cells and PD1⁺ Tregs transduced with shFOXP3 and control shRNA, *—p = 0.015. (D) TGFβ levels in media of PD1⁻ and PD1⁺ Tregs transduced with shFOXP3 and the control, ****—p < 0.0001.

Figure 4

Cytometry measurements of CD4⁺ PD1⁺ T lymphocytes co-cultured with U937-tagGFP and transduced with 4AHc∆24AA-102c3-pseudotyped (A) VSVG_mut+102c3R-pseudotyped (B) and 4AHc∆24-pseudotyped (C) LVs carrying the tagRFP sequence. (D) FOXP3 expression in PD1⁺ Tregs co-cultured with U937 cells and transduced with different pseudotypes of LVs carrying shFOXP3 or control shRNA. *—p = 0.0485, ns—p > 0.05.