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. 2026 Jul 30;17:1867422. doi: 10.3389/fimmu.2026.1867422

Figure 2.

Figure composed of six panels illustrating experimental workflow and results for ZsG-expressing 4T1 tumor cells. Panel A displays a scatter plot of protein intensity versus protein number, highlighting ZsG. Panel B presents a schematic workflow: ZsG is introduced to 4T1 cells, which are engrafted in mice, followed by lymphocyte extraction and hybridoma generation for screening monoclonal antibodies. Panel C shows two flow cytometry contour plots comparing CD19 and ZsG expression in 4T1 and 4T1-ZsG tumors. Panel D is a bar graph quantifying CD19⁺ZsG⁺ lymphocytes, showing increased frequency in 4T1-ZsG samples with statistical significance. Panel E consists of two histograms illustrating IgM and IgG1 hybridoma BCR expression. Panel F displays three histograms at three time points after hybridization showing relative cell count and increasing IgG1 BCR expression at three conditions, with rising percentages.

Characterization of 4T1-ZsG tumors and their TDLNs. (A) Proteomic analysis of the abundance of ZsG in 4T1-ZsG cells, relative to other detected cellular proteins, as measured by intensities of 2D mass spectrometry (LC-MS/MS). (B) Experimental scheme depicting the generation of cloned hybridomas from TDLN-derived lymphocytes of mice engrafted orthotopically with 4T1-ZsG cells. (C) Representative flow cytometry analysis of binding of soluble extract prepared from 4T1-ZsG tumor cells by lymphocytes in the TDLN of mice orthotopically engrafted with 4T1-ZsG cells. (D) Proportion of CD19+ ZsG+ B cells in the TDLN of 4T1 vs. 4T1-ZsG, on days 14–18 after tumor engraftment. n=4. Error bars represent standard error. *p<0.05, two-tailed t-test. (E) Representative flow cytometry analysis measuring the expression of IgM BCR on hybridoma (orange) versus an IgG1 BCR (blue), in two monoclonal hybridomas generated from TDLN lymphocytes. Each panel represents a prior mixture of the 2 hybridomas, stained either with mouse IgM antibody (left panel) or mouse IgG1 antibody (right panel). (F) Representative flow cytometry analysis tracking the temporal appearance of BCRs (IgG1) on TDLN-derived hybridomas at the indicated days (d) post hybridization. Mouse myeloma cell line (NS0) served as control.