Journal: bioRxiv

Article Title: Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4 + T cell responses

doi: 10.64898/2026.02.18.706639

Figure Lengend Snippet: TcdB 1918 and TcdB 1961 are immunodominant epitopes following vaccination. (A) Amino acid sequences from TcdB peptide library . 15mers were analyzed using the Immune epitope database MHC-II peptide binding prediction for H2-IAb. Data were represented as adjusted rank which is generated by comparing each peptide’s score to a database of 15mers. Lower numbers are predicted to be better binders to H2-IAb. (B) Representative FACS plots showing frequency of TcdB pooled peptide library, individual peptides, or DMSO stimulated CD4 + T cells expressing IFN-γ. (C) The total number of IFN-γ + CD4 + T cells responding to TcdB peptide library or individual peptides. To account for the background cytokine production observed, the total number of IFN-γ + CD4 + T cells in the DMSO group was subtracted from the total number of peptide-responsive IFN-γ + CD4 + T cells. Naïve n=5 and TcdB-CROPs mRNA vaccinated n=5 per timepoint. Two-way ANOVA with Tukey’s multiple comparison test. Statistical significance is indicated as follows: ** P < 0.01; **** P < 0.0001. Representative FACS plots were pre-gated based on the following parameters: Singlets, Live, Lymphocytes, CD45 + , CD3/5 + , CD4 + CD44 + , CD62L lo . Frequency of parental gate +/− standard error of the mean is shown in green. Cells were stimulated with peptide or DMSO for 1 hour before the addition of BFA-M. Cells were incubated for an additional 4 hours before intracellular cytokine staining and analysis by flow cytometry.

Article Snippet: Potential immunodominant epitopes in our TcdB peptide library were identified using predicted immunogenicity analysis from the immune epitope database (IEDB) , on the TcdB-CROPs peptide library generated previously .

Techniques: Binding Assay, Generated, Expressing, Comparison, Incubation, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4 + T cell responses

doi: 10.64898/2026.02.18.706639

Figure Lengend Snippet: TcdB 1918 and TcdB 1961 are immunodominant epitopes following vaccination. (A) Amino acid sequences from TcdB peptide library . 15mers were analyzed using the Immune epitope database MHC-II peptide binding prediction for H2-IAb. Data were represented as adjusted rank which is generated by comparing each peptide’s score to a database of 15mers. Lower numbers are predicted to be better binders to H2-IAb. (B) Representative FACS plots showing frequency of TcdB pooled peptide library, individual peptides, or DMSO stimulated CD4 + T cells expressing IFN-γ. (C) The total number of IFN-γ + CD4 + T cells responding to TcdB peptide library or individual peptides. To account for the background cytokine production observed, the total number of IFN-γ + CD4 + T cells in the DMSO group was subtracted from the total number of peptide-responsive IFN-γ + CD4 + T cells. Naïve n=5 and TcdB-CROPs mRNA vaccinated n=5 per timepoint. Two-way ANOVA with Tukey’s multiple comparison test. Statistical significance is indicated as follows: ** P < 0.01; **** P < 0.0001. Representative FACS plots were pre-gated based on the following parameters: Singlets, Live, Lymphocytes, CD45 + , CD3/5 + , CD4 + CD44 + , CD62L lo . Frequency of parental gate +/− standard error of the mean is shown in green. Cells were stimulated with peptide or DMSO for 1 hour before the addition of BFA-M. Cells were incubated for an additional 4 hours before intracellular cytokine staining and analysis by flow cytometry.

Article Snippet: Potential immunodominant epitopes in our TcdB peptide library were identified using predicted immunogenicity analysis from the immune epitope database (IEDB) , on the TcdB-CROPs peptide library generated previously .

Techniques: Binding Assay, Generated, Expressing, Comparison, Incubation, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4 + T cell responses

doi: 10.64898/2026.02.18.706639

Figure Lengend Snippet: 37 ° C incubation and high concentrations are required for optimal tetramer signal. (A) Representative FACS plots showing frequency and (B) total number of TcdB 1961 tetramer binding CD4 + T cells from naïve and TcdB-CROPs mRNA-LNP-vaccinated mice at various incubation temperatures. (C) Frequency and (D) total number of TcdB 1961 tetramer binding CD4 + T cells at various tetramer concentrations. High: 18µg/mL Med: 7µg/mL Low: 3µg/mL. (E) Frequency and (F) total number of TcdB 1961 tetramer binding CD4 + T cells at various incubation times. Splenocytes from naïve and TcdB-CROPs mRNA-LNP-vaccinated mice were isolated on day 7 post-boost. Cells were fixed using 2% PFA following flow cytometry staining. Representative data of two experiments Naïve n=6 and TcdB-CROPs mRNA vaccinated n=6 per timepoint. Two-way ANOVA with Tukey’s multiple comparison test. Statistical significance is indicated as follows: ** P < 0.01; **** P < 0.0001. Representative FACS plots were pre-gated based on the following parameters: Singlets, Live, Lymphocytes, CD45 + , CD3/5 + , CD4 + CD44 + , CD62L lo . Frequency of parental gate +/− standard error of the mean is shown in green.

Article Snippet: Potential immunodominant epitopes in our TcdB peptide library were identified using predicted immunogenicity analysis from the immune epitope database (IEDB) , on the TcdB-CROPs peptide library generated previously .

Techniques: Incubation, Binding Assay, Isolation, Flow Cytometry, Staining, Comparison

Journal: bioRxiv

Article Title: Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4 + T cell responses

doi: 10.64898/2026.02.18.706639

Figure Lengend Snippet: Intranuclear permeabilization increases signal to noise ratio among tetramer + CD4 + T cells. (A) Representative FACS plots showing frequency and (B) total number of TcdB 1961 tetramer binding CD4 + T cells from naïve and TcdB-CROPs mRNA-LNP-vaccinated mice with different fixation protocols. (C) Signal to noise ratio is the total number of TcdB 1961 tetramer binding CD4 + T cells divided by the total number of CLIP tetramer binding CD4 + T cells among TcdB-CROPs mRNA-LNP-vaccinated mice. Splenocytes from naïve and TcdB-CROPs mRNA-LNP-vaccinated mice were isolated on day 9 post-boost. Representative data of two experiments Naïve n=6 and TcdB-CROPs mRNA vaccinated n=6 per timepoint. (B) Two-way ANOVA with Tukey’s multiple comparison test. Statistical significance is indicated as follows: **** P < 0.0001. (C) Paired t test. Statistical significance is indicated as follows: ** P < 0.01. Representative FACS plots were pre-gated based on the following parameters: Singlets, Live, Lymphocytes, CD45 + , CD3/5 + , CD4 + CD44 + , CD62L lo . Frequency of parental gate +/− standard error of the mean is shown in green.

Article Snippet: Potential immunodominant epitopes in our TcdB peptide library were identified using predicted immunogenicity analysis from the immune epitope database (IEDB) , on the TcdB-CROPs peptide library generated previously .

Techniques: Binding Assay, Isolation, Comparison

Journal: bioRxiv

Article Title: Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4 + T cell responses

doi: 10.64898/2026.02.18.706639

Figure Lengend Snippet: TcdB-CROPs mRNA-LNP vaccination induces TcdB 1961 -specific Tfh cells. (A) Representative FACS plots showing frequency and (B) total number of Tfh cells from the spleens of naïve and TcdB-CROPs mRNA-LNP-vaccinated mice. (C) Frequency and (D) total number of TcdB 1961 tetramer binding Tfh cells. Splenocytes from naïve and TcdB-CROPs mRNA-LNP-vaccinated mice were isolated on day 9 post-boost. Representative data of two experiments Naïve n=6 and TcdB-CROPs mRNA vaccinated n=6 per timepoint. Cells were fixed using 2% PFA or an intranuclear (IN) fixation/permeabilization buffer following surface staining. (B) Unpaired t test. (D) Two-way ANOVA with Tukey’s multiple comparison test. Statistical significance is indicated as follows: **** P < 0.0001. Representative FACS plots were pre-gated based on the following parameters: Singlets, Live, Lymphocytes, CD45 + , CD3/5 + , CD4 + CD44 + , CD62L lo . Frequency of parental gate +/− standard error of the mean is shown in green.

Article Snippet: Potential immunodominant epitopes in our TcdB peptide library were identified using predicted immunogenicity analysis from the immune epitope database (IEDB) , on the TcdB-CROPs peptide library generated previously .

Techniques: Binding Assay, Isolation, Staining, Comparison