Murine Pancreatic Adenocarcinoma Reduces Ikaros Expression and Disrupts T Cell Homeostasis

Background Maintenance of T cell immune homeostasis is critical for adequate anti-tumor immunity. The transcription factor Ikaros is essential for lymphocyte development including T cells. Alterations in Ikaros expression occur in blood malignancies in humans and mice. In this study, we investigated the role of Ikaros in regulating T cell immune balance in pancreatic cancer mouse models. Methodology and Principal Findings Using our Panc02 tumor-bearing (TB) mouse model, western blot analysis revealed a reduction in Ikaros proteins while qRT-PCR showed no differences in Ikaros mRNA levels in TB splenocytes compared to control. Treatment of naïve splenocytes with the proteasomal inhibitor, MG132, stabilized Ikaros expression and prevented Ikaros downregulation by Panc02 cells, in vitro. Western blot analyses showed a reduction in protein phosphatase 1 (PP1) and protein kinase CK2 expression in TB splenocytes while CK2 activity was increased. Immunofluorescence microscopy revealed altered punctate staining of Ikaros in TB splenocytes. Flow cytometry revealed a significant decrease in effector CD4+ and CD8+ T cell percentages but increased CD4+CD25+ regulatory T cells in TB splenocytes. Similar alterations in T cell percentages, as well as reduced Ikaros and CK2 but not PP1 expression, were observed in a transgenic, triple mutant (TrM) pancreatic cancer model. Ikaros expression was also reduced in enriched TB CD3+ T cells. MG132 treatment of naïve CD3+ T cells stabilized Ikaros expression in the presence of Panc02 cells. Western blots showed reduced PP1 and CK2 expression in TB CD3+ T cells. Conclusions/Significance The results of this study suggest that the pancreatic tumor microenvironment may cause proteasomal degradation of Ikaros, possibly via dysregulation of PP1 and CK2 expression and activity, respectively. This loss of Ikaros expression may contribute to an imbalance in T cell percentages. Ikaros may potentially be a therapeutic target to restore T cell homeostasis in pancreatic cancer hosts, which may be critical for effective anti-tumor immunity

Antilymphocytic Antibodies and Marrow Transplantation X. T Cell Depletion in Marrow Donors with C1q High and Low Affine Antibodies for Suppression of GVHD in Fully Mismatched Mice.

Rat monoclonal antibodies (mAbs) of the same specificity (anti-Thy-1) but different immunoglobulin (Ig) subclass were investigated for their effect on suppression of graft-versus-host disease (GVHD) by depleting the marrow donors of T cells in vivo. Transplantation to homozygous, fully mismatched mice of spleen and bone marrow cells from unthymectomized mice injected with the mAbs revealed that two rat anti-Thy-1 mAbs with high affinity for C1q (IgG2b) suppressed and prevented acute and chronic mortality of GVHD. In contrasts, rat mAbs with low affinity for C1q (IgM, IgG2c) barely delayed acute mortality. This correlated with findings on the degree of splenic T-cell depletion in donor mice with the IgG2b mAb, able to deplete 97%, and the IgG2c and IgM mAbs, only 83% and 75% of T cells, respectively. An effect akin to the one achieved with IgG2b was seen, however, when donor mice were thymectomized and then treated with three injections of IgG2c isotype. The rat IgM mAb was not immunosuppressive even under such conditions. Immunocytochemical and immunohistochemical examination of the donor lymph nodes after a single injection of either mAb showed that only 84% of T cells were eliminated, and in contrast to the spleen, none of the tested antibodies could deplete T cells further. The thymus did not appear depleted at all, although the cortical thymocytes were coated with either of the injected mAbs

Distribution of injected anti-thy-1 monoclonal antibodies in mouse lymphatic organs: evidence for penetration of the cortical blood-thymus barrier, and for intravascular antibody-binding onto lymphocytes.

The localization of monoclonal anti-Thy-1 binding in mouse thymus, spleen, and lymph nodes was studied at early intervals after intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) injection of a single dose of the monoclonal antibody (MAb). Five minutes after i.v. injection, anti-Thy-1 was bound to cortical thymocytes surrounding capillaries in the thymic cortex, to thymic cells beneath the thymic capsule and to medullary thymocytes around venules of the thymus medulla. When anti-Thy-1 was injected i.p. or s.c. the MAb was first deposited in capillary walls in the thymus cortex and did not appear on thymocytes outside of capillaries until 60 min after injection. These findings suggest that thymic cortical capillaries are permeable for anti-Thy-1 MAb contrary to the generally accepted principle of a blood thymus barrier to antigens in thymic cortex. Some cortex capillaries also became permeable for peroxidase when injected 15 min after anti-Thy-1 MAb. Anti-Thy-1 MAb penetration into spleen white pulp and lymph node paracortex occurs along the circulatory pathway of the vascular system in the spleen and of lymphatics in lymph nodes. But those lymphocytes with a strong anti-Thy-1 MAb loading always appeared along the pathways of lymphocyte circulation indicating that the most intense contact between anti-Thy-1 MAb and T-lymphocytes occurs not in the lymphatic organs but during the intravascular period of recirculation of lymphocytes

Simultaneous demonstration of two antigens on single T cells using antibodies with contrasting labels.

We describe a double labeling method for the discrimination of 2 antigens on single cells. It consists of a combination of 3H-immunoautoradiography and immunocytochemistry applied to cells previously fixed on poly-l-lysine (PLL)-coated multispot slides. The method has been applied to various mouse cells contemporaneously labeled with 2 different monoclonal antibodies. In order to distinguish the attached antibodies unambiguously, they were labeled with contrasting markers. One of the antibodies was marked with tritium blackening the photographic film that covers the slide. The other was detected with the peroxidase-anti-peroxidase (PAP) method forming a reddish precipitate. The contrast between the reddish reaction product of the PAP-labeled antibody and the black silver grains allows cells, specifically labeled with both antibodies, to be distinguished from cells labeled with only one or neither of the antibodies. Tritium-labeled antibodies were introduced because of their advantage over antibodies labeled with iodine in the closer localization of the silver grains to the bound antibody and their much longer halflife (60 days versus 12 years). In this study we applied a tritium-labeled anti-Thy-1.1 together with anti-Lyt-1 monoclonal antibody for studying the distribution of the corresponding antigens on lymphocytes in the mouse thymus and lymph node cells