Transplantation of mouse HSCs genetically modified to express a CD4-restricted TCR results in long-term immunity that destroys tumors and initiates spontaneous autoimmunity
Sung P. Ha, … , Hal E. Broxmeyer, Christopher E. Touloukian
Sung P. Ha, … , Hal E. Broxmeyer, Christopher E. Touloukian
Published November 15, 2010
Citation Information: J Clin Invest. 2010;120(12):4273-4288. https://doi.org/10.1172/JCI43274.
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Research Article Genetics

Transplantation of mouse HSCs genetically modified to express a CD4-restricted TCR results in long-term immunity that destroys tumors and initiates spontaneous autoimmunity

Abstract

The development of effective cancer immunotherapies has been consistently hampered by several factors, including an inability to instigate long-term effective functional antitumor immunity. This is particularly true for immunotherapies that focus on the adoptive transfer of activated or genetically modified mature CD8+ T cells. In this study, we sought to alter and enhance long-term host immunity by genetically modifying, then transplanting, mouse HSCs. We first cloned a previously identified tumor-reactive HLA-DR4–restricted CD4+ TCR specific for the melanocyte differentiation antigen tyrosinase-related protein 1 (Tyrp1), then constructed both a high-expression lentivirus vector and a TCR-transgenic mouse expressing the genes encoding this TCR. Using these tools, we demonstrated that both mouse and human HSCs established durable, high-efficiency TCR gene transfer following long-term transplantation into lethally irradiated mice transgenic for HLA-DR4. Recipients of genetically modified mouse HSCs developed spontaneous autoimmune vitiligo that was associated with the presence of a Th1-polarized memory effector CD4+ T cell population that expressed the Tyrp1-specific TCR. Most importantly, large numbers of CD4+ T cells expressing the Tyrp1-specific TCR were detected in secondary HLA-DR4–transgenic transplant recipients, and these mice were able to destroy subcutaneously administered melanoma cells without the aid of vaccination, immune modulation, or cytokine administration. These results demonstrate the creation of what we believe to be a novel translational model of durable lentiviral gene transfer that results in long-term effective immunity.

Authors

Sung P. Ha, Nicholas D. Klemen, Garrett H. Kinnebrew, Andrew G. Brandmaier, Jon Marsh, Giao Hangoc, Douglas C. Palmer, Nicholas P. Restifo, Kenneth Cornetta, Hal E. Broxmeyer, Christopher E. Touloukian

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Figure 1

DR4-restricted, Tyrp1-specific TCR is expressed and functional.

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DR4-restricted, Tyrp1-specific TCR is expressed and functional. (A) At a...
(A) At an increasing viral MOI, lentivector containing the F2A gene was superior to IRES in generating stable TCR heterodimers in human PBMCs. Cell surface expression of the Tyrp1-specific TCR was determined by flow cytometry using a PE-labeled DR4 Ultimer containing the Tyrp1277–297 epitope. At MOI 30: 37.5% ± 1.5% versus 56.4% ± 1.1% (P = 0.0008). hCD45, human CD45. (B) PBMCs transduced with LV-TRP1-F2A (LV-TCR) were functionally specific. Specific IFN-γ production detected by ELISA to the specific peptide (Tyrp1277–297), lysate (B16 lysate), and intact tumors (B16-DR4, 624 Mel pretreated with IFN-γ, and 1102 Mel stably transduced with Tyrp1), but not to the control peptide (HA306–318), lysate (MC38), or tumors (wild-type B16, MC38-DR4, and 1102 Mel stably transduced with GFP). Interactions with Tyrp1277–297–pulsed targets were blocked by L243 (HLA-DR) and L3T4 (CD4), but not W6/32 (class I) or HIT8a (CD8) antibodies. Peptides were pulsed onto DR4+ 1088 EBV-B cells at 50 μM for 3 hours. Txp, transplant. (C) TCR Tg mice crossed with DR4 Tg mice strongly expressed the gene-specific TCR. Flow cytometry analysis (CD3/CD4/Tyrp1 Ultimer) of TCR+DR4+ and TCR+DR4– mice. Control staining of TCR+DR4+ with a gp100 Ultimer is shown. (D) Summary of PB TCR gene expression in TCR+DR4+ and TCR+DR4– mice within CD3 and CD4 compartments: CD4, 79% ± 8.3% versus 6.4% ± 1.0%; P < 0.0001. Data (mean ± SEM with P value; n = 5–7 per group) and flow cytometry are representative of 2–3 independent experiments.