Generating a Model of Human CD8+ T-Cell Lymphoma from Genetically Modified Normal T-Cells

Background: JAK2 fusions with multiple partners have been recurrently and frequently detected in the primary cutaneous CD8⁺ aggressive epidermotropic T-cell lymphoma (PCAETCL), in which CDKN2A mutation/deletion also commonly co-occurred¹. The pathogenesis role of JAK2 fusion and CDKN2A loss in this CD8⁺ T-cell lymphoma is yet to be determined.

Methods: We used CRISPR/Cas9 to knockout CDKN2A in normal human pan T-cells followed by introduction of a common JAK2 fusion, KHDRBS1-JAK2^1,2, through lentiviral transduction. Cell growth was monitored in vitro, while we also injected the modified cells intravenously into immunodeficient mice to determine the in vivo proliferative potential of these cells. The KHDRBS1-JAK2 fusion was also introduced into Jurkat cells to study the localization of the fusion protein and identification of its phosphorylation targets.

Results: The expression of p14 was barely detectable in normal T-cells but we could clearly demonstrate the deletion of p16 not only genetically but also by Western blot. CDKN2A-KO cells showed much superior growth than their wild-type counterpart. After ectopic expression of KHDRBS1-JAK2 fusion via a GFP-expressing lentiviral construct in either wild-type or CDKN2A-null T-cells, they did not exhibit any growth advantage in vitro based on GFP tracking. However, all three mice injected with the modified cells showed the presence of human CD45⁺ cells after two months with variable GFP⁺ percentage (9-67%). Importantly, the mice looked healthy and did not have signs of graft-versus-host disease (GvHD).

Interestingly, one of the three mice that received the human T-cells with JAK2 fusion and CDKN2A KO developed lymphoma after 133 days. The mouse displayed splenomegaly, with tumor nodules in the spleen, liver, and kidney. Peripheral blood examination showed the presence of large pleomorphic lymphoid cells. H & E staining revealed large abnormal cells in the affected organs forming tumor nodules in the spleen, kidney, and Liver. Immunohistochemistry showed that the malignant cells in the spleen were mostly human CD4 and CD8 double positive, whereas they were predominantly CD8⁺ cells in the kidney. In the liver, human CD3⁺ cells were detected in the hepatic sinus, with clusters of malignant cells showing strong CD4 staining. Although no tumor nodules were seen in the lung during autopsy, interstitial infiltration of tumor cells were found in tissue sections. Flow cytometric analysis of isolated cells from the spleen, liver, and kidney right after autopsy detected 40-88% of GFP⁺ cells, indicating an enrichment of the cells with JAK2 fusion in the tumor infiltrate. Furthermore, TCR clonotyping indicated a monoclonal phenotype of the tumor.

In Jurkat cells, the KHDRBS1-JAK2 fusion protein was predominantly in the cytoplasm. We are investigating the gene expression profile and the kinase substrates of KHDRBS1-JAK2 through phosphoproteomics to further understand the functional changes induced by the fusion protein. We are currently testing the penetrance of the tumors and their transplantability.

Conclusions: By introducing CDKN2A KO and JAK2 fusion, we generated a human T-cell lymphoma model, which is the first human T-cell lymphoma model generated from normal human T-cells to our knowledge. As the modified human T-cells expanded in the mouse without causing GvHD, we are not clear what factors were stimulating and sustaining the modified T-cells in vivo. The CD4/8 phenotype of the tumor cells was variable and there was no epidermotropism, thus the tumor does not entirely recapitulate the phenotype and tropism of PCAETCL, but further modifications may improve its authenticity as a PCAETCL model. The availability of this model will greatly facilitate the understanding of the pathogenesis of PCAETCL.

Reference

1. Lee K, Evans MG, Yang L, et al. Primary cytotoxic T-cell lymphomas harbor recurrent targetable alterations in the JAK-STAT pathway. Blood. 2021;138(23):2435-2440.
2. Bastidas Torres AN, Cats D, Out-Luiting JJ, et al. Deregulation of JAK2 signaling underlies primary cutaneous CD8(+) aggressive epidermotropic cytotoxic T-cell lymphoma. Haematologica. 2022;107(3):702-714.