HUMAN BONE MARROW CELLS RETROVIRALLY TRANSDUCED WITH THE ALLOGENEIC CLASS II GENE, HLA–DR3&bgr;, DOWN REGULATE ANTI–ALLOGENEIC RESPONSES OF AUTOLOGOUS LYMPHOID CELLS

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TITLE: HUMAN BONE MARROW CELLS RETROVIRALLY TRANSDUCED WITH THE ALLOGENEIC CLASS II GENE, HLA–DR3&bgr;, DOWN REGULATE ANTI–ALLOGENEIC RESPONSES OF AUTOLOGOUS LYMPHOID CELLS

Bonnie B Blomberg,^1,2 J. M. Mathew,¹ H. Fainman,¹ S. Hussini,¹ M. Carreno,¹ H. J. Hnatyszyn,² R. Garcia–Morales,¹ L. Fuller,^1,2 T. Vallone,^1,3 A. Rosen,¹ V. Esquenazi,^1,2,3 C. Ricordi,^1,2,4 A. Tzakis,¹ J. Miller,^1,2,3

¹Surgery, University of Miami School of Medicine, Miami, Florida; ²Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida; ³VA Medical Center, Miami, Florida; ⁴Diabetes Research Institute, Miami, Florida

The purpose of the current study is to generate enhanced donor–specific allo–immune inhibitory activity in recipient (autologous) human bone marrow by transducing the cells with donor HLA genes. Our laboratory has previously demonstrated (strong) allo–immune inhibitory capacity of donor bone marrow cells (DBMC) in MLR in vitro and (weaker) allo–immune inhibitory activity of autologous (recipient) bone marrow cells (RBMC). Others have shown that the transduction and expression of allo MHC class I or II genes in infused autologous bone marrow yielded improved graft acceptance in animal models (Emery et al., 1997). We chose a representative human class II DR allele, DR3&bgr;, to use for transduction experiments. PBMC were first used for transduction, with a Moloney murine leukemia retrovirus expressing the HLA DR3&bgr; allele, to determine the feasibility of this system. Autologous MLR was generated to DR3&bgr;–transduced PBMC.

Retrovirally transduced PBMC as well as RBMC showed more than 50% of class II positive cells being positive for DR3&bgr;. There was an increased inhibition of an MLR of PBMC to allogeneic PBMC expressing the endogenous HLA–DR3&bgr; allele when RBMC transduced with DR3 were added to the culture as compared to inhibition by vector control transduced or untransduced RBMC. This increased inhibition occurred in 8 out of 9 experiments. We also attempted to assay for the ability of transduced bone marrow cells to suppress the cytolytic activity (CTL assay) of PBMC towards a (autologous) transduced target. Interestingly, a cytolytic response was absent from PBMC stimulated with autologous DR3–transduced PBMCs (4/4 times), whereas a control anti–allo response to endogenous DR3 was positive. Further phenotypic and functional analyses of the DR3–transduced subsets of RBMC will enable us to determine which populations might contribute to the observed suppressive activity.

Supported by NIH R01–DK25243–20 and the Medical Research Services of Miami Veterans Affairs Medical Center