Cutting Edge: Genetic Variation Among 129 Substrains: Practical Consequences loan M. G. Sechler,' Jason C. Yip, and Amy S. Rosenberg We designed a series of experiments to define the role of IFN-y in cellular interactions mediating graft rejection by as- sessing the rejection of H-Y disparate grafts in both ligand and receptor knockout mice and their control inbred strain. In the course of these studies it became apparent that neither knockout strain is histocompatible with the putative control and that the putative control is not histocompatible with ei- therknockoutstrain. In the process of deducing why this might be so, it became apparent that the putative control is not an inbred strain of mouse. Thus, in the absence of rigor- ous genetic control, the utility of suchknockoutstrains of miceforassessingtheeffects of cytokines and receptors in transplantation and autoimmunity is limited. Thelournal of Immunology, 1997,159: 5766-5768. I FN-y is a potent modulator of immune function produced by T cells and NK cells. Although it has been demonstrated that IFN--y is required for rejection of class I1 MHC disparate grafts but not for rejection of class I MHC disparate grafts (1) or acute rejection of heart allografts (2), its role in the rejection of grafts requiring interactions between T cell subsets is not known. Because rejection of H-Y disparate grafts requires interaction be- tween CD4' Th and CD8+ TK cells (3), we studied the require- ment for IFN-y in the rejection of such grafts in 129 (H-2b)strain mice with an interruption in the gene for IFN--y ligand or with an interruption in the gene for IFN-y receptor. Materials and Methods Animals 129/SvEvTac mice were purchased as controls from Taconic Farms (Ger- mantown, NY). The progeny of 129/SvEvTac mice that had been blasto- cyst injected with AB-I embryonic stem (ES)2 cells containing an inter- rupted gene encoding IFN-y ligand (4) and were homozygous for the interrupted gene (a gift from Dr. Edouard Cantin), and homozygous IFN-y receptor knockout mice made with AB-I ES cells (5) and bred onto a 129ISvEvTac background (a gift from Dr. Edouard Cantin, who obtained Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 8, 1997. Receivedfor publication August 11, 1997. Accepted for publication October The costs of publication of this article were defrayedin part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Address correspondence and reprint requests to JoanM. C. Sechler, Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Dr., Bldg. 29N2B12, Bethesda, MD 20892. * Abbreviations used in this paper: ES, embryonic stem. Copyright 0 1997 by The American Association of Immunologists them from Dr. Michel Aguet) were bred in our specific pathogen-free facility. Skin grafting Mice were engrafted on the left flank with tail skin grafts according to an adaptation o f the method of Billingham and Medawar (6). The grafts were scored daily until rejection, defined as the loss of >SO% o f engrafted tissue or the study end point. Mice grafted a second time were grafted above the scar from the first graft. Results and Discussion Experiments in which GKO and GRKO females were engrafted with control 129/SvEvTac female skin and vice versa were star- tling, in thatbothknockout strains rejected control female skin grafts with median survival times of 22 days, and control mice rejected skin from both knockout lines with a median survival time of 20 days for GKO and 23 days for GRKO (Table I and Fig. 1). These studies demonstrate that 129/SvEvTac, the purported appro- priatecontrolanimal,is not histocompatible with either of the knockout strains. Rejection times of control male grafts by GKO and GRKO fe- males were significantly accelerated with respect to rejection time by 129 females (p < O.OOO1, by Wilcoxon analysis; Table I1 and Fig. 2) and thus are consistent with responses to multiple minor disparities, but not with those to H-Y alone. In addition, the male grafts from the 129/SvEvTac mice primed the knockout mice to rapidly reject subsequently placed female grafts (Table 111), pro- viding additional support for the presence of multiple minor Ag disparities. Although these data effectively demonstrate that nei- ther IFN-y nor the IFN-yR is required for a primed response to minor Ags, the role of IFN-y or IFN-yR in the rejection of H-Y disparate grafts cannot be addressed with these mice. Studies within each knockout line, i.e., GKO male onto GKO female or GRKO male onto GRKO female, would be difficult to Table 1. Median survival of female 129, GKO, and GRKO skin grafts on 129 female hosts and median survival of 129 female skin grafts on GKO and GRKO female hosts" Host Donor MST (female) (female) (days) 129 (5) 129 >70b 129 (5) CKO 20 129 (5) GRKO 23 CKO (5) 129 22 CRKO (5) 129 22 .' MST is median survival time for the grafts. Number of mice grafted is given age of the graft without a complete loss of the grafted tissue. in parentheses. Chronic rejection is characterized by loss of hair from and shrink- " One chronic rejection. 0022-1 767/97/$02.00