IMMUNOSELECTION BY T LYMPHOCYTES GENERATES REPEATED MHC CLASS I-DEFICIENT METASTATIC TUMOR VARIANTS Angel GARCIA-LORA 1 , Ignacio ALGARRA 2 , Jose J. GAFORIO 2 , Francisco RUIZ-CABELLO 1 and Federico GARRIDO 1 * 1 Servicio de Ana ´lisis Clı ´nicos, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Granada, Spain 2 Departamento de Ciencias de La Salud, Universidad de Jae ´n. Jae ´n, Spain Alteration of MHC class I molecule expression is a wide- spread mechanism used by tumor cells to evade T cell re- sponses. It has long been proposed that the origin of these MHC class I-negative or -deficient tumor variants is T cell immune selection. However, there are no experimental or clinical data to substantiate this hypothesis, and this issue is currently the subject of debate. Here we report that an H-2 class I-negative fibrosarcoma tumor clone generated MHC class I-negative spontaneous lung metastases in immuno- competent syngeneic BALB/c mice. Interestingly, the same B9 clone generated MHC class I-positive metastatic nodes, under basal conditions, in athymic nu/nu BALB/c mice. This phenomenon was observed in the metastatic nodules gener- ated after a period of in vivo growth but not in the primary tumors growing locally in the footpad. These findings support the hypothesis that the H-2 phenotype of metastatic nodes is influenced by the T cell repertoire of the host, since in the absence of this T cell pressure (i.e., in nude mice) the meta- static nodes ‘recovered’ H-2 class I expression. In addition, 2 different phenotypes were found when the metastatic nod- ules obtained from immunocompetent mice were treated with IFN-. One phenotype, present in 83% of the colonies, was characterized by resistance of the L d molecule to IFN- induction, due to a deletion involving the L d gene. The second phenotype (17% of the colonies) was similar to the original B9 clone and was characterized by the response of K, D and L class I genes to IFN-. These data provide evidence that the changes in MHC class I expression during tumor develop- ment might not be random but could be predictable. © 2001 Wiley-Liss, Inc. Key words: T lymphocytes, tumor variants MHC class I downregulation is a important escape mechanism that occurs in many mouse and human tumors derived from MHC class I-positive epithelia. 1–4 These variations (absence, selective losses, absence of induction and qualitative changes) can affect tumor aggressiveness and can modify antigen presentation and sensitivity to cytotoxic immune effectors. 5,6 Multiple mechanisms are responsible for the altered MHC class I phenotypes found in tumors. These mechanisms include mutations of class I 7 or 2 microglobulin genes, 8 MHC haplotype loss associated with loss of heterozygosity (LOH), 9,10 alterations in the peptide processing or transport machinery 2 and alterations in the regulation of MHC class I gene transcription. 11 In principle, any step required for the synthesis of a functional class I molecule can be the target for a particular mechanism able to produce an altered MHC class I tumor phenotype. It has been proposed that the major factor contributing to the appearance of MHC class I negative tumor clones is T cell immune selection; 12–14 nevertheless, the origin of these MHC-negative tumor variants remains unknown. 15,16 In the experiments reported here, we used a well-characterized mouse tumor model system generated in our laboratory: the methylcholantrene (MCA)induced fibrosarcoma GR9. This tumor is composed of different clones with different MHC class I expression, ranging from H-2-negative to strongly H-2-positive. 17–20 This tumor system was adapted to tissue culture directly from the original tumor mass with no in vivo step, in order to avoid immune selection. Clones with low or negative H-2 expression are locally highly tumorigenic but have low metastatic potential (only 1 to 4 lung colonies produced in a spontaneous metastasis assay). In contrast, the high H-2 class I expressing clones are pooly tumorigenic but have high metastatic potential (more than 90 lung colonies). In vitro NK studies exhib- ited that there is an inverse correlation between the NK sensitivity of tumor clones and H-2 class I expression. The B9 clone is one of the most thoroughly studied clones in this tumor system. This clone is H-2-negative, with no cell surface expression of K d ,D d or L d class I molecules, whereas H-2 expression of all these mole- cules recovers with IFN treatment. Compared with other tumor clones from the GR9 tumor system, the B9 clone has low meta- static potential in immunocompetent mice. The B9 tumor clone was injected in syngeneic immunocompe- tent and T cell-deficient (nu/nu) Balb/c mice, and spontaneous metastasis assays were performed. Then we asked the following questions: 1) are the metastatic MHC profiles similar in immuno- competent and athymic mice? and 2) do the metastatic colonies present the same H-2 class I phenotype as the primary tumor clone? The data obtained indicated that immunocompetent BALB/c mice generated metastatic nodes that were H-2 class I-negative under basal conditions, like the injected B9 primary clone. In contrast, in athymic nu/nu BALB/c mice, the metastatic nodes produced were H-2 class I-positive. After IFN- treatment we observed that 83% of the metastases generated in immunocom- petent mice repeatedly exhibited a phenotype different to the B9 clone, with no induction of the L d antigen. These results indicated that these metastatic nodules exhibited qualitative changes in MHC antigens compared with the B9 primary tumor clone, and that these alterations were not random but identical in different colonies obtained in different syngeneic BALB/c mice. Interest- ingly, the H-2 class I expression of the primary tumor growing locally in the footpad of immunocompetent or nude BALB/c mice was always H-2 class I-negative under basal conditions. MATERIAL AND METHODS Mice Immunocompetent 6- to 8-week-old BALB/c mice were ob- tained from the Animal Center of our institution, and athymic nu/nu BALB/c mice were purchased from IFFA-Credo (CRIFFA, Barcelona, Spain). The average weight of the mice was 20 g. Mice were housed under specific pathogen-free conditions, and all work with the animals conformed to guidelines approved by our insti- tution. B9 cell lines used for spontaneous metastasis assays For spontaneous metastasis assays we used the H-2-negative B9 clone derived from the chemically-induced fibrosarcoma GR9. 17 Grant sponsor: Fondo de Investigaciones Sanitarias (FIS); Grant spon- sor: Plan Andaluz de Investigaci´ on; Grant sponsor: Servicio Andaluz de Salud (SAS), Spain. *Correspondence to: Federico Garrido, Servicio de Ana ´lisis Clı ´nicos, Hospital Universitario Virgen de las Nieves, Av. de las Fuerzas Armadas 2, 18014 Granada, Spain. Fax: 34 958 283147. E-mail: fgarrido@hvn.sas.cica.es Received 24 February 2000; Revised 18 April, 23 June 2000; Accepted 3 August 2000 Published online 24 November 2000 Int. J. Cancer: 91, 109 –119 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer