Anti-STAT6 CTL activity in Stat6 (-/-) mice: A cautionary tale.

The generation of germline gene mutations in mice has been an invaluable tool for experimental biology. However, studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations. We observed that CD8(+) T cells from Stat6 (-/-) mice displayed "autoreactivity" to STAT6-expressing cells, associated with specific STAT6 peptides binding to MHC class I molecules. These results suggest caution in interpreting experiments where STAT6-expressing cells are transferred into Stat6 (-/-) mice, or where adoptive transfer of Stat6 (-/-) lymphocytes is performed. Our results further highlight additional considerations when studying immune responses involving cell transfer into gene-deficient mice.

The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase.

Amino acid starvation by asparaginase (ASNase) enhances phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible 2 (GCN2) kinase, leading to reduced global mRNA translation rates. This conserves energy and allows cells time to reprogram stress-related gene expression to alleviate cell injury. This study addressed the importance of GCN2 for the immune system to adapt to amino acid starvation by ASNase. GCN2(+/+) and GCN2(-/-) mice were injected once daily with ASNase or saline for up to 7 d. In both thymus and spleen, activation of amino acid stress response genes to ASNase, such as asparagine synthetase and CAAT enhancer binding protein homologous protein, required GCN2. ASNase reduced food intake and body weight in both genotypes, but spleen and thymus wet weights and total cell numbers in thymus, spleen, bone marrow, and mesenteric lymph nodes were less in GCN2(-/-) mice treated with ASNase (genotype x ASNase, P < 0.05). In the thymus, GCN2(-/-) mice treated with ASNase demonstrated enhanced apoptosis and fewer cells in all subpopulations examined (CD3+, CD4-8-, CD4+8+, CD4+8-, CD4-8+) compared with GCN2(+/+) mice treated with ASNase (genotype x ASNase, P < 0.05). In the spleen, GCN2 deletion magnified ASNase-induced reductions in CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ leukocytes (genotype x ASNase, P < 0.05). These results indicate that loss of GCN2 enhances immunosuppression by ASNase and that this eIF2 kinase is broadly required for amino acid stress management in the immune system.

Alanyl-glutamine consumption modifies the suppressive effect of L-asparaginase on lymphocyte populations in mice.

Asparaginase (Elspar) is used in the treatment of acute lymphoblastic leukemia. It depletes plasma asparagine and glutamine, killing leukemic lymphoblasts but also causing immunosuppression. The objective of this work was to assess whether supplementing the diet with glutamine modifies the effect of asparaginase on normal lymphocyte populations in the spleen, thymus, and bone marrow. Mice consuming water ad libitum with or without alanyl-glutamine dipeptide (AlaGln; 0.05 mol/L) were injected once daily with 0 or 3 international units/g body weight Escherichia coli L-asparaginase for 7 d. Tissue expression of specific immune cell surface markers was analyzed by flow cytometry. Asparaginase reduced B220+ and sIgM+ cells in the bone marrow (P < 0.05) and diminished total cell numbers in thymus (-42%) and spleen (-53%) (P < 0.05). In thymus, asparaginase depleted double positive (CD4+ CD8+) and single positive (CD4+ CD8-, CD4-CD8+) thymocytes by over 40% (P < 0.05). In spleen, asparaginase reduced CD19+ B cells to 33% of controls and substantially depleted the CD4+ and CD8+ T cell populations. CD11b-expressing leukocytes were reduced by 50% (P < 0.05). Consumption of AlaGln did not lessen the effects of asparaginase in bone marrow or thymus but mitigated cellular losses in the CD4+, CD8+, and CD11b+ populations in spleen. AlaGln also blunted the increase in eukaryotic initiation factor 2 (eIF2) phosphorylation by asparaginase in spleen, whereas eIF2 phosphorylation did not change in thymus in response to asparaginase or AlaGln. In conclusion, asparaginase reduces maturing populations of normal B and T cells in thymus, bone marrow, and spleen. Oral consumption of AlaGln mitigates metabolic stress in spleen, supporting the peripheral immune system and cell-mediated immunity during asparaginase chemotherapy.

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase.

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.

Polymorphism and conservation of the genes encoding Qa1 molecules.

To evaluate the polymorphism and conservation of the major histocompatibility complex class Ib molecule Qa1 in wild mouse populations, we determined the nucleotide sequence of exons 1-3 of Qa1 of eight mouse haplotypes derived from wild mice, including Mus musculus domesticus, M. m. castaneus, M. m. bactrianus, and M. spretus, as well as two t haplotypes. Our data identify eight new alleles of Qa1. Taken together with previously published data on Qa1 among the common laboratory inbred strains, and in agreement with cytotoxic T-lymphocyte, serological, and biochemical data, these results further confirm the existence of two families of Qa1 molecules, Qa1(a)-like and Qa1(b)-like, and illuminate the extreme conservation of the peptide-binding region of these molecules, even across species.

Differential requirement for tapasin in the presentation of leader- and insulin-derived peptide antigens to Qa-1b-restricted CTLs.

The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm.

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.