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The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood, and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic regeneration, centered on detecting the loss of dying thymocytes that are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 via TAM receptor signaling and activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. However, after damage, when profound thymocyte depletion occurs, this TAM-Rac1-NOD2-miR29c pathway is attenuated, increasing production of IL-23 and BMP4. Notably, pharmacological inhibition of Rac1-GTPase enhanced thymic function after acute damage. These findings identify a complex trigger of tissue regeneration and offer a regenerative strategy for restoring immune competence in patients whose thymic function has been compromised., Competing Interests: Declaration of interests S.K. and J.A.D. have filed a patent application on this work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide: MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of E alpha52-68: I-A(b) and class II-associated invariant chain peptide (CLIP): I-A(b) complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of E alpha52-68: I-A(b) complexes in thymic epithelial cells is approximately 5-10% of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A(b) complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP:MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP:MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Li peptide bound to the peptide-binding groove.

To define the intracellular site of assembly of endogenous peptide-MHC class II complexes, an immunochemical approach was undertaken employing a monoclonal antibody specific for an endogenous peptide-class II complex in combination with subcellular fractionation. Here, we show that newly synthesized MHC class II molecules, upon exit from the Golgi, are delivered into a dense endocytic compartment (MIIC) distinct from late endosomes and lysosomes. Endogenous peptide-class II complexes are initially formed in this compartment and subsequently traffic through late endosomal vesicles prior to cell surface expression. Exogenous antigen delivered via immunoglobulin receptors is targeted to MIIC en route to lysosomes after passing through early and late endosomes. Processing of an endocytosed antigen was observed in this compartment. Our results suggest a specific role for MIIC in the processing of endogenous and exogenous proteins as well as the assembly of peptide-MHC class II complexes.

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.