Collateral Damage: a Mechanism for Hematopoiesis-Restricted Mhag-Specific T Cells to Play a Role in the Effector Phase of GvHD.

Abstract 3552

Poster Board III-489

Graft versus host disease (GvHD) after allogeneic stem cell transplantation (SCT) may occur when donor T cells respond to antigens specifically expressed on cells of the recipient, such as minor histocompatibility antigens (mHags). It has been hypothesized that ubiquitously expressed mHags, like HY and ADIR-1F, may be targets for both GvHD and graft versus leukemia (GvL), whereas hematopoiesis-restricted mHags, such as HA-1, may give rise to a more specific GvL effect without GvHD. However, it has been reported that mismatching for HA-1 is correlated with the development of GvHD. In addition, there is clinical evidence that the presence of a high tumor load leading to a profound GvL reaction may be associated with the induction of GvHD. To explain these observations, we analyzed whether T cells directed against hematopoiesis-restricted antigens, like HA-1, are able to contribute to destruction of the surrounding non-hematopoietic tissues in the effector phase of the immune response. First, we investigated whether HA-1 specific T cells could directly attack non-hematopoietic tissues, such as human primary skin fibroblasts from HLA-A2⁺, HA-1⁺ individuals using a ⁵¹Cr-release assay. In accordance with the absent gene expression of HA-1 in fibroblasts, no direct antigen-specific kill of the fibroblasts was seen. In contrast, exposure of ADIR-1F mHag positive fibroblasts to ADIR-specific CTLs resulted in 70% lysis after 20 hours. Even in a more inflammatory environment, induced by pretreatment with interferon gamma (IFNg), primary human fibroblasts from HLA-A2⁺, HA-1⁺ individuals were not directly killed by HA-1 specific CTLs. However, based on the clinical observation that the induction of a profound GvL effect in patients with a high tumor load correlated with the induction of GvHD, we hypothesized that these hematopoiesis-restricted T cells may have the capacity to induce collateral damage to surrounding non-hematopoietic tissues during an ongoing profound immune response against the hematopoietic target cells. To test this hypothesis, we developed an in vitro system in which monolayers of ⁵¹Cr-labeled primary human fibroblasts from HLA-A2⁺, HA-1⁺ individuals were co-cultured with HA-1-specific CTLs in the presence or absence of HA-1-expressing hematopoietic targets (EBV-LCL) for 4, 8 and 20hrs. Activation of HA-1-specific T cells by the EBV-LCL was shown to be dose dependent and was found to be optimal at stimulator/responder (S/R) ratios higher than 1/1 as measured by their specific IFNg production. Despite optimal activation of the HA-1-specific CTLs by the EBV-LCL (S/R ratio 10/1), initially no collateral damage to surrounding fibroblast was observed. However, under pro-inflammatory conditions, mimicked by the treatment of the fibroblasts with IFNg, HA-1-specific CTLs optimally activated by the HA-1⁺ EBV-LCL which were seeded between the fibroblasts (S/R ratio 10/1), exerted 40% (25-60%) bystander cytotoxicity to the surrounding fibroblasts at a 10/1 T cell/fibroblast-ratio after 20 hrs. Interestingly, there was a strict correlation between the amount of collateral damage to the surrounding fibroblasts and the strength of the T cell activation by the hematopoietic cells expressing the relevant target antigen, being optimal at S/R ratios higher than 1/1, possibly reflecting the clinical situation of a GVL response in the presence of a high tumor load. Similarly, the magnitude of the immune response, reflected by the number of T cells, was associated with the amount of collateral damage, since no collateral damage could be observed at CTL/fibroblast ratios below 3/1. In conclusion, these data indicate that a profound immune response against hematopoietic targets, mediated by T cells directed against hematopoiesis-restricted antigens, may result in the induction of GvHD via collateral damage to the surrounding non-hematopoietic tissues without the necessity for these tissues to express the specific target antigen.