In this report, we show that Ceacam1, which is found on both donor alloreactive T cells as well as non-hematopoietic tissues such as gastrointestinal and hepatic epithelium, can regulate both donor T cell function and the sensitivity of allo-BMT recipients to radiation-containing preparative regimens. In addition, Ceacam1 on donor T cells and tumors may modulate GVT activity. Ceacam1 on both the donor allograft and recipient tissues thus appears to represent an important regulator of GVHD and GVT morbidity and mortality via both T cell dependent and independent mechanisms, suggesting that therapeutic approaches which modulate Ceacam1 may need to assess and balance GVHD vs. GVT.
Ceacam1 on T cells has previously been shown to restrain CD4 T cell polarization, cytokine secretion and cytotoxicity. In our GVHD model systems however, we found similar T cell polarization and cytokine secretion when we analyzed donor alloreactive T cells ex vivo (). We ascribe this to the strongly proinflammatory cytokine milieu found in recipients following myeloablative radiation treatment, as well as the ubiquitous presence of alloantigen, which together promote strong Th1 differentiation regardless of Ceacam1 expression. However, in our model systems Ceacam1 regulated T cell activation, and numbers of donor alloactivated T cells in both lymphoid tissues and GVHD target tissues, in patterns that generally correlated with levels target organ damage ( and ).
We also assessed the role of Ceacam1 in allo-BMT recipients. In our model systems, WT T cells in a Ceacam1-deficient environment showed a phenotype similar to that of Ceacam1−/−alloactivated T cells: both showed increased activation, selective damage to the large intestines, and preferential accumulation in the MLN and intestinal parenchyma of mice with GVHD, and correspondingly decreased infiltration of the liver and PLN, ultimately leading to exacerbation of disease, with accelerated mortality in the first two weeks post-transplant. This suggests that Ceacam1 on donor T cells interacts with recipient tissues, and that Ceacam1 “fraternal” interactions between cells of the donor graft, were not sufficient to restrain GVHD in Ceacam1−/− recipients. However, the increased early mortality of Ceacam1−/− allo-BMT recipients with GVHD also led us to ask whether Ceacam1−/− mice were sensitive to radiation injury. While Ceacam1−/− mice were not significantly defective for hematopoiesis after sublethal irradiation at 3.5 and 4.5 Gy (data not shown), they did exhibit significantly increased damage to the small intestines after lethal irradiation ().
Ceacam1 also directly regulates intestinal epithelia. Due to enhanced Wnt/β-catenin signaling, Ceacam1−/−
jejunal and ileal enterocytes exhibit higher levels of the positive cell cycle regulators c-Myc and cyclin D1 
. Dysregulated c-Myc may sensitize cells to apoptosis 
, and higher levels of these proteins may render Ceacam1−/−
enterocytes more sensitive to radiation injury. Finally, Ceacam1 also regulates cell-cell adhesion 
under normal and pathological conditions; it may therefore also be possible that loss of Ceacam1 regulates radiation-induced sloughing of intestinal epithelium.
It is difficult to directly assess the relative importance of gastrointestinal radiation sensitivity versus increased GVHD in Ceacam1−/−
allo-BMT recipients, as radiation-induced gut damage may both be directly manifested in intestinal pathology, yet transmural migration of bacterial superantigens is an important first step for the initiation of GVHD
, and increased damage to the intestines of Ceacam1−/−
mice may thus amplify the development of GVHD in these mice, and also explain in part the specifically increased large intestinal GVHD we observed.
In experiments with Ceacam1−/−
donor T cells, we also observed a trend for splenic donor CD8 alloactivated T cells to express higher levels of α4
. Although integrin α4
is important for GVHD pathogenesis, and we have previously shown that β7−/−
T cells cause a sustained decrease in acute systemic and intestinal GVHD 
, differential expression of integrin α4
T cells is almost certainly only one part of how Ceacam1 regulates target organ GVHD. Indeed, donor alloactivated CD4 T cells expressed comparable levels of integrin α4
as wildtype cells, yet were also found in increased numbers in the gut (). This suggests that other mechanisms, such as Ceacam1 regulation of donor T cell activation () may also contribute to its regulation of GVHD target organ damage.
Moreover, recipients of Ceacam1-Tg T cells also had reduced intestinal infiltrates despite similar integrin α4β7 expression, suggesting that Ceacam1 regulates the accumulation of donor T cells in target tissues via multiple mechanisms. Thus, our results on donor lymphocyte infiltrates into GVHD target tissues and secondary lymphoid tissues must be interpreted cautiously, as they must be influenced by T cell proliferation, retention and apoptosis, in addition to trafficking.
Although Ceacam1−/− and Ceacam1-Tg T cells displayed overall symmetric and opposite phenotypes, we also noted differences. Ceacam1-Tg T cells primarily showed decreased proliferation, whereas Ceacam1−/− T cells showed changes in proliferation, but also trafficking and activation. Some of these differences may be due to our models: on WT T cells, Ceacam1 is only briefly and transiently upregulated during activation. Consequently, Ceacam1−/− T cells are “missing” Ceacam1 only transiently, while Ceacam1-Tg T cells constitutively over-express Ceacam1. Furthermore, while Ceacam1−/− T cells are effectively insensitive to all Ceacam1 ligands and interactions, Ceacam1-Tg T cells which over-express the protein may have increased fraternal Ceacam1 interactions with other donor T cells, but may not necessarily experience increased Ceacam1 interactions with donor BM or host hematopoietic and non-hematopoietic components. These differences may explain why their activation and trafficking phenotypes are not directly opposed.
We were interested to note that in our GVT experiments, recipients of Ceacam1−/− T cells had significantly improved survival when challenged with A20 lymphoma but not renal cell carcinoma. Although both A20 lymphoma and renal cell carcinoma express Ceacam1, A20 cells uniformly expressed Ceacam1 at high levels, while only a subset of RENCA cells showed (somewhat lower) expression. Indeed, a number of hematologic tumors, including EL4 leukemia, P815 mastocytoma, and C1498 myeloid leukemia all express substantial levels of Ceacam1 (data not shown), whereas some solid tumors, such as mouse 4T1 breast epithelial cancer and CT51 colon tumor normally express only lower or even minimal levels of Ceacam1, similar to the lower level of expression we found with RENCA (not shown).
Therefore, one possibility is that the GVT activity of T cells can be negatively regulated by tumors expressing high levels of Ceacam1, but is less important for tumors that express low levels or only on a subset of cells in the first place. However, RENCA in our GVT model systems is found primarily in the liver, and to a lesser extent, the lungs. Since donor allografts with Ceacam1−/− T cells showed decreased numbers of donor alloreactive T cells in the liver as compared with wildype in GVHD experiments (), interpretation of GVT activity against RENCA with respect to Ceacam1 on T cells must also consider this aspect of its biology.
In conclusion, our results show that Ceacam1 on both donor T cells and allo-BMT recipients controls the proliferation, activation, and trafficking of donor alloreactive T cells, and the sensitivity of gastrointestinal tissues to irradiation. Consequently, Ceacam1 may represent a viable target for reducing radiation-associated gastrointestinal toxicity, for the control of GVHD and GVT activity after allo-BMT.