Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.NWO15.002.Metals in Catalysis, Biomimetics & Inorganic MaterialsBio-organic Synthesi

Lipid antigen-dependent and -independent recognition of CD1 by human T cells

Cluster of differentiation 1 (CD1) molecules are Major Histocompatibility Complex (MHC) class I-like proteins that present diverse lipid antigens to T cells instead of peptides. This thesis centers around the question what the biological function of the CD1 isoforms CD1b and CD1c is in T cell immunity. Part I of this thesis explores how CD1b and CD1c present lipid antigens from Mycobacterium tuberculosis to antibacterial T cells. Chapter 2 describes the total synthesis of three mycobacterial diacyltrehalose (DAT) lipids. The small differences in the chemical structure of the lipidic parts of DAT1, DAT2, and DAT3 led to drastic differences in binding by the innate immune receptor Mincle, which could only be activated by DAT3. In chapter 3 we investigated the recognition of mycobacterial DAT lipids by human T cell receptors. Whereas the Mincle receptor only recognizes DAT3, T cells follow a different pattern as observed by binding of DAT1- and DAT2- treated CD1b tetramers, but not DAT3- treated CD1b tetramers. Chapter 4 describes the immunological recognition of three different synthetic mycobacterial mannosyl phosphomycoketide (MPM) analogs designed to withstand hydrolysis to phosphomycoketide. One analog, MPM-3, was hydrolysis-resistant and recognized by human T cells that are cross-reactive to natural MPM. Part II of this thesis describes research on lipid antigen-independent recognition of CD1, also known as CD1 autoreactivity. CD1-mediated T cell autoreactivity has been shown previously, but it is unclear whether that represented a rare event revealed by the methods used in the past, or whether autoreactivity is common in the CD1 system. In chapters 5 and 6 tetramers were used to study CD1 autoreactivity patterns of T cells. Chapter 5 describes the discovery that CD1b is recognized by several human Vd1 gamma delta T cell receptors and stimulates autoreactive responses. The different gamma delta T cell receptors interacted with different surfaces of CD1b and showed different requirements for lipid antigens and co-recognition of butryophilin-like proteins. In chapter 6 we investigated the recognition of CD1c itself by human T cells. Tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self-lipids that were fully seated within the antigen binding cleft, suggesting that the small lipid size is a determinant of autoreactive T cell responses via CD1c. In conclusion, this thesis focuses on recognition of the antigen presenting molecules CD1b and CD1c by the human immune system. Two types of T cell-CD1 interaction are covered here: 1) recognition of CD1 presented mycobacterial lipids by human T cells and 2) the recognition of the CD1 itself, independent of the lipid presented

Lipid antigen-dependent and -independent recognition of CD1 by human T cells

Cluster of differentiation 1 (CD1) molecules are Major Histocompatibility Complex (MHC) class I-like proteins that present diverse lipid antigens to T cells instead of peptides. This thesis centers around the question what the biological function of the CD1 isoforms CD1b and CD1c is in T cell immunity. Part I of this thesis explores how CD1b and CD1c present lipid antigens from Mycobacterium tuberculosis to antibacterial T cells. Chapter 2 describes the total synthesis of three mycobacterial diacyltrehalose (DAT) lipids. The small differences in the chemical structure of the lipidic parts of DAT1, DAT2, and DAT3 led to drastic differences in binding by the innate immune receptor Mincle, which could only be activated by DAT3. In chapter 3 we investigated the recognition of mycobacterial DAT lipids by human T cell receptors. Whereas the Mincle receptor only recognizes DAT3, T cells follow a different pattern as observed by binding of DAT1- and DAT2- treated CD1b tetramers, but not DAT3- treated CD1b tetramers. Chapter 4 describes the immunological recognition of three different synthetic mycobacterial mannosyl phosphomycoketide (MPM) analogs designed to withstand hydrolysis to phosphomycoketide. One analog, MPM-3, was hydrolysis-resistant and recognized by human T cells that are cross-reactive to natural MPM. Part II of this thesis describes research on lipid antigen-independent recognition of CD1, also known as CD1 autoreactivity. CD1-mediated T cell autoreactivity has been shown previously, but it is unclear whether that represented a rare event revealed by the methods used in the past, or whether autoreactivity is common in the CD1 system. In chapters 5 and 6 tetramers were used to study CD1 autoreactivity patterns of T cells. Chapter 5 describes the discovery that CD1b is recognized by several human Vd1 gamma delta T cell receptors and stimulates autoreactive responses. The different gamma delta T cell receptors interacted with different surfaces of CD1b and showed different requirements for lipid antigens and co-recognition of butryophilin-like proteins. In chapter 6 we investigated the recognition of CD1c itself by human T cells. Tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self-lipids that were fully seated within the antigen binding cleft, suggesting that the small lipid size is a determinant of autoreactive T cell responses via CD1c. In conclusion, this thesis focuses on recognition of the antigen presenting molecules CD1b and CD1c by the human immune system. Two types of T cell-CD1 interaction are covered here: 1) recognition of CD1 presented mycobacterial lipids by human T cells and 2) the recognition of the CD1 itself, independent of the lipid presented

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use