Human-engineered Treg-like cells suppress FOXP3-deficient T cells but preserve adaptive immune responses in vivo

Abstract

Objectives: Genetic or acquired defects in FOXP3+ regulatory T cells (Tregs) play a key role in many immune-mediated diseases including immune dysregulation polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previously, we demonstrated CD4+ T cells from healthy donors and IPEX patients can be converted into functional Treg-like cells by lentiviral transfer of FOXP3 (CD4LVFOXP3). These CD4LVFOXP3 cells have potent regulatory function, suggesting their potential as an innovative therapeutic. Here, we present molecular and preclinical in vivo data supporting CD4LVFOXP3 cell clinical progression.

Methods: The molecular characterisation of CD4LVFOXP3 cells included flow cytometry, qPCR, RNA-seq and TCR-seq. The in vivo suppressive function of CD4LVFOXP3 cells was assessed in xenograft-versus-host disease (xeno-GvHD) and FOXP3-deficient IPEX-like humanised mouse models. The safety of CD4LVFOXP3 cells was evaluated using peripheral blood (PB) humanised (hu)- mice testing their impact on immune response against pathogens, and immune surveillance against tumor antigens.

Results: We demonstrate that the conversion of CD4+ T cells to CD4LVFOXP3 cells leads to specific transcriptional changes as compared to CD4+ T-cell transduction in the absence of FOXP3, including upregulation of Treg-related genes. Furthermore, we observe specific preservation of a polyclonal TCR repertoire during in vitro cell production. Both allogeneic and autologous CD4LVFOXP3 cells protect from xeno-GvHD after two sequential infusions of effector T cells. CD4LVFOXP3 cells prevent hyper-proliferation of CD4+ memory T cells in the FOXP3-deficient IPEX-like hu-mice. CD4LVFOXP3 cells do not impede in vivo expansion of antigen-primed T cells or tumor clearance in the PB hu-mice.

Conclusion: These data support the clinical readiness of CD4LVFOXP3 cells to treat IPEX syndrome and other immune-mediated diseases caused by insufficient or dysfunctional FOXP3+ Tregs.

Keywords: CRISPR/Cas9; FOXP3; IPEX syndrome; gene therapy; lentiviral vector; regulatory T cells.

Conflict of interest statement

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© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

Figures

Figure 1

CD4LVFOXP3 cells have a Treg‐like phenotype. Expression of bona fide Treg molecules measured by FACS in (a) healthy donors (n = 8, mean + SEM) and (b) patients with IPEX syndrome (n = 2, mean + SEM). (c) Expression of other Treg‐related molecules measured by FACS (n = 6, mean + SEM). (d) Expression of Treg‐related molecules measured by real‐time PCR. HPRT mRNA was used as internal control. Relative expression was calculated by ∆CT (n = 8, mean + SEM). UT, untransduced CD4+ T cells cultured in parallel with CD4LVFOXP3 and CD4LVNGFR cells. P‐values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 2

CD4LVFOXP3 cells maintain a polyclonal TCR repertoire. CDR3 length of CD4+ T cells (prior to the culture), CD4LVNGFR and CD4LVFOXP3 cells from (a) healthy donors (n = 2) and (b) IPEX patients (n = 2). (c) TCRVβ usage of CD4+ T cells, CD4LVNGFR and CD4LVFOXP3 cells from the same healthy donors (n = 2) and IPEX patients (n = 2). HD = healthy donors, GD0037 and GD0064 = IPEX patients.

Figure 3

CD4LVFOXP3 cells have a Treg‐like gene expression profile. (a) Principal component analysis (PCA), (b) hierarchical cluster analysis (HCA), (c) heat map of differentially expressed genes (DEGs) between CD4LVFOXP3 and CD4LVNGFR cells (n = 3), (d) shared DEGs (upregulated) between freshly isolated FOXP3 Tregs and CD4LVFOXP3 cells of the same donors. (e) Heat map of the seven shared upregulated genes between freshly isolated FOXP3 Tregs and CD4LVFOXP3 cells. (f) DEGs (downregulated) in freshly isolated FOXP3 Tregs and CD4LVFOXP3 cells.

Figure 4

CD4LVFOXP3 cells suppress xeno‐GvHD in autologous condition. (a) Survival of xeno‐GvHD mice with Teff alone or in the presence of CD4LVFOXP3 cells (allogeneic or autologous) or autologous CD4LVNGFR cells (n = 7 or 8). (b) Weight loss of xeno‐GvHD mice under the same conditions (n = 7 or 8, mean ±  SEM). (c) Percentage of human (h) CD45+ cells in peripheral blood (PB; n = 7 or 8, mean ±  SEM). (d) Percentage of NGFR+ cells in hCD45+ cells in PB of the mice (n = 7 or 8, mean ±  SEM). (e) Percentage of hCD45+ cells in the spleen between weeks 2 and 3 (n = 4, mean ±  SEM). (f) Percentage of CD25, CD69, CD71 and HLA‐DR‐positive cells in hCD45+cells in the spleen between weeks 2 and 3 (n = 4, mean ±  SEM). Data are representative of two independently repeated experiments. All phenotyping was performed by FACS. P‐values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 5

CD4LVFOXP3 cells prevent xeno‐GvHD reaction after re‐challenge. (a) Survival of xeno‐GvHD mice with Teff alone (day 0), Teff and CD4LVNGFR/CD4LVFOXP3 cells co‐injection (day 0), Teff‐late (day 16) and re‐challenge conditions (day 0 and day 16). Re‐challenge mice received the same responding Teff (day 16) after the initial co‐injection of autologous CD4LVFOXP3 cells (n = 8 or 9). (b) Weight loss of xeno‐GvHD mice (n = 8 or 9, mean ±  SEM). (c) Percentage of human (h) CD45+ cells in PB (n = 8 or 9, mean ±  SEM). (d) Percentage of NGFR+ cells in hCD45+ cells in PB (n = 8 or 9, mean ±  SEM). Data are representative of two independently repeated experiments. All phenotyping was performed by FACS. P‐values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 6

CD4LVFOXP3 cells can ameliorate CD4 dominant lymphoproliferation in FOXP3 KO hu‐mice. (a) Survival of FOXP3 KO hu‐mouse model comparing mice transplanted with unmodified HSPC (UM), FOXP3 KO HSPC alone or plus CD4LVFOXP3 cells (n = 16). (b) Percentage of NGFR+ cells in human CD4+ cells in PB (n = 16, mean ± SEM). (c) Percentage of hCD45+/CD4+/CD8+ cells in PB (n = 12–16, mean ± SEM). (d) Percentage of hCD45+/CD4+/CD8+ cells in the spleen (n = 12–16, mean ± SEM). (e) Absolute numbers of hCD45+/CD4+/CD8+ cells in the spleen (n = 12–16, mean + SEM). (f) Frequency of INDEL was measured by TIDE analysis of PB from FOXP3 KO hu‐mice (n = 8–10, mean ± SEM). Data are representative of two independently repeated experiments. All phenotyping was performed by FACS. P‐values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 7

Immunity against various pathogens is preserved after CD4LVFOXP3 cells administration.(a) Design of the experiment to test immune response against fungal (Candida albicans) or viral antigens (Adenovirus). Results after in vitro restimulation in the presence of mDC pulsed with different antigens are shown. (b) Frequency of IFN‐γ‐positive cells counted by ELISpot (n = 4 or 5, mean ± SEM). (c) Proliferation of CD4+ T cells (n = 8 or 9, mean ± SEM). (d) Frequency of IFN‐γ‐positive cells counted by ELISpot (n = 8 or 9, mean ± SEM). (e) Frequency of IFN‐γ‐positive cells (n = 8 or 9, mean ± SEM). Data are representative of two independently repeated experiments. no = T cells only, NO = no antigen, CA = Candida albicans, TT = tetanus toxoid (negative control), Adv = Adenovirus, EBV = Epstein‐Barr virus (negative control), CMV = Cytomegalovirus (negative control). P‐values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 8

Immune surveillance against tumor antigen is preserved after CD4LVFOXP3 cells administration. (a) Design of the experiment to test immune surveillance against tumor antigens. (b) Tumor size measured every 3 days by direct measurement (n = 8 or 9, mean ± SEM). (c) Luminescence intensity (ROI) of skin sarcoma measured weekly by in vivo imaging system (IVIS) (n = 8 or 9, median). (d) Tumor weight (mg) measured after isolation of skin sarcoma (n = 8 or 9, mean ± SEM). (e) Tumor volume (mm3) calculated after isolation of skin sarcoma (n = 8 or 9, mean ± SEM). Data are representative of two independently repeated experiments. P‐values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.