Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

Abstract

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both alpha and beta chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

Fig. 1.

Imparting the desired CD8 cytotoxic or CD4 helper T cell specificity to the mouse T cell repertoire by genetic modification of HSCs. HSCs from RAG1^–/– or B6 mice were infected with MOT1 or MOT2 retroviruses and transferred into either RAG1^–/– (denoted as RAG1/MOT1 or RAG1/MOT2) or B6 (denoted as B6/MOT1 or B6/MOT2) recipient mice, respectively. RAG1^–/– (denoted as RAG1) or B6 mice were included as controls. OT1 and OT2 TCRs were detected by costaining of TCR Vα2 and Vβ5. (a) Schematic representation of the MOT1 and MOT2 retrovirus constructs. IRES, internal ribosomal entry site; WRE, woodchuck responsive element. (b) Expression of OT1 or OT2 TCRs in BM detected by intracellular staining. (c) Thymic development of OT1 or OT2 T cells. (Upper) Thymocyte expression of OT1 or OT2 TCRs was detected by intracellular staining. (Lower) Distribution of developmental markers CD4 and CD8 is shown. (d) Detection of mature OT1 CD8 or OT2 CD4 T cells in periphery.

Fig. 2.

Comparison of the efficacy of generating OT2 T cells in RAG1/MOT2 mice and the conventional RAG1/OT2 Tg mice. OT2 TCRs were detected by costaining of TCR Vα2 and Vβ5. (a) OT2 TCR expression in BM as measured by intracellular staining. (b) OT2 TCR expression and T cell development in thymus (Thy). T cell development was assessed by distribution of developmental markers CD4 and CD8. OT2 TCR expression at each development stage is shown. DN, double negative; DP, double positive; SP, single positive. (c) TCR expression in peripheral OT2 T cells. Mice were immunized with OVAp2 antigen and complete Freund's adjuvant for 6 days. Both intracellular and surface expressions of OT2 TCR was measured. (d) Total OT2 T cells in unchallenged (naïve) and immunized mice.

Fig. 3.

Characterization of the OT1 CD8 T cells generated by genetic programming of WT HSCs. OT1 T cells harvested from B6/MOT1 mice [denoted as OT1(BMT), where BMT is BM transfer] 8 weeks after HSCs transfer were considered to be naïve. They were stimulated with OVAp1 in vitro for 3 days to generate effector OT1 T cells, which were then transferred into host mice for memory study. (a) Patterns of surface activation markers on OT1(BMT) T cells at the naïve, effector, or memory stages. (b) Functional analysis of the naïve OT1(BMT) T cells (▪). Proliferation and IFN-γ production in response to OVAp1 stimulation are shown. The responses were compared with OT1 T cells from the conventional OT1 TCR transgenic mice, denoted as OT1(Tg) (•). B6 spleen cells were included as negative control (B6 Ctrl, ▴). Equal numbers of OT1(BMT) and OT1(Tg) T cells were used in all of the experiments. (c) Functional analysis of memory OT1(BMT) T cells [denoted as OT1(BMT, Memory), ▪]. Dosage response (Left) and time-course response (Center) to OVAp1 stimulation as measured by IFN-γ production and proliferation response to cytokine stimulation (Right) are shown. The responses were compared with those of the naïve OT1(BMT) T cells [denoted as OT1(BMT, Naïve), •). B6 spleen cells were included as a negative control (B6 Ctrl, ▴). Equal numbers of OT1(BMT, Memory) and OT1(BMT, Naive) T cells were used in all of the experiments.

Fig. 4.

In vivo constitution of anti-tumor T cell immunity. E.G7 mouse tumor model was used. Tumor size is shown as the product of the two largest perpendicular diameters a × b (mm²). Each line represents one mouse. Four mice were included in each group, and experiments were performed three times. Results from one representative experiment are shown. (a) Constitution in mouse of both arms of anti-tumor T cell immunity. FACS staining showed the detection of mature OT1 CD8 and OT2 CD4 T cells in the spleen of B6/MOT1+MOT2 mice (B6 mice receiving both MOT1- and MOT2-transduced WT HSCs). (b) Suppression of syngenic tumor growth by in vivo constitution of anti-tumor T cell immunity. EL.4 and its OVA-expressing derivative, E.G7, were the two mouse tumor types examined. DC/OVAp1, DCs loaded with OVAp1. (c) Eradication of established solid tumors by in vivo constitution of both arms of anti-tumor T cell immunity. E.G7 tumor cells were used. DC/OVAp1+2, DCs loaded with both OVAp1 and OVAp2.