1. by William J. Grossman, and Lee Ratner
Cytokine Expression and Tumorigenicity of Large Granular Lymphocytic Leukemia Cells From Mice Transgenic for the tax Gene of Human T-Cell Leukemia Virus Type I
by William J. Grossman, and Lee Ratner
Blood
Volume 90(2):
July 15, 1997
©1997 by American Society of Hematology

2. Flow cytometric analysis of cell surface markers on Tax-transgenic LGL cell lines.
Flow cytometric analysis of cell surface markers on Tax-transgenic LGL cell lines. Reactivity of antibodies against cell surface markers is indicated with solid lines. Reactivity of a control antibody, indicated by dashed lines, is depicted in all panels.
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

3. RT-PCR cytokine analysis of GzmB-Tax transgenic tumors and derived LGL cell lines.
RT-PCR cytokine analysis of GzmB-Tax transgenic tumors and derived LGL cell lines. The first and third panels show mRNA expression of IL-1α, IL-1β, IFNγ, and GM-CSF in the peripheral tumors of two different transgenic mice (m388 Tumor and f197 Tumor). The second and fourth panels show that the tumor-derived LGL cell lines express mRNAs for only IFNγ and GM-CSF (m388 LGL Cell Line and f197 LGL Cell Line). Negative and positive RT-PCR controls are shown in each panel and are labeled as HPGRT(−) and HPGRT(+), respectively. Excess primer for each RT-PCR reaction is indicated at the right side of each panel (primer).
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

4. MTT cell proliferation assay on LGL cell lines.
MTT cell proliferation assay on LGL cell lines. (A) LGL cell lines were incubated with or without IL-2 and measured for their proliferation. As shown, the F8 and SC LGL cell lines have become independent of IL-2 for proliferation, but have retained their capability to respond to IL-2 (*P < .05). In contrast, the f197 and m388 cell lines are highly dependent on exogenous IL-2 for their proliferation (**P < .01). (B) The IL-2–dependent LGL cell lines, f197 and m388, were incubated with the cytokines found to be expressed in the peripheral tumor from which they were derived (IL-1α and IL-1β, IFNγ, and GM-CSF ) and examined for their proliferative effects. Incubation with IL-2 and media alone served as positive and negative controls, respectively. As shown, none of these cytokines was able to support the proliferation of these IL-2–dependent LGL cell lines.
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

5. ELISAs for GM-CSF and IFNγ production of tumor-derived LGL cell lines.
ELISAs for GM-CSF and IFNγ production of tumor-derived LGL cell lines. (A) GM-CSF ELISA showing production of soluble GM-CSF by all four cell lines (f197, 867± 75 pg/mL; m388, 620± 56 pg/mL; F8, 127 ± 9 pg/mL; and SC, 141 ± 9 pg/mL). The production of GM-CSF between IL-2–dependent cell lines (f197 and m388) is significantly different from that of the IL-2–independent cell lines (F8 and SC; **P < .01). (B) IFNγ ELISA showing production of soluble IFNγ by all four cell lines (f197, 288 ± 18 U/mL; m388, 212 ± 15 U/mL; F8, 119 ± 12 U/mL; and SC, 108 ± 11 U/mL). Similarly, the production of IFNγ between IL-2–dependent cell lines and IL-2–independent cell lines is significantly different (**P < .01).
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

6. Immunoblot analysis of Tax expression in LGL cell lines.
Immunoblot analysis of Tax expression in LGL cell lines. Tax expression is indicated by the arrow on the left, with molecular mass markers shown on the right. LGL cell line dependency for IL-2 is indicated below each sample lane. Jurkat and MT2 cells were used for negative and positive controls of Tax expression, respectively.
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

7. Pathohistological features of the tissues of SCID mice engrafted with the transformed Tax-transgenic F8 LGL cell line.
Pathohistological features of the tissues of SCID mice engrafted with the transformed Tax-transgenic F8 LGL cell line. (A) Section of a peritoneal tumor located at the site of injection, showing that it is comprised primarily of LGLs (original magnification × 720). (B) Section of a mesenteric lymph node showing massive LGL infiltration, characteristic of diffuse large-cell type lymphoma (original magnification × 1,080). (C) Section of a spleen showing infiltration of LGLs in the left portion with generalized disruption of both the white and red pulp (original magnification × 720). (D) Section of a liver showing infiltration of LGLs into the sinusoid and portal region (open arrows; original magnification × 360). (E) Section of a mock-injected SCID lung showing an intrapulmonary bronchus with associated epithelium and normal alveolar ducts and alveoli (original magnification × 360). (F ) Lung section from an LGL-engrafted SCID mouse showing massive infiltration of LGLs into the bronchus epithelium and alveoli septa (original magnification × 360). (G) Peripheral blood smear from a mock-injected SCID mouse showing a paucity of circulating lymphocytes and neutrophils (original magnification × 360). (H) Peripheral blood smear from an LGL-engrafted SCID mouse demonstrating LGL lymphocytosis and neutrophilia (original magnification × 720). (I) Higher magnification of (H) showing morphologic characteristics of the LGLs (original magnification × 2,100).
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

8. Flow cytometric analysis of adhesion molecules on LGL cell lines.
Flow cytometric analysis of adhesion molecules on LGL cell lines. Reactivity of antibodies against adhesion molecules is indicated with solid lines. Reactivity of a control antibody, indicated by dashed lines, is depicted in all panels.
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology

9. Hypothetical model for the role of cytokines in the process of peripheral tumor development in GzmB-Tax transgenic mice.
Hypothetical model for the role of cytokines in the process of peripheral tumor development in GzmB-Tax transgenic mice. This model would suggest that there is a need for an initial injury to peripheral skin tissue (eg, ear puncture, tail clipping, and fighting). This injury would subsequently start an inflammatory response by skin epithelial cells (eg, keratinocytes [KC]) that release inflammatory cytokines such as IL-1α and IL-1β. These inflammatory cytokines could then act on ECs of nearby vessels to cause the extravasation of circulating LGLs and neutrophils (PMN) by upregulating adhesion molecules such as ICAM-1 and VCAM-1 on ECs. Circulating LGLs also constitutively express adhesion molecules on their surface (LFA-1 and VLA-4), which promote their binding to activated ECs and extravasation. Once at the site of inflammation, these LGLs would become activated and release cytokines such as GM-CSF, which could then cause a systemic increase in PMN production and PMN extravasation into the tumors. In addition, these LGLs produce large quantities of IFNγ that could potentially initiate/exacerbate this inflammatory process by upregulating the expression of adhesion molecules such as ICAM-1 and VCAM-1 at this peripheral site.
William J. Grossman, and Lee Ratner Blood 1997;90:
©1997 by American Society of Hematology