Highly lytic CD8+, alpha beta T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin, MHC class I-deficient mice.

Abstract

Targeted disruption of the f2-microglobulin (p2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance offunctional CD8+ cytotoxic T lymphocytes (CTLs) in 13m-deficient (132m -/-) mice. We asked whether the 32m -/-mice are able to reject Intraperitoneal (i.p.) injection of allogeneic major histocompatibility complex (MHC) class I-bearing tumor cells is followed by destruction of injected cells and survival of mice and is routinely used to obtain peritoneal exudate leukocytes (PELs) containing a,B T-cell receptor-positive (TCR+) cytotoxic T lymphocytes (CTLs) with high, MHC class I-specific lytic activity toward injected tumor cells (1-3).Cell-surface expression of MHC class I requires 12-microglobulin (32m) since no MHC class I antigens were detected on cell mutants lacking expression of 132m (4), and it is believed that 3m is needed for stabilization of peptide-H-2 complex, its correct folding, and cell membrane expression (5,6). Expression of MHC class I is considered instrumental in thymic education of MHC class I-restricted CD8+ CTLs and many studies have supported this view (7,8). It was reported that gene-targeted disruption of 2m results in the virtual disappearance of surface-expressed native MHC class I antigens and that 2m-deficient (/2m -/-) animals lack functional CD8+ CTLs (9-12). A very low percentage of or no CD8+CD4-cells were found in the thymus or periphery of (2m -/-mice with no significant effect on the development or survival of such animals, raising doubts about the functional significance of CD8+ CTLs. We confirmed in this study the original reports that naive 32m-deficient mice, in contrast to normal mice, have dramatically decreased levels of MHC class I antigen (Fig. lA a and b) and no more than 0.5% of CD8+a/3TCR+ cells were found in the thymus or peripheral organs, such as lymph nodes (Fig. lA c and d) and spleen (data not shown). We were trying to address the issue of the role of CD8+ CTLs by using an in vivo model, in which highly lytic and antigen-specific CD8+ PEL CTLs are implicated in tumor cell rejection (1-3) and could be easily recovered from peritoneal cavities of i.p. tumor-injected mice. MATERIALS AND METHODSMice. C57BL/6, 129/J (129J x C57BL/6)F1, DBA/2, and 12m -/-mice were 6-8 weeks old at the beginning of the experiments and were maintained in a pathogen-free environment. 32m -/ -mice used in the study were derived from embryonic stem cells (129/ola) in which the P2m gene was disrupted by gene targeting. Chimeras obtained from these cells were mated with C57BL/6 and offspring carrying the defective gene were crossed to obtain animals homozygous for the mutation (10).Cells. Thymocytes and lymph node cells were obtained as a single cell suspension after lymphoid organs were taken from control or immunized mice. The routine protocol was used to obtain a PEL suspension (1, 2). Briefly, (32m +/+ and/or 82m -/ -mice were i.p. injected with 6-25 x 106 P815 CTLs during immunization of P2m -/-mice) or with tumor cells gr...

αβTCR+ Cells Are a Minimal Fraction of Peripheral CD8+ Pool in MHC Class I-Deficient Mice

“…Immunization of ␤ 2 m Ϫ/Ϫ mice with ␤ 2 m ϩ/ϩ cells results in increased accumulation of TCR␤ ϩ CD8␣ ϩ cells (14,15,31) and induction of MHC class I-directed CTL activity (12, 14 -16, 31-33). In addition, CD8␣ ϩ cell lines can be generated by repeated in vitro restimulations with ␤ 2 m ϩ/ϩ tumors (15).…”

“…Despite impairment of both thymic and peripheral selection, residual CD8 ϩ T cells are present in ␤ 2 -microglobulin (␤ 2 m) 4 -deficient (␤ 2 m Ϫ/Ϫ ) mice as evidenced by rejection of ␤ 2 m ϩ/ϩ allogeneic tumors in a CD8-dependent manner (12,13) and detection by flow cytometry or by the CD8-dependent cytotoxicity to MHC class I-positive targets of expanded TCR␤ ϩ CD8␣ ϩ cells upon immunization with ␤ 2 m ϩ/ϩ tumor or spleen cells (12,14,15). CD8␣ ϩ cells are present in ␤ 2 m Ϫ/Ϫ mice even before tumor cell injection, and their frequency is about 50-fold lower in the peripheral blood (16) or 10-to 20-fold lower in the spleens (10) compared with wild-type mice.…”

“…First, alloreactive CD8 ϩ responses cannot be raised from ␤ 2 m Ϫ/Ϫ lymphoid tissues without prior in vivo immunization (12,14,15). Assuming a similar frequency of alloreactive TCRs within ␤ 2 m Ϫ/Ϫ and wild-type CD8 ϩ cells (the frequency of alloreactive cells is on average 1-10% in wild-type mice (18)), the frequency of alloreactive CD8 ϩ cells in total ␤ 2 m Ϫ/Ϫ spleen should be 5-100/10 4 cells.…”

MHC Class I Exon 4 in the Multiocellated Racerunners (Eremias multiocellata): Polymorphism, Duplication and Selection

“…T-cells (Otten et al, 1992;Apasov and Sitkovsky, 1993), we detected some functional amino acid sites according to the structure of HLA-A2 (Saper et al, 1991) and MHC class I gene of some other species (e.g., Siddle et al, 2006;Murphy et al, 2009). …”

Skin Graft Rejection Elicited by β2-Microglobulin as a Minor Transplantation Antigen Involves Multiple Effector Pathways: Role of Fas-Fas Ligand Interactions and Th2-Dependent Graft Eosinophil Infiltrates

“…As a consequence, the number of CD8 T cells is also decreased in ␤ 2 m Ϫ/Ϫ knockout mice (12). Nevertheless, the remaining CD8 T cells are able to expand after in vivo challenge, and they can develop CD8-associated cytotoxicity (13)(14)(15). CD4 T cells and B cell number and function are normal (16,17).…”