Intracellular glutathione determines bortezomib cytotoxicity in multiple myeloma cells.

Multiple myeloma (myeloma in short) is an incurable cancer of antibody-producing plasma cells that comprise 13% of all hematological malignancies. The proteasome inhibitor bortezomib has improved treatment significantly, but inherent and acquired resistance to the drug remains a problem. We here show that bortezomib-induced cytotoxicity was completely dampened when cells were supplemented with cysteine or its derivative, glutathione (GSH) in ANBL-6 and INA-6 myeloma cell lines. GSH is a major component of the antioxidative defense in eukaryotic cells. Increasing intracellular GSH levels fully abolished bortezomib-induced cytotoxicity and transcriptional changes. Elevated intracellular GSH levels blocked bortezomib-induced nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2)-associated stress responses, including upregulation of the xCT subunit of the Xc- cystine-glutamate antiporter. INA-6 cells conditioned to increasing bortezomib doses displayed reduced bortezomib sensitivity and elevated xCT levels. Inhibiting Xc- activity potentiated bortezomib-induced cytotoxicity in myeloma cell lines and primary cells, and re-established sensitivity to bortezomib in bortezomib-conditioned cells. We propose that intracellular GSH level is the main determinant of bortezomib-induced cytotoxicity in a subset of myeloma cells, and that combined targeting of the proteasome and the Xc- cystine-glutamate antiporter can circumvent bortezomib resistance.

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.

gp96-peptide vaccination of mice against intracellular bacteria.

This work demonstrates that gp96 preparations isolated from cells infected with intracellular bacteria induce cytotoxic T-lymphocyte responses and confer protection. Our findings extend previous reports on the immunogenicity of gp96-associated peptides to antigens derived from intracellular bacteria. Immunization with gp96 may therefore represent a promising vaccination strategy against bacterial pathogens.

Effects of different antigenic microenvironments on the course of CD8+ T cell responses in vivo.

The influence of microenvironment on the course of CD8 + T cell responses in vivo was investigated by injecting H-2Kb-specific T cells from donor TCR transgenic (TCR-Tg) mice into H-2kb-Tg mice. H-2Kb expression in recipients was either ubiquitous (CBK mice) or restricted to myeloid and erythroid cells (K beta mice). Donor T cells proliferated as extensively and acquired similar surface phenotypes in spleen of both recipient types. Thus, neither the restricted pattern of H-2Kb expression nor the significantly reduced level of H-2Kb expression by myeloid cells in Kbeta recipients affects the ability of the splenic microenvironment to prime T cell proliferation in vivo. However, an unsustained burst of cytolytic activity was generated rapidly in spleen of CBK recipients, whereas relatively little cytolytic activity was generated in K beta spleen. This indicates that effector T cells were not generated efficiently in spleen of Kbeta recipients even though extensive T cell proliferation was taking place in this microenvironment. Furthermore, activated donor T cells dispersed rapidly throughout primary and secondary lymphoid organs of Kbeta recipients, whereas few T cells migrated from spleen in CBK recipients. Consequently, the course of CD8+ T cell responses and the anatomical distribution of activated T cells are profoundly influenced by the nature of the antigenic microenvironment encountered in vivo. We conclude that T cells rapidly proliferate and acquire new tissue-homing characteristics but do not differentiate into cytolytic effector cells at the site of priming when they encounter myeloid cells expressing low levels of antigen in vivo.

Tolerance induction by elimination of subsets of self-reactive thymocytes.

Immature thymocytes expressing TCRs which confer reactivity to self-MHC molecules are subject to efficient elimination as a result of negative selection. Previously, we have identified a lineage of H-2Kb Tg mice, CD2Kb-3, which fails to reject skin grafts from mice expressing H-2Kb even though H-2Kb-specific cytotoxic T cells can be generated in vitro. We now show that bone marrow derived cells are responsible for tolerance induction and that tolerance is acquired, at least in part, by negative selection in CD2Kb-3 mice. Thymocytes expressing two different transgenic TCR (TCR-Tg) clonotypes conferring reactivity to H-2Kb are eliminated prior to the CD8+CD4+ stage of differentiation in double Tg (CD2Kb-3 x TCR-Tg)F1 mice. As in other cases where thymocytes from TCR-Tg mice develop in the presence of deleting ligands, large numbers of TCR+ CD8-CD4- T cells accumulate in double Tg mice. However, these T cells fail to respond to H-2Kb in vitro but can be activated with immobilized anti-clonotypic antibody. Consequently, thymocytes expressing these types of TCR molecules represent a fraction of H-2Kb-reactive thymocytes which are unable to mature into T cells capable of mounting H-2Kb-specific cytotoxic responses. Presumably, precursors of H-2Kb-specific cytotoxic T cells found in the periphery of CD2Kb-3 mice express a distinct repertoire of TCR molecules conferring reactivity to H-2Kb. We consider potential explanations to account for this discrepancy and their wider implications, including the possibility that the repertoire of thymocytes able to recognize self-H-2Kb molecules in CD2Kb-3 mice is divided into distinct subsets; those which are, and those which are not, subject to negative selection.

Induction of tolerance to self MHC class I molecules expressed under the control of milk protein or beta-globin gene promoters.

We have studied tolerance induction in transgenic CBA mice expressing H-2Kb genes under the influence of guinea-pig alpha-lactalbumin (KAL) or human beta-globin gene promoter (K beta). KAL radio-resistant cells, but not bone marrow derived cells, induce tolerance to H-2Kb in chimeric mice. In contrast, bone marrow derived and radio-resistant cells of K beta mice induce tolerance. Although appropriate, tissue-specific, expression of H-2Kb molecules occurs in KAL and K beta mice, H-2Kb is expressed at low levels in thymus of transgenic mice. In addition, dendritic cells and macrophages express H-2Kb molecules when K beta, but not when KAL bone marrow is cultured in vitro. The mode of tolerance induction was examined in double transgenic mice by mating KAL or K beta mice to mice expressing TCR transgenes (Tg-TCR) derived from a H-2Kb specific, CD8-independent cytotoxic T cell clone. In both cases, a large number of Tg-TCR+ CD8+CD4+ thymocytes develop but mature CD8+CD4- thymocytes fail to appear suggesting that thymocytes are eliminated late in development. Some CD8-CD4- and CD8-CD4+ Tg-TCR+ T cells develop in double transgenic mice and respond to activation through their TCR-CD3 complex in vitro, although no responses to stimulation with H-2Kb expressing cells were detected. Thus, tolerance induction in KAL and K beta mice proceeds via a deletional mechanism that is inefficient due either to low numbers of H-2Kb expressing thymic cells or to the low levels of H-2Kb expressed by thymic cells, or to a combination of these factors.

MHC-unrestricted T-cell cytotoxicity against tumour cells.

F9 embryonal carcinoma cells (EC) grow as tumours in their strain of origin, 129/Sv, but can be rejected by mouse strains differing at the H-2 and/or non-H-2 loci. The presence of H-2 class I and/or minor H antigens on F9 and other EC cells is implied by (i) the rejection of EC cells by mice immunized with appropriate H-2 class I transfectants, and (ii) the ability of appropriate EC cells to prime mice for second-set in vivo skin-graft rejection responses to H-Y, and secondary MLC responses to multiple minor H antigens. However, EC cells express no H-2 class I antigens in vitro, and for in vivo rejection by T-cell responses directed either at allogeneic class I molecules or at minor H antigens restricted by self class I molecules, one would need to postulate that EC cells growing in vivo could express sufficient class I antigens for recognition by T cells. In the course of investigating this question, we found evidence for class I expression but also evidence for an additional antigen(s), shared by EC and tumour cells and recognized in a non-MHC-restricted manner.