Systemic T cell-independent tumor immunity after transplantation of universal receptor-modified bone marrow into SCID mice

Abstract

Gene modification of hematopoietic stem cells (HSC) with antigen-specific, chimeric, or "universal" immune receptors (URs) is a novel but untested form of targeted immunotherapy. A human immunodeficiency virus (HIV) envelope-specific UR consisting of the extracellular domain of human CD4 linked to the zeta chain of the T cell receptor (CD4 zeta) was introduced ex vivo into murine HSC by retroviral transduction. After transplantation into immunodeficient SCID mice, sustained high level expression of CD4 zeta was observed in circulating myeloid and natural killer cells. CD4 zeta-transplanted mice were protected from challenge with a lethal dose of a disseminated human leukemia expressing HIV envelope. These results demonstrate the ability of chimeric receptors bearing zeta-signaling domains to activate non-T cell effector populations in vivo and thereby mediate systemic immunity.

Figure 1

Analysis of CD4ζ expression in transplanted mice. (a) Flow cytometric analysis. PB leukocytes from mice 3 wk after transplant were incubated with the following murine-specific mAbs conjugated to FITC: anti–GR-1, anti–Mac-3, anti-5E6 in addition to anti–human CD4-PE. Background human CD4 expression in various lineages in a control mouse (top) is compared to human CD4 expression in a CD4ζ transplanted mouse (bottom). Specific cell staining was measured on gated populations containing myeloid cells (Gr-1 and Mac-3) and lymphoid cells (NK5E6), as determined by forward and side scatter characteristics. Results are representative of those observed in 80 additional mice. (b) QC-PCR analysis. CD4ζ-expressing PB cells from a mouse 3 wk after transplant (e ⁻) were combined with known titrations of CD4ζ-transduced competitor cells (e ⁺). The lane in which the e ⁻ and e ⁺ amplification products are equivalent represents the percentage of PB cells containing the CD4ζ gene; in this case, ∼10%. The corresponding expression level by FACS^® analysis in this mouse was 20%. PB from an untransplanted mouse serves as a control. (c) The structure of the integrated CD4ζ retroviral vector and the competing template used in the QC-PCR assay. The CD4ζ construct used to generate the e ⁺ competitor cells contains an additional 102-bp sequence to enable differential separation of the PCR products on gel electrophoresis. The location of the PCR primers (black arrows) and the expected competing transcripts (e ⁻ and e ⁺) are shown. PGK, human phosphoglycerate kinase promoter.

Figure 2

Survival of transplanted mice after Raji-env cell infusion. In two separate experiments, SCID mice were transplanted with UR-transduced bone marrow (CD4ζ or SAbζ). 3 wk after transplant, UR-transduced and control mice were injected via the tail vein with Raji-p or Raji-env tumor cells. (a) Survival of CD4ζ mice (10/group) receiving 10⁵ Raji-env (CD4ζ+R-env) or Raji-p (CD4ζ+R-p) cells compared to historical control untransplanted mice (5/group) receiving 10⁵ Raji-env (ctrl+R-env) or Raji-p (ctrl+R-p) cells. Between 4 and 8 mo after transplant, four of the Raji-env survivors died from the spontaneous development of endogenous thymic lymphomas, which is a known complication of sublethal irradiation in SCID mice (51). (b) Survival of CD4ζ and SAbζ mice (5/group) challenged with 10⁵ Raji-env or Raji-p cells compared with concurrent control untransplanted mice infused with either tumor (5/group). SAbζ mice infused with 10⁵ Raji-p all died within 50 d.

Figure 3

Death of CD4ζ-transplanted mice after Raji-env infusion is associated with a loss of gp120 expression in vivo. (a) Flow cytometric analysis. Raji-env cells sorted from the bone marrow of a CD4ζ-transplanted (CD4ζ sort) and a control mouse (control sort), as well as Raji-p and Raji-env cells maintained in liquid culture, were incubated with mouse anti-gp120 mAb to detect surface expression of HIV-env or the isotype-negative control, followed by incubation with goat anti–mouse biotin F(ab′)₂ and APC (Molecular Probes). (b) Immunoblot analysis. Sorted Raji-env cells (CD4ζ sort and control sort) and cultured Raji-p and Raji-env cells were lysed and subjected to SDS-PAGE, followed by immunoblotting with anti-gp120 mAb to detect the presence of the env protein.

Figure 4

PCR analysis of surviving mice shows no evidence of residual Raji-env. Lysates of PB were prepared from eight surviving CD4ζ-transduced mice challenged with Raji-env at 4 mo after transplant. DNA was amplified using primers specific for a 905-bp human endogenous retroviral sequence (Herv-H) to detect Raji-env DNA (Mouse PB, lanes 1–8). Results are compared to titrations of cultured Raji-env cells in a background of 10⁴ murine cells amplified with Herv-H primers (Raji) and Herv-H–amplified PB from a control mouse (con). Samples were amplified with murine β-actin primers (447-bp product) as a positive control (Mouse PB, lanes 1–3).

Figure 5

CD4ζ-expressing neutrophils lyse Raji-env targets in vitro. Cytotoxicity assays were performed using neutrophils isolated from CD4ζ-transplanted or control mice as effectors and Raji-p or Raji-env cells as targets. E/T ratios were corrected for the percentage of the bulk neutrophil population expressing CD4ζ by FACS^® analysis. FcR-mediated ADCC was measured by incubation of neutrophils with Raji-p in the presence of polyclonal anti–human lymphocyte serum. Maximal cytolysis of Raji-env cells by CD4ζ-expressing neutrophils in three experiments was 10%, 9%, and 8% at corrected E/T ratios of 20:1, 30:1, and 25:1, respectively. Background cytolysis by controls (CD4ζ neutrophils+Raji-p and untransduced (ctrl) neutrophils+Raji-env) was always <1%, even at E/T ratios as high as 400:1.