pMHCII Complexes Research Articles

Ubiquitous MHC class II peptides shape regulatory T cell development (P1039)

Abstract T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regulatory T cells (Tregs) that mature in the medullar compartment. We have achieved Treg-dependent tolerance of fully allogeneic transplants via the transfer of donor MHC class II genes (MHCII) that induced newly made Tregs specific of donor MHCII peptides. Given that gene therapy for tolerance was effective with MHCII but not MHCI genes, the goal of the study was to identify natural MHCII peptides able to impact Treg development and function. Using experimental models that either recapitulated MHCII peptide presentation - the IEα 52-68 peptide presented on I-Ab molecules - or promoted Treg-mediated tolerance to heart grafts via MHCII gene transfer, we confirmed that high amounts of IEα peptide were presented by MHCII from most if not all I-Ab+ CD11c+ dendritic cells of the thymic medulla, suggesting its role in Treg maturation. This hypothesis was confirmed by demonstrating that the introduction of the IEα peptide through hematopoietic chimerism converted host IEα-specific CD4 T cells into CD25hiFoxp3+ suppressive Tregs. Treg suppression for tolerance to transplants required prior activation by cognate pMHCII complexes as MHCII-treated recipients rejected MHCII-deficient while accepting MHCII-sufficient grafts. These data are consistent with a prominent role of “MHCII self-presentation” in shaping thymic Treg TCR specificities and controlling Treg function in the periphery.

Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display.

Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2-MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs.

ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling

AbstractImmature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)–containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

The membrane distribution of MHC-II-peptide complexes and its role in T cell activation (78.18)

Abstract The efficiency of the interaction between MHC-II-peptide complexes (pMHC-II) and T cell receptor depends on the amount of antigen present at the surface of DCs. MHC-II molecules are constitutively present in plasma membrane lipid rafts, membrane microdomains that are important to induce T cell activation at low antigen doses. In this work we investigated if DCs can possess a specific pMHC-II distribution at the surface that can enhance T cell activation. In murine bone marrow derived mature DCs (mDCs), MHC-II molecules displayed a clustured phenotype. Using an antibody that recognizes specific IAk-HEL pMHC-II complexes, we observed that pMHC-II also showed a clustered distribution on the surface of antigen-loaded mDCs. While DCs treated with LPS for only 12 h had 50% less IAk-HEL pMHC-II complexes at the cell surface than DCs treated for 30 h, both cell types showed a clustured distribution of these complexes. Despite the fact that 12 h DCs expressed less IAk-HEL pMHC-II complexes and less CD86/CD40 molecules at the cell surface, T cell activation was similar using 12 h- or 30 h-mature DCs. We are currently investigating the relationship between pMHC-II complex number and cluster distribution over time with the antigen presenting cell function to elucidate the role of microdomain association of pMHC-II in T cell activation. This work is supported by NCI.

Major Histocompatibility Complex Class II-Peptide Complexes Internalize Using a Clathrin- and Dynamin-independent Endocytosis Pathway

Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6(+)Rab35(+)EHD1(+) tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.

T cell-induced secretion of MHC class II–peptide complexes on B cell exosomes

Antigen-specific interactions between B cells and T cells are essential for the generation of an efficient immune response. Since this requires peptide-MHC class II complexes (pMHC-II) on the B cell to interact with TCR on antigen-specific T cells, we have examined the mechanisms regulating the persistence, loss, and secretion of specific pMHC-II complexes on activated B cells. Using a mAb that recognizes specific pMHC-II, we found that activated B cells degrade approximately 50% of pMHC-II every day and release 12% of these pMHC-II from the cell on small membrane vesicles termed exosomes. These exosomes directly stimulate primed, but not naïve, CD4 T cells. Interestingly, engagement of antigen-loaded B cells with specific CD4 T cells stimulates exosome release in a manner that can be mimicked by pMHC-II crosslinking. Biochemical studies revealed that the pMHC-II released on exosomes was previously expressed on the plasma membrane of the B cells, suggesting that regulated exosome release from activated B cells is a mechanism to allow pMHC-II to escape intracellular degradation and decorate secondary lymphoid organs with membrane-associated pMHC-II complexes.

Stage-dependent reactivity of thymocytes to self-peptide–MHC complexes

In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2C(+)RAG(-/-) mice) most of the peripheral T cells are CD8(+), a few are CD4(+), and a significant fraction are CD4(-)CD8(-) [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other alphabeta TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide-MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD8(+) 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD8(+) thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD4(+)CD8(+) [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature naïve T cells in the periphery.

Structure of a covalently stabilized complex of a human alphabeta T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1.

An alphabeta T-cell receptor (alphabetaTCR)/hemagglutinin (HA) peptide/human leukocyte antigen (HLA)-DR1 complex was stabilized by flexibly linking the HA peptide with the human HA1.7 alphabetaTCR, to increase the local concentration of the interacting proteins once the peptide has been loaded onto the major histocompatibility complex (MHC) molecule. The structure of the complex, determined by X-ray crystallography, has a binding mode similar to that of the human B7 alphabetaTCR on a pMHCI molecule. Twelve of the 15 MHC residues contacted are at the same positions observed earlier in class I MHC/peptide/TCR complexes. One contact, to an MHC loop outside the peptide-binding site, is conserved and specific to pMHCII complexes. TCR gene usage in the response to HA/HLA-DR appears to conserve charged interactions between three lysines of the peptide and acidic residues on the TCR.