Carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM-1) is a transmembrane protein found on leukocytes, endothelium, and epithelium. Its activation can attenuate colitis in murine models. Microarray analysis revealed that CEACAM-1 is increased in the small bowel during intestinal graft-versus-host-disease (GVHD). We studied the role of CEACAM-1 in mouse models for allogeneic bone marrow transplantation. We found that CEACAM-1-/- donor T cells caused significantly more GVHD (p<0.05), while CEACAM-1-Tg donor T cells caused significantly less GVHD (p<0.01). Administration of a CEACAM-1 agonistic antibody CC1 also significantly attenuated GVHD (p<0.01) Histopathological analysis revealed significantly increased GVHD of the large bowel in recipients of CEACAM-1-/- T cells (p<0.05), while recipients of CEACAM-1-Tg T cells had decreased GVHD in all organs (p<0.01). We performed an extensive analysis and found that alloactivated CEACAM-1-/- T cells (a) have increased CD25 and decreased CD62L expression (b) have increased expression of the gut- homing integrin ?4?7 (LPAM) and (c) preferentially infiltrate the intestines, while CEACAM-1-Tg T cells (d) have decreased infiltration of all organs. Therefore the major hypothesis of this application is: CEACAM-1 is an important negative regulator of donor T cells during GVHD. We will test the following specific hypotheses: (1) CEACAM-1 regulates tumor growth and the graft-versus-tumor activity;(2) CEACAM-1 regulates alloreactive T cell trafficking and integrin ?4?7 expression;(3) CEACAM-1 regulates DC-mediated imprinting of gut-specific homing of alloreactive T cells;(4) CEACAM-1 regulates T cell polarization toward the Th1, Th2, Th17, and regulatory T cells;and (5) the administration of the CEACAM-1 agonist CC1 can ameliorate GVHD.
Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for hematological malignancies, but its efficacy is severely limited by graft-versus-host-disease (GVHD). Our preliminary studies show that Carcinoembryonic Antigen Associated Cell Adhesion Molecule 1 (CEACAM-1) could negatively regulate GVHD, which prompts us to propose studies in clinically-relevant mouse allo-BMT models to analyze how CEACAM-1 regulates GVHD. This could lead to the development of CEACAM-1 activation as a novel strategy for GVHD prevention or treatment. Supplement 1 RO1HL095075-01A2, PI Marcel R.M. van den Brink, MD PhD Introduction to the Revised Application. In accordance with criteria set out by the American Recovery and Reinvestment Act of 2009, this application has undergone major revisions, including a) prioritization of analyses to reduce the application?s scope and accomplish its major goals in a two-year funding period, including a revised timeline, b) improved focus in Specific Aims 1 and 2 by deleting low-priority Goals (detailed below), and c) where appropriate, indication of where the additional personnel to be hired for this project will contribute efforts to allow for the project?s timely completion within the funding period. We detail below our specific modifications to our two Specific Aims Aim 1. To study the effects of CEACAM-1 inhibition or over-expression on graft-versus- host-disease and graft-versus-tumor activity. In this Aim, we propose to study how modulating CEACAM-1 function by inhibiting or over- expressing the protein, or CEACAM-1 ligation can regulate graft-versus-host-disease (GVHD). This Aim was initially subdivided into four linked objectives, which were a) to assess the mechanisms and importance of CEACAM-1 in regulating CD4 vs. CD8 T cells, b) analysis of whether CEACAM-1 functions via cell autonomous versus heterologous signaling mechanisms, c) characterization of the effects and mechanisms of the CEACAM-1 agonistic antibody CC1, and d) evaluating the role of interventions which influence the biology of CEACAM-1 on the graft-versus-tumor (GVT) effect and tumor biology. Prioritization of sub-aims: Based on feedback from the reviewers? comments and the two-year funding period, we will select and prioritize our studies in Aim 1 according to the following hierarchy: 1. We will perform in-depth studies of how manipulating CEACAM-1, including CEACAM-1 activation, can influence GVT activity and tumor biology (section D.1.11). 2. We will characterize the effects and mechanisms of CEACAM-1 activation with the monoclonal antibody CC1 on attenuating GVHD (sections D.1.9-D.1.10). However, based on additional preliminary studies, we have observed relatively limited benefit to CC1 agonist administration for GVHD prophylaxis. Therefore, we will begin with limited studies to analyze the efficacy of CC1 and perform further studies only if promising preliminary data are obtained. 3. If time remains within the funding period, we will perform studies analyzing a) the differential role of CEACAM-1 in CD4 vs. CD8 T cells (section D.1.7);b) whether CEACAM-1 acts in a cell autonomous vs. heterologous fashion to regulate T cell function (section D.1.8);and c) the effect of CEACAM-1 in MHC-matched models of GVHD (section D.1.6). We continue to plan on using the techniques and reagents as described in the introduction to this Aim (sections D.1.2-D.1.5). Role of additional personnel requested: We are requesting to hire one additional laboratory technician to expedite the completion of our Goals within the two-year funding period. The sub- aims that we have selected will require in-depth analysis: because of the wide expression of CEACAM-1 across many cell types, particularly leukocytes, this will require comprehensive evaluation of GVHD pathophysiology and alloreactive T cell function as described in Table 6, and evaluation of NK cells, dendritic cells, macrophages, and monocytes as described in Table 7. In addition to assistance with animal husbandry and routine experimental analyses, the additional laboratory technician we are requesting will focus on the detailed analysis of non-T cell leukocyte function after administration of the CC1 CEACAM-1 agonist, as shown in Table 7. Similarly, we expect the mechanisms and effects of manipulating CEACAM-1 on tumor biology and GVT activity to be complex, because of complex interactions between CEACAM-1-bearing tumors and donor T cells, and the protean and unpredictable effects of direct CEACAM-1 ligation on tumor biology. In addition to assisting with the analyses of GVT experiments, the additional personnel we are requesting will be responsible for characterizing the direct effects of CC1 administration on tumor biology in T cell depleted allogeneic bone marrow transplantation experiments (described in section D.1.11). Aim 2. To study the cellular and molecular mechanisms by which CEACAM-1 regulates alloactivated T cells. In this Aim, we proposed several series of studies to elucidate the cellular and molecular mechanisms by which CEACAM-1 regulates alloactivated T cells. This Aim was initially subdivided into four linked objectives, which were a) understanding how CEACAM-1 could regulate the gut-homing integrin ?4?7, b) probing the role of CEACAM-1 in regulating the imprinting of alloreactive T cells by organ-specific dendritic cells, c) assessing how CEACAM-1 can regulate T cell polarization and activation, and d) elucidating a role for CEACAM-1 in the suppressive function of regulatory T cells. Prioritization of sub-aims: Based on feedback from the reviewers? comments, and the two-year funding period, we will select and prioritize our studies in Aim 2 according to the following hierarchy: 1. We will study the role of CEACAM-1 and its regulation of the gut-homing integrin ?4?7 (sections D.2.4-D.2.5, and D.2.7-D.2.8). 2. We will assess how CEACAM-1 may regulate T cell polarization and activation (sections D.2.9-D.2.10). 3. We will evaluate whether CEACAM-1 may regulate alloreactive T cell imprinting by organ- specific dendritic cells (section D.2.6). 4. We will test whether CEACAM-1 is important for the suppressive function of regulatory T cells (section D.2.11) We will initially focus on the role of CEACAM-1 in regulating alloreactive T cell trafficking. Apart from the preliminary data regarding the regulation of integrin ?4?7 by CEACAM-1, we have recently found that recipients of ?7-/- donor alloreactive T cells suffer diminished thymic GVHD, indicating a role for integrin ?4?7 on donor T cells in target organ damage to the thymus (not shown). As CEACAM-1-/- alloreactive T cells upregulate levels of integrin ?4?7 (Figure 7), our recent finding prompts us to analyze in-depth the observation that recipients of CEACAM-1-/- donor T cells and CEACAM-1-Tg donor T cells both suffer attenuated thymic GVHD (Figures 5 and 11). We will use techniques and reagents as described in sections D.1.2-D.1.5 and D.2.2. Role of additional personnel requested: The additional laboratory technician we are requesting will be central to ensure timely breeding and creation of B6.CEACAM-1-/-?7-/- mice for our studies of the importance of CEACAM-1 in regulating the expression of trafficking molecules versus other T cell functions (section D.2.7), as well as assistance in performing additional transplants and tissue analyses (similar to Aim 1). Revised Timeline. We plan to complete the majority of studies proposed in Aim 1 during the first year. Studies proposed in Aim 2 will begin during the first year and continue for the duration of the funding period. All animal breeding will be completed by the end of year one.
