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. 2021 Jun 14;10:e62927. doi: 10.7554/eLife.62927

Figure 3. NC410 therapy promotes human T cell expansion in a xenogeneic-graft versus-host disease model.

In a non-tumor model, 1 × 107 total human peripheral blood mononuclear cells were adoptively transferred intravenously to NSG mice (N = 6/group) on day 0. Mice were treated with indicated doses of NC410 by intravenous injection on days 0 and 2. On day 6, mice were euthanized and spleens were analyzed for naïve (CD45RA+CCR7+), central memory (CM, CD45RA-CCR7+), effector memory (EM, CD45RA-CCR7-) and effector (CD45RA+CCR7-) CD4+ (A) and CD8+ (B) T cell populations. The graph shows the percentage of T cell subpopulations as a percentage of total human T cells. (C–E) Cell counts of (C) CD4+EM, (D) CD8+EM and (E) CD8+ effector T cells in the spleen. The graphs show the means ± SD (error bars). Asterisks indicate statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey’s multiple comparisons.

Figure 3.

Figure 3—figure supplement 1. Gating strategy to identify human naïve, memory and effector memory T cell subsets in an NSG non-tumor mouse model.

Figure 3—figure supplement 1.

Human peripheral blood mononuclear cells were transferred into NSG mice, and at day 6 post transfer human T cells subsets were analyzed in the spleen based on CD45RA and CCR7 expression (naïve CD45RA+CCR7+; effector memory CD45RA-CCR7-; central memory CD45RA-CCR7+ and effector CD45RA+CCR7-).
Figure 3—figure supplement 2. HT-29 mRNA collagen expression by RNA sequencing.

Figure 3—figure supplement 2.

For collagen gene expression analysis of HT-29 cells, data was acquired from the public dataset GSE41586 (https://pubmed.ncbi.nlm.nih.gov/23902433/). Raw count data of untreated HT-29 cells was retrieved and normalized using the DESeq2 package (v1.28.1) in R (v4.0.2). Data was then log2 transformed and plotted using the ggplot package (v3.3.2).
Figure 3—figure supplement 3. NC410 binds to collagens on HT-29 cells but does not induce antibody-dependent cellular cytotoxicity.

Figure 3—figure supplement 3.

Immunofluorescence analysis of HT-29 cells stained with (A) isotype control, (B) NC410 or (C) pan-collagen antibody. 40× magnification (D) HT-29 cells were removed from culture flasks using increasing concentrations of EDTA for 10 min. Mean fluorescence intensity (MFI) of NC410 staining is shown, demonstrating that EDTA-treated HT-29 cells keep surface collagen expression. (E) HT-29 cells treated with 0.1 mM collagenase lose NC410 binding. Data from three independently performed experiments. (F) In vitro chromium release assay after 24 hr using HT-29 and peripheral blood mononuclear cells at three different effector to target ratios. 20:1 and 50:1 n = 16 and 100:1 n = 25 in 17 independently performed experiments. Closed circles indicate NC410 treatment, and open circles indicate control treatment. The graphs show the means ± SD (error bars). ***p<0.001, two-way ANOVA with Dunnett’s correction.
Figure 3—figure supplement 4. NC410 anti-tumor activity is dependent on T cells and an active IgG1 Fc.

Figure 3—figure supplement 4.

(A) Humanized tumor model (A) of HT-29 tumor injected subcutaneously in the presence of human peripheral blood mononuclear cells (PBMCs). 1 × 107 total human PBMCs with or without T cell depletion were adoptively transferred intravenously to NSG mice (N = 5 or 6/group) on day 0. 1 × 106 HT-29 tumor cells were injected subcutaneously with Matrigel on day 1. Mice were treated with NC410, LAIR-2-FES (Fc dead FES mutant NC410) or controls by intraperitoneal injection, Q4D × 4 doses followed by Q7D until endpoint. Tumor growth was monitored two times a week. (B) Analysis of tumor growth. Asterisks indicate statistical significance compared to control. The graphs show the means ± SD (error bars). ****p<0.0001, two-way ANOVA followed by Tukey’s multiple comparisons. (C) The images of tumors obtained from PBMC-transferred mice on day 53.