Biology subjects

Stern, L. J.

Publications and source records attributed to Stern, L. J..

5 recordsLinked to original sources

bioRxiv2026

DNA-barcode labelled MHCII multimers for detection of antigen-specific CD4 T cells across large libraries of epitopes

The role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.

immunology↗

bioRxiv2022

Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but identification and characterization of the T cell response to seasonal human coronaviruses remain largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal human coronavirus OC43. We identified MHC-I and MHC-II bound peptides derived from the viral spike, nucleocapsid, hemagglutinin-esterase, 3C-like proteinase, and envelope proteins. Only three MHC-I bound OC43-derived peptides were observed, possibly due to the potent MHC-I downregulation induced by OC43 infection. By contrast, 80 MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. These peptides elicited low-abundance recall T cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T cell lines. Among the validated epitopes, S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. N54-68 and HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow T cell cross-reactivity after infection or vaccination and could aid in the selection of epitopes for inclusion in pan-coronavirus vaccines. Author SummaryThere is much current interest in cellular immune responses to seasonal common-cold coronaviruses because of their possible role in mediating protection against SARS-CoV-2 infection or pathology. However, identification of relevant T cell epitopes and systematic studies of the T cell responses responding to these viruses are scarce. We conducted a study to identify naturally processed and presented MHC-I and MHC-II epitopes from human cells infected with the seasonal coronavirus HCoV-OC43, and to characterize the T cell responses associated with these epitopes. We found epitopes specific to the seasonal coronaviruses, as well as epitopes cross-reactive between HCoV-OC43 and SARS-CoV-2. These epitopes should be useful in following immune responses to seasonal coronaviruses and identifying their roles in COVID-19 vaccination, infection, and pathogenesis.

immunology↗

bioRxiv2022

Peptide Avidity for TCR on CD4 Effectors Determines the Extent of Memory Generation

The impact of initial peptide antigen affinity for TCR in driving memory fate has been studied previously, however its contributions when effectors contract to memory is unclear. To become memory, effector CD4 T cells must recognize antigen at 5-8 days post-infection, at what we call the "effector checkpoint." We examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We made an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP-peptide variants that bind FluNP TCR with a broad range of avidity. To vary avidity in vivo, we primed naive donor FluNP in IAV-infected hosts, purified 6d FluNP effectors and co-transferred them with peptide-pulsed APC into uninfected second hosts. Higher affinity peptides yielded higher numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN, and drove better protection against lethal influenza infection. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors. IL-2R expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory.

immunology↗

bioRxiv2022

Broadly-recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can impact the T cell response in COVID-19. We studied T cells recognizing SARS-CoV-2 and HCoVs in convalescent COVID-19 donors, and identified a highly conserved SARS-CoV-2 sequence S811-831, with two overlapping epitopes presented by common MHC-II proteins HLA-DQ5 and HLA-DP4. These epitopes were recognized by CD4+ T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and by low-abundance CD4+ T cells in uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. CD4+ T cell cross-reactivity was driven by the remarkably strong conservation of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC-II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly-conserved CD4+ T cell epitope broadly recognized across human populations.

immunology↗

bioRxiv2018

High diversity, turnover, and structural constraints characterize TCR α and β repertoire selection

Recognition modes of individual T-cell receptors (TCR) are well studied, but how TCR repertoires are selected during acute through persistent human virus infections is less clear. Here, we show that persistent EBV-specific clonotypes account for only 9% of unique clonotypes but are highly expanded in acute infectious mononucleosis, and have distinct antigen-specific public features that drive selection into convalescence. The other 91% of highly diverse unique clonotypes disappear and are replaced in convalescence by equally diverse \"de-novo\" clonotypes. These broad fluctuating repertoires lend plasticity to antigen recognition and potentially protect against T-cell clonal loss and viral escape.

immunology↗