Constitutive reductions in mTOR alter cell size, immune cell development, and antibody production

Abstract

Mammalian TOR (mTOR) regulates cell growth, proliferation, and migration. Because mTOR knock-outs are embryonic lethal, we generated a viable hypomorphic mouse by neo-insertion that partially disrupts mTOR transcription and creates a potential physiologic model of mTORC1/ TORC2 inhibition. Homozygous knock-in mice exhibited reductions in body, organ, and cell size. Although reductions in most organ sizes were proportional to decreased body weight, spleens were disproportionately smaller. Decreases in the total number of T cells, particularly memory cells, and reduced responses to chemokines suggested alterations in T-cell homing/homeostasis. T-cell receptor-stimulated T cells proliferated less, produced lower cytokine levels, and expressed FoxP3. Decreased neutrophil numbers were also observed in the spleen, despite normal development and migration in the bone marrow. However, B-cell effects were most pronounced, with a partial block in B-cell development in the bone marrow, altered splenic populations, and decreases in proliferation, antibody production, and migration to chemokines. Moreover, increased AKT Ser473 phosphorylation was observed in activated B cells, reminiscent of cancers treated with rapamycin, and was reduced by a DNA-pk inhibitor. Thus, mTOR is required for the maturation and differentiation of multiple immune cell lineages. These mice provide a novel platform for studying the consequences of constitutively reduced mTORC1/TORC2 activity. IntroductionThe mammalian target of rapamycin (mTOR) is part of a conserved pathway regulating fundamental physiologic functions, including nutrient sensing and metabolism, and cell growth, proliferation, and migration. mTOR forms 2 protein complexes: one with RAPTOR, mLST8(G␤L), and PRAS40 to form TOR complex 1 (mTORC1) involved in phosphorylating S6K and 4EBP1, 1,2 and a second with RICTOR, mLST8(G␤L), SIN1, and PROTOR to form TOR complex 2 (mTORC2), which phosphorylates AKT on Ser473. [2][3][4] In yeast, TOR controls cell proliferation and size. 5,6 In Drosophila, inactivation of dTOR results in lethality and reduced embryo size. 7,8 Genetically targeting the kinase domain of murine mTOR for inactivation results in embryonic lethality, 9-11 although deletions in the C terminal portion yield mice that are normal and fertile. 11 ENU-mutagenesis screens uncovered an additional embryonic lethal mutation of mTOR, resulting in flat-top embryos lacking telencephalons resulting from limited neuroectodermal cell proliferation. 10,12 Knockouts of Raptor or Sin1 13 result in early embryonic lethality, whereas those of Rictor and mLST8(G␤L) lead to late embryonic lethality and defective vascular development. mTOR signaling/function has been deduced from studies with rapamycin, which associates with FKBP12, 14 and together binds mTOR to destabilize mTORC1. Although originally thought to affect only mTORC1, long-term treatment with rapamycin can also affect mTORC2. 15 Rapamycin and numerous rapalogs are potent immunosuppressants used in cancer chemotherapy and bone marro...

Constitutive Akt1 signals attenuate B‐cell receptor signaling and proliferation, but enhance B‐cell migration and effector function

“…Mice deficient for p85α or with inactive p110δ PI3K subunit show a similar phenotype to our Akt1 overexpressing mice resulting in a partial block at the pro-B-cell stage, reduced numbers of splenic B cells, and a strong reduction in peritoneal B1 cells [36,37]. A partial block in B-cell development and reduced B-cell proliferation was also found in mice with reduced mTOR levels [38]. Our results from Akt1 Tg mice seem contradictory to above findings, but the lower BCR signaling strength suggests that constitutively enhanced Akt1 signals induce a feedback that resets BCR responsiveness, "mimicking," at least in part, loss of PI3K signaling and, thereby, influencing B-cell maturation and function [13,14,39].…”

Cyclic adenosine monophosphate involvement in low-dose cyclophosphamide-reversed immune evasion in a mouse lymphoma model

“…The cAMP pathway participates in the PI3K-AKT-mTOR axis, which is used by Tregs to control FOXP3 levels. 53,54 With their potential roles in reversing tumor immune evasion and promoting apoptosis, and due to the potential clinical cooperation between the cAMP pathway and the CY-Tregs pathway, cAMP analogs/PDE4B inhibitors and lowdose CY may be good candidate agents for lymphoma therapy. …”

Effect of Immunosuppressive Drugs on Humoral Allosensitization after Kidney Transplant

“…Sirolimus and everolimus engage FK binding proteins, forming a complex that inhibits mTOR. This inhibition prevents the translation of mRNA-encoding proteins needed to enter the cell cycle 68 (thus reducing T cell proliferation) and cytokine production 40,69 (Figure 1). Interestingly, engagement of the BCR activates PI3K, which beyond activation of NF-kBdependent transcription, also initiates a distinct signaling pathway involving the Akt and mTOR serine/threonine kinases.…”

“…Homozygous knockin mice for hypomorphic mTOR showed decreased ability to produce antibodies. 69 To determine whether these effects were caused by a direct mTOR blockade in the B cells or only reflected T cell deficiency, the same group examined the humoral responses in mTOR conditional B cell knockout mice. 71 Mice with mTOR deleted in their B cell lineage produced fewer splenic germinal centers and also, exhibited a decrease in switched highaffinity antibody responses in contrast to their wild-type littermates.…”