Doctor of Philosophy, Massachusetts Institute of Technology, Microbiology (2020)
Master of Science, Swiss Federal Institute of Technology (EPFL), Life Sciences and technologies (2012)
Bachelor of Science, Swiss Federal Institute of Technology (EPFL), Life Sciences and technologies (2010)
Michael Fischbach, Postdoctoral Faculty Sponsor
Antigen discovery tools for adaptive immune receptor repertoire research
Current Opinion in Systems Biology
View details for DOI 10.1016/j.coisb.2020.10.002
Antigen-specific induction of CD4 CD8αα intraepithelial T lymphocytes by Bacteroidetes species
View details for DOI 10.1101/2020.08.05.236513
Recognition of Class II MHC Peptide Ligands That Contain beta-Amino Acids
JOURNAL OF IMMUNOLOGY
2019; 203 (6): 1619–28
Proteins are composed of α-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from β-amino acids) have biological properties that differ starkly from those of their conventional counterparts. How changes in the structure of the peptide backbone affect the loading of peptides onto MHC-II or recognition of the resulting complexes by TCRs has not been widely explored. We prepared a library of analogues of MHC-II-binding peptides derived from OVA, in which at least one α-amino acid residue was replaced with a homologous β-amino acid residue. The latter contain an extra methylene unit in the peptide backbone but retain the original side chain. We show that several of these α/β-peptides retain the ability to bind tightly to MHC-II, activate TCR signaling, and induce responses from T cells in mice. One α/β-peptide exhibited enhanced stability in the presence of an endosomal protease relative to the index peptide. Conjugation of this backbone-modified peptide to a camelid single-domain Ab fragment specific for MHC-II enhanced its biological activity. Our results suggest that backbone modification offers a method to modulate MHC binding and selectivity, T cell stimulatory capacity, and susceptibility to processing by proteases such as those found within endosomes where Ag processing occurs.
View details for DOI 10.4049/jimmunol.1900536
View details for Web of Science ID 000484842100021
View details for PubMedID 31391235
View details for PubMedCentralID PMC6736755
Internalization of Influenza Virus and Cell Surface Proteins Monitored by Site-Specific Conjugation of Protease-Sensitive Probes
ACS CHEMICAL BIOLOGY
2019; 14 (8): 1836–44
Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.
View details for DOI 10.1021/acschembio.9b00493
View details for Web of Science ID 000481979400018
View details for PubMedID 31348637
One-step generation of monoclonal B cell receptor mice capable of isotype switching and somatic hypermutation
JOURNAL OF EXPERIMENTAL MEDICINE
2018; 215 (10): 2686–95
We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation.
View details for DOI 10.1084/jem.20172064
View details for Web of Science ID 000447673200017
View details for PubMedID 30181412
View details for PubMedCentralID PMC6170169
One-Pot Dual Labeling of IgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination of Sortase A and Butelase 1
2018; 29 (10): 3245–49
Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule.
View details for DOI 10.1021/acs.bioconjchem.8b00563
View details for Web of Science ID 000447953500003
View details for PubMedID 30231608
View details for PubMedCentralID PMC6429940
The studies of ParA and ParB dynamics reveal asymmetry of chromosome segregation in mycobacteria
2017; 105 (3): 453–68
Active segregation of bacterial chromosomes usually involves the action of ParB proteins, which bind in proximity of chromosomal origin (oriC) regions forming nucleoprotein complexes - segrosomes. Newly duplicated segrosomes are moved either uni- or bidirectionally by the action of ATPases - ParA proteins. In Mycobacterium smegmatis the oriC region is located in an off-centred position and newly replicated segrosomes are segregated towards cell poles. The elimination of M. smegmatis ParA and/or ParB leads to chromosome segregation defects. Here, we took advantage of microfluidic time-lapse fluorescent microscopy to address the question of ParA and ParB dynamics in M. smegmatis and M. tuberculosis cells. Our results reveal that ParB complexes are segregated in an asymmetrical manner. The rapid movement of segrosomes is dependent on ParA that is transiently associated with the new pole. Remarkably in M. tuberculosis, the movement of the ParB complex is much slower than in M. smegmatis, but segregation as in M. smegmatis lasts approximately 10% of the cell cycle, which suggests a correlation between segregation dynamics and the growth rate. On the basis of our results, we propose a model for the asymmetric action of segregation machinery that reflects unequal division and growth of mycobacterial cells.
View details for DOI 10.1111/mmi.13712
View details for Web of Science ID 000405994500010
View details for PubMedID 28517109
Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2017; 114 (12): 3157-3162
Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4(+) and CD8(+) T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.
View details for DOI 10.1073/pnas.1701746114
View details for Web of Science ID 000396893600066
View details for PubMedID 28270614
Tissue-specific emergence of regulatory and intraepithelial T cells from a clonal T cell precursor
2016; 1 (2): eaaf7471
Peripheral Foxp3+ regulatory T cells (pTregs) maintain immune homeostasis by controlling potentially harmful effector T cell responses toward dietary and microbial antigens. Although the identity of the T cell receptor (TCR) can impose commitment and functional specialization of T cells, less is known about how TCR identity governs pTreg development from conventional CD4+ T cells. To investigate the extent to which TCR identity dictates pTreg fate, we used somatic cell nuclear transfer to generate a transnuclear (TN) mouse carrying a monoclonal TCR from a pTreg (pTreg TN mice). We found that the pTreg TCR did not inevitably predispose T cells to become pTreg but instead allowed for differentiation of noninflammatory CD4+CD8αα+ intraepithelial lymphocytes (CD4IELs) in the small intestine. Only when we limited the number of T cell precursors that carried the TN pTreg TCR did we observe substantial pTreg development in the mesenteric lymph nodes and small intestine lamina propria of mixed bone marrow chimeras. Small clonal sizes and therefore decreased intraclonal competition were required for pTreg development. Despite bearing the same TCR, small intestine CD4IEL developed independently of precursor frequency. Both pTreg and CD4IEL development strictly depended on the resident microbiota. A single clonal CD4+ T cell precursor can thus give rise to two functionally distinct and anatomically segregated T cell subsets in a microbiota-dependent manner. Therefore, plasticity of the CD4 T cell compartment depends not only on the microbiota but also on specialized environmental cues provided by different tissues.
View details for DOI 10.1126/sciimmunol.aaf7471
View details for Web of Science ID 000434298000003
View details for PubMedID 28783695
View details for PubMedCentralID PMC6296461
Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria
2013; 4: 2470
During the bacterial cell cycle, chromosome replication and cell division must be coordinated with overall cell growth in order to maintain the correct ploidy and cell size. The spatial and temporal coordination of these processes in mycobacteria is not understood. Here we use microfluidics and time-lapse fluorescence microscopy to measure the dynamics of cell growth, division and chromosome replication in single cells of Mycobacterium smegmatis. We find that single-cell growth is size-dependent (large cells grow faster than small cells), which implicates a size-control mechanism in cell-size homoeostasis. Asymmetric division of mother cells gives rise to unequally sized sibling cells that grow at different velocities but show no differential sensitivity to antibiotics. Individual cells are restricted to one round of chromosome replication per cell division cycle, although replication usually initiates in the mother cell before cytokinesis and terminates in the daughter cells after cytokinesis. These studies reveal important differences between cell cycle organization in mycobacteria compared with better-studied model organisms.
View details for DOI 10.1038/ncomms3470
View details for Web of Science ID 000325533900017
View details for PubMedID 24036848