Journal: bioRxiv

Article Title: A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2

doi: 10.1101/2025.10.16.682991

Figure Lengend Snippet: HLA-DR3 and HLA-DR15 mice were infected with influenza B/Brisbane/60/08 virus. Draining lymph nodes were harvested 12 days post infection and used as the source of CD4 T cells in IFN-γ ELISpot assays. Enriched CD4 T cells were restimulated with pools of peptides encompassing the entire translated regions of HA (red), NA (orange), NP (blue), M1 (yellow) and NS1 (green) to assess the total response to infection and allow exclusion of non-stimulatory proteins from further evaluation. Shown in A and B are the frequencies of IFN-γ producing cells per million CD4 T cells as a bar graphs, with the percent of the response to each protein shown as a pie chart with the total number of spot forming cells (SFC) shown beneath each pie. Panels C (HLA-DR3) and D (HLA-DR15) illustrate the responses to the HA peptide matrix, where peptides are grouped into pools denoted as rows (“R”) and columns (“C”) with no overlapping peptides contained in a row or column. The peptide composition of each pool is indicated with each of the 144 overlapping peptides represented by peptide numbers 1-144. Pools were considered positive with a response at least 3-fold over background (typically >50 spots). Non-stimulatory pools are indicated in gray, stimulatory pools are indicated in yellow and pools in white were stimulatory, but weak responses. Peptides at the intersection points are potentially the immunodominant epitopes, which were further tested in single peptide analyses.

Article Snippet: Briefly, CD8- and CD56-depleted PBMCs (300,000-400,000 cells per well) were cultured with single peptides on plates coated with 10 μg/ml anti-human IFNγ (clone 1-D1K, MabTech) for 36 hr at 37°C, 5% CO 2 .

Techniques: Infection, Virus, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2

doi: 10.1101/2025.10.16.682991

Figure Lengend Snippet: Shown here are representative plots of single peptide analyses for identification of CD4 T cell peptide epitopes in HA (left) and M1 (right) proteins of influenza B/Brisbane/60/08 after infection. The peptides selected for analysis for HA-B are based on the stimulatory peptides and peptides eliminated in the matrices shown in . M1 peptides were tested individually without using a matrix pooling approach. Shown are the responses of splenic CD4 T cells with the bar graphs represent the average frequency of IFN-γ producing cells per million CD4 T cells with background subtracted.

Article Snippet: Briefly, CD8- and CD56-depleted PBMCs (300,000-400,000 cells per well) were cultured with single peptides on plates coated with 10 μg/ml anti-human IFNγ (clone 1-D1K, MabTech) for 36 hr at 37°C, 5% CO 2 .

Techniques: Infection

Journal: bioRxiv

Article Title: A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2

doi: 10.1101/2025.10.16.682991

Figure Lengend Snippet: PBMCs from healthy adults that were collected between 2018 and 2023 and typed for HLA-DRB1*01:01 (far left), HLA-DRB1*04:01 (left middle), HLA-DRB1*03:01 (right middle) or HLA-DRB1*15:01 (far right) were enriched for CD4 T cells and APC by depletion of CD8 and CD56+ cells. CD4 T cell reactivity to each of the individual influenza derived peptides (top, A) and SARS-CoV-2 derived peptides (bottom, B) previously identified by epitope mapping in HLA-DR transgenic assessed in IFN-γ ELISpot assays is shown. Individual subjects are represented by unique symbols , and the median response is shown as a grey bar. All data are represented as spots per million CD4 T cell-enriched PBMC, with background subtracted.

Article Snippet: Briefly, CD8- and CD56-depleted PBMCs (300,000-400,000 cells per well) were cultured with single peptides on plates coated with 10 μg/ml anti-human IFNγ (clone 1-D1K, MabTech) for 36 hr at 37°C, 5% CO 2 .

Techniques: Derivative Assay, Transgenic Assay, Enzyme-linked Immunospot

Journal: Clinical Cancer Research

Article Title: HLA Class II Loss and JAK1/2 Deficiency Coevolve in Melanoma Leading to CD4 T-cell and IFNγ Cross-Resistance

doi: 10.1158/1078-0432.ccr-23-0099

Figure Lengend Snippet: Figure 3. Melanoma cell–intrinsic HLA-II expression sensitizes to cytotoxic CD4 T cells. A, Activation of CD4 T cells in the presence of autologous APC or Ma-Mel-61b cells measured by IFNg ELISpot assay. Numbers indicate mean IFNg spots of duplicate determinations. Representative data from one of two independent experiments. B, Stimulation of T cells by HLA-II–negative (Ma-Mel-61a) and constHLA-II (Ma-Mel-61b) melanoma cells measured by ICS (IFNg and TNFa) in the presence or absence of anti–HLA-II blocking antibodies (aHLA-II). Representative dot plots from one of two independent experiments; numbers indicate frequencies. C, Cytotoxic activity of CD4 T cells against autologous melanoma cells. Impedance-based real-time killing of Ma-Mel-61b cells by autologous CD4 T cells at indicated target to effector ratios. Representative data of duplicate determinations from one of two independent experiments. D, Activation of CD4 T cells by autologous Ma-Mel-61b cells measured by Granzyme B ELISpot assay. Numbers indicate Granzyme B spots. Representative data from one of two independent experiments. E, Intracellular expression of indicated molecules in Ma–Mel-61b–reactive CD4 T-cell cultures (MLTC) and blood T-cell subsets from different patients with melanoma (n ¼ 4, ex vivo) measured by flow cytometry analysis. F, Ma-Mel-61a cells analyzed by Western blot analysis for expression of STAT1, IRF1, HLA-II after IFNg, IFNb, or IFNa treatment (48 hours); GAPDH, loading control. Representative data from one of two independent experiments. G, Activation of CD4 T cells by IFNg-treated (48 hours) melanoma cells and control cells measured by IFNg ELISpot assay.Data represent means of two independent experiments (þSEM). H, HLA-DR cell surface expression measured by flow cytometry analysis of Ma-Mel-61a cells treated with IFNg (48 hours; black) and Ma-Mel-61b cells (gray). Thin dashed lines represent unstained controls. Representative data from one of three independent experiments.

Article Snippet: Briefly, multiscreenHA plates (Millipore) were coated with capture antibodies for IFNg (anti-hIFNg , clone 1-D1K) or Granzyme B (anti-Granzyme B, clone MT28;MABTECH).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunospot, Blocking Assay, Activity Assay, Ex Vivo, Cytometry, Western Blot, Control