Adult T-cell leukemia/lymphoma development in HTLV-1-infected humanized SCID mice

Abstract

The molecular and genetic factors induced by human T-lymphotropic virus type-1 (HTLV-1) that initiate adult T-cell leukemia/lymphoma (ATLL) remain unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. HTLV-1-infected humanized nonobese diabetic severe combined immunodeficiency (HU-NOD/SCID) mice were generated by inoculation of NOD/SCID mice with CD34(+) hematopoietic progenitor and stem cells (CD34(+) HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCID mice exclusively developed CD4(+) T-cell lymphomas with characteristics similar to ATLL and elevated proliferation of infected human stem cells (CD34(+)CD38(-)) in the bone marrow were observed in mice developing malignancies. Purified CD34(+) HP/HSCs from HTLV-1-infected patient peripheral blood mononuclear cells revealed proviral integrations suggesting viral infection of human bone marrow-derived stem cells. NOD/SCID mice reconstituted with CD34(+) HP/HSCs transduced with a lentivirus vector expressing the HTLV-1 oncoprotein (Tax1) also developed CD4(+) lymphomas. The recapitulation of a CD4(+) T-cell lymphoma in HU-NOD/SCID mice suggests that HSCs provide a viral reservoir in vivo and act as cellular targets for cell transformation in humans. This animal model of ATLL will provide an important tool for the identification of molecular and cellular events that control the initiation and progression of the lymphoma and potential therapeutic targets to block tumor development.

Figure 1

Lymphomagenesis in HTLV-1 HU-NOD/SCID mice. Representative histologic analysis of HTLV-1-HU-NOD/SCID mice and mock mice killed 14 to 16 weeks after reconstitution. Development of lymphomas localized to the mesenteric (MLN) and lymph nodes surrounding the pancreas (PLN; red arrows) and spleen (A-B,D) in HTLV-1-HU-NOD/SCID mice in comparison with mock-infected HU-NOD/SCID mice (C,E). H&E staining of MLN (F), pancreatic lymph nodes (G), and infiltrating lymphocytes in spleen (I) of HTLV-1-HU-NOD/SCID mice in contrast to lymph nodes (H) and spleen (J) of mock-infected HU-NOD/SCID mice. H&E staining shows diffuse large scale lymphomas in the kidney, pancreas, liver, and lungs (K,M,O,Q) of HTLV-1-HU-NOD/SCID mice in comparison with kidney, pancreas, liver, and lungs (L,N,P,R) of mock-infected HU-NOD/SCID mice. Insets (K,M,O,Q) show immunohistochemical analysis for human CD45.

Figure 2

Phenotype analysis of lymphomas from HTLV-1-HU-NOD/SCID and Tax1-HU-NOD/SCID mice. Phenotype analysis of lymphocytes from the MLN (A,E,I,L) and the spleen (B,F,J,M) of HTLV-1-HU-NOD/SCID mouse (#7762, 16 weeks after injection) and MLN of Tax1-HU-NOD/SCID (#7749, 14 weeks after injection) mouse (C,G,K,N). Human CD3⁺ cells are gated and analyzed in panels I, J, and K. CD4⁺/CD8⁻ are the predominant populations in HTLV-1-HU-NOD/SCID mice MLN (I) and spleen (J) and Tax1-HU-NOD/SCID mice MLN (K). Human lymphocytes in the mesenteric lymph nodes (D) of mock-infected donor-tissue matched HU-NOD/SCID (#7762, 16 weeks after injection) mice are predominantly of CD19⁺ (B-cell) lineage (H). HTLV-1 p19^gag expression in CD4⁺ T cells from the MLN (L) and spleen (M) of the HTLV-1-HU-NOD/SCID mouse. Expression of GFP in the CD4⁺ T cells from (N) the MLN of the Tax1-HU-NOD/SCID mouse and (O) in cells from the MLN of mock-infected mouse. (P) Proliferation of CD4⁺/CD8⁻ subpopulation among gated human lymphocytes in the thymus of the HTLV-1-HU-NOD/SCID mouse (#7241, 9 weeks after injection; iii) in comparison with mock HU-NOD/SCID mouse (#7241, 8 weeks after injection; ii). Comparative Giemsa staining of lymphoma cells derived from HTLV-1-HU-NOD/SCID mouse (Q) and RV-ATL tumor cell line (R).

Figure 3

Hyperproliferation of infected HSCs in the BM of HTLV-1-HU-NOD/SCID mice. (A) Predominance of CD34⁺/CD38⁻ human stem cell subpopulation among human lymphocytes in the BM of the HTLV-1-HU-NOD/SCID mouse (iii) in comparison with mock-infected HU-NOD/SCID mouse (ii) when gated on human CD45 subpopulation (i). (B) Cumulative quantization of CD34⁺/CD38⁻ and CD34⁺/CD38⁺ subpopulations in BM of HTLV-1 infected (n = 7) and mock-infected HU-NOD/SCID mice (n = 8).

Figure 4

HTLV-1 infection skews hematopoiesis to the T-cell lineage in HU-NSG mice. Expression of CD19 and CD3 on lymphocytes (gated on human CD45) from the BM (A-B), thymus (C-D), spleen (E-F), and MLN (G-H) of HTLV-1-HU-NSG mouse (14 weeks after injection) in comparison with mock-infected HU-NSG mouse reconstituted with tissue-matched CD34⁺ donor cells. Human CD3⁺ cells were then gated and subsequently analyzed for CD4 and CD8 expression (i-viii). CD4⁺/CD8⁻ single-positive T cells are the predominant population in HTLV-1-HU-NSG mice BM (ii), thymus (iv), spleen (vi), and MLN (viii) in comparison with mock-infected HU-NSG mice. Human lymphocytes in mock-infected donor tissue–matched HU-NSG mice (14 weeks after injection) mice show broader and more diverse hematopoietic lineage development, including the presence of B cells (CD19⁺), mature single CD8⁺ T cells, and immature CD4⁺/CD8⁺ T cells (i,iii,v,vii).

Figure 5

Hyperproliferation of CD3⁺ T cells in HTLV-1/HU-NSG mice. Representative histologic analysis of the spleen of HTLV-1/HU-NSG mice and mock HU-NSG mice killed 14 weeks after reconstitution. (A-B) Immunohistochemical analysis of human CD3 expression in the spleen of HTLV-1-HU-NSG mice in comparison with mock-infected HU-NSG mice (×40 magnification). (C) Quantification of hyperproliferation of CD3⁺ T cells in the BM, thymus, spleen, and MLN of HTLV-1-HU-NSG mice in comparison with mock mice reconstituted with the same donor CD34⁺ cell preparation (HTLV-1, n = 6; mock, n = 4).

Figure 6

Serial transplantation and engraftment of lymphomas cells from HTLV-1-HU-NOD/SCID mice. (A) Pooled cells from the spleen and MLNs of HTLV-1-HU-NOD/SCID mouse (#7749) were serially transplanted intraperitoneally into naive NOD/SCID mice. Cells from lymphomas in the mesenteric lymph nodes (B), in the secondary recipient mice (#7749-2) as visualized by H&E (C), and immunohistochemistry staining for human CD45 (Hu-CD45; D). Phenotypic analysis of lymphoma cells in the MLN of secondary recipients demonstrate CD4⁺ staining (E), coexpression of HTLV-1 gag antigen p19^gag (F), and expression of CD25 (G). H&E staining demonstrating diffuse large-scale lymphomas in the spleen (H), kidney (I), and small intestine (J) of secondary recipient NOD/SCID mice. (K) Immunohistochemical analysis for human CD45 in the spleen of secondary recipient mice that developed lymphoma.