TCR-dependent transformation of mature memory phenotype T cells in mice

Abstract

A fundamental goal in cancer research is the identification of the cell types and signaling pathways capable of initiating and sustaining tumor growth, as this has the potential to reveal therapeutic targets. Stem and progenitor cells have been implicated in the genesis of select lymphoid malignancies. However, the identity of the cells in which mature lymphoid neoplasms are initiated remains unclear. Here, we investigate the origin of peripheral T cell lymphomas using mice in which Snf5, a chromatin remodelling-complex subunit with tumor suppressor activity, could be conditionally inactivated in developing T cells. In this model of mature peripheral T cell lymphomas, the cell of origin was a mature CD44hiCD122loCD8⁺ T cell that resembled a subset of memory cells that has capacity for self-renewal and robust expansion, features shared with stem cells. Further analysis showed that Snf5 loss led to activation of a Myc-driven signaling network and stem cell transcriptional program. Finally, lymphomagenesis and lymphoma proliferation depended upon TCR signaling, establishing what we believe to be a new paradigm for lymphoid malignancy growth. These findings suggest that the self-renewal and robust proliferative capacities of memory T cells are associated with vulnerability to oncogenic transformation. Our findings further suggest that agents that impinge upon TCR signaling may represent an effective therapeutic modality for this class of lethal human cancers.

Figure 1. Schematic of T cell development.

CLP cells originate in the bone marrow and migrate to the thymus. Cells at the CD4^–CD8^– DN stage are divided into 4 sequential subsets (DN1–DN4) according to expression of CD44 and CD25. Expression of the pre-TCR at DN3 leads to progression to DN4, followed by expression of CD4 and CD8 and progression to the CD4⁺CD8⁺ DP stage. Positive selection then leads to commitment toward the CD4 or the CD8 T cell lineage. Mature CD4 and CD8 single-positive cells then migrate to the periphery. The hCD2-Cre transgene is expressed at the CLP stage, leading to Snf5 deletion prior to arrival in the thymus. The Lck-Cre transgene is initially activated during early intrathymic development. The CD4-Cre transgene results in loss of Snf5 protein only in mature (CD4 and CD8) T cells in the periphery.

Figure 2. Deletion of Snf5 in T cells results in mature T cell lymphoma.

(A) Flow cytometric analysis of thymocytes from WT (CD2-Cre Snf5^+/+), Lck-Cre Snf5^fl/fl, hCD2-Cre Snf5^fl/fl, and CD4-Cre Snf5^fl/fl mice. The numbers above the plots indicate the total numbers of thymocytes averaged from 9 WT mice, 6 Lck-Cre Snf5^fl/fl mice, 9 hCD2-Cre Snf5^fl/fl mice, and 6 CD4-Cre Snf5fl/fl mice. The top row shows CD4 versus CD8 staining for all thymocytes. The bottom row is gated to show only the CD4^–CD8^– DN population, and CD25 versus CD44 staining is used to resolve the DN1, DN2, DN3, and DN4 populations, as labeled in blue. The percentage of cells within each quadrant is labeled in red. (B) CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells were sorted from the spleens of Lck-Cre Snf5^fl/fl mice (n = 8) or hCD2-Cre Snf5^fl/fl mice (n = 12), and deletion of the Snf5 allele was quantified by quantitative real-time PCR. Due to known variable penetrance of Lck-Cre expression, some T cells in Lck-Cre Snf5^fl/fl mice do not undergo deletion until later stages of development. Consequently, the Lck-Cre Snf5^fl/fl mice contain a population of mature peripheral T cells that are Snf5 deficient, a key distinction from the hCD2-Cre Snf5^fl/fl mice in which the deleted allele was undetectable (U/D) in peripheral T cells. (C) Tumor-free survival curve of hCD2-Cre Snf5^fl/fl mice (n = 24), Lck-Cre Snf5^fl/fl mice (n = 11), and CD4-Cre Snf5^fl/fl (n = 16) mice.

Figure 3. CD44^hiCD122^lo cells are the tumor-propagating cells.

(A) CD3⁺CD8⁺ splenocytes from 4-week-old Lck-Cre GFP Snf5 WT (black lines) or Lck-Cre GFP Snf5^fl/fl mice (red lines) were isolated, stained with antibodies for CD122 and CD44, and analyzed by FACS. Representative plots are shown. (B) Immunoblot of Snf5 expression in WT CD8 T cells and a lymphoma cell line. (C) Cells from the Snf5-deficient lymphoma cell line uniformly expresses CD3 and CD8, lack CD122, but consist of 2 populations with respect to CD44, CD44^hi and CD44^lo. The percentage of cells within each gate is indicated. (D) Snf5-deficient lymphoma cells were double sorted into CD44^hi and CD44^lo populations and then maintained in culture. CD44 and CD122 staining is shown from cells 5, 15, and 40 days after sorting. Only the CD44^hi cells were capable of recapitulating the parent cell line phenotype by giving rise to both the CD44^hi and the CD44^lo populations. The percentage of cells within each quadrant is indicated. (E) Survival curve of mice injected with specified number of CD44^hi or CD44^lo cells derived from Snf5-deficient lymphomas. Each group contains 4 mice. (F) Flow cytometry analysis of tumors arising from recipient mice injected with 100 CD44^hi cells. The percentage of cells within each gate is indicated.

Figure 4. The CD44^hi subset of Snf5-deficient lymphoma cells has memory-like features.

(A) Evaluation of global gene expression using KNN analysis. The signatures of CD44^hi and CD44^lo subsets from 4 independent Snf5-deficient lymphomas were compared with previously published gene signatures for naive and memory cell populations. Enrichment for the memory signature is indicated by an upward positive deflection of the bars, while enrichment for the naive signature is indicated by a downward negative deflection. (B) GSEA of a set of genes highly expressed in memory cells. Upward deflection of the green line indicates enrichment of the memory signature within the CD44^hi population (P < 0.0001). FDR, false discovery rate; NES, normalized enrichment score. (C) Relative expression of RANTES (top) and Actb (bottom) by sorted CD44^hi and CD44^lo Snf5^–/– lymphoma subpopulations determined by array-based gene expression analysis. Horizontal bars indicate the mean.

Figure 5. Snf5 loss leads to activation of Myc module.

(A) Average gene expression values (log₂) of core stem cell modules and Myc regulatory network are tested in WT CD8⁺ T cells and Snf5-deficient CD8⁺ lymphoma cells. (B) Myc protein levels are elevated in Snf5-deficient lymphomas. Immunoblot analysis of Myc in WT CD8⁺ T cells and Snf5-deficient CD8⁺ lymphoma cells. (C–G) GSEA of defined (C, E, and G) Myc module or (D and F) Myc target genes in expression data from (C and D) purified Snf5-deficient CD8⁺ lymphoma cells compared with WT CD8⁺ T cells, (E and F) Snf5-deficient MEFs compared with WT MEFs, and (G) human SNF5-deficient malignant rhabdoid tumor (MRT) samples compared with normal cerebellum.

Figure 6. Oncogenesis caused by Snf5 loss is TCR signaling dependent.

(A) CD3⁺CD8⁺ T cells were sorted from the spleens of hCD2-Cre Snf5^fl/fl mice (n = 6) or hCD2-Cre OT-1 Snf5^fl/fl mice (n = 4), and deletion of the Snf5 allele was quantified by quantitative real-time PCR. The deleted allele was undetectable in CD3⁺CD8⁺ T cells from the hCD2-Cre Snf5^fl/fl mice. (B) Tumor-free survival curve of hCD2-Cre Snf5^fl/fl mice (n = 24) and hCD2-Cre OT-1⁺ Snf5^fl/fl (n = 8) mice. (C) Tumor-free survival curve of Lck-Cre Snf5^fl/fl mice (n = 11) and Lck-Cre Snf5^fl/fl Rag2^–/– (n = 5) mice.

Figure 7. Blockade of TCR signaling leads to growth arrest of Snf5-deficient lymphoma cells.

(A) CD8-enriched cells from Lck-Cre Snf5^+/+ mice (indicated as Snf5 WT CD8 T) or tumor-bearing Lck-Cre Snf5^fl/fl mice (indicated as Snf5^–/– lymphoma) were stimulated for 60 hours in vitro in increasing numbers in the presence of syngeneic APCs. Proliferation was measured by 3H-Thymidine incorporation. (B) The Snf5-deficient lymphoma cell line was cultured for 2 days or 5 days in vitro in the presence of 1 μg/ml anti-CD8α or isotype control. Proliferation was measured by 3H-Thymine incorporation. (C) The Snf5-deficient lymphoma cell line or a control cell line was cultured for 5 days in vitro in the presence or absence of 5 μg/ml CsA. Proliferation was measured by 3H-Thymine incorporation. (D) The Snf5^–/– lymphoma cell line was cultured for 5 days in vitro in the presence of 1 μg/ml anti-CD8α and 5 μg/ml CsA. Proliferation was measured by 3H-Thymine incorporation. (E) Tumor-free survival of sublethally irradiated B6 or MHC class I–deficient K^b–/–D^b–/– mice after intravenous transfer of 106 CD8-enriched cells from tumor-bearing Lck-Cre Snf5^fl/fl mice.

Figure 8. SNF5 is expressed at low levels in human PTCLs.

Heat map analysis of SNF5 in 33 PTCLs, 8 immature lymphoblastic T cell leukemia/lymphomas, and normal T cells isolated from either peripheral blood (C1 and C2) or reactive lymph nodes. The average expression of SNF5 across all the PTCLs is significantly lower than that in either normal cells (P < 0.003) or immature lymphoblastic T cell leukemia/lymphomas (P < 0.007). Horizontal bars indicate the mean.