Fig. 2. Functional characterization of human immune cells in humanized mice.

a–c T- and B-cell response to hTERT vaccine. a Schema for hTERT DNA vaccination. Hu-mice, as described in Fig. 1, received a total of three injections of hTERT vaccine (hTERT DNA [50 μg; i.m] followed by electroporation) every 2 weeks and the mice were euthanized by CO₂ inhalation/cervical dislocation and organs harvested 1 week after the last injection to determine T- and B-cell responses. b Anti-TERT T-cell responses. Spleen cells from Hu-mice (n = 3) were stimulated overnight (18 h) with pools of overlapping hTERT peptides (15 mer; 2 μg/ml/peptide) spanning the entire hTERT protein. Human IFNγ was detected in ELISPOT assay using a kit. Data are represented as SFU (spot forming units; mean ± SE) per 10⁶ splenocytes. hTERT-specific T-cell (IFNγ) response from vaccinated mice was compared to age-matched Hu-mice that received pMV101, a modified pVAX1 vector, as control or untreated age, sex-matched NSG mice controls. c Anti-TERT antibody (IgG) responses. Endpoint binding titer was determined in sera of hTERT vaccinated mice (n = 3) after 3 immunizations and compared to sera from NSG mice as controls. Data are presented as mean values ± SD. d–h Functional ability of immune T cells to restrict tumor growth. d Schema for Hu-mice tumor challenge experiment. e Hu-mice with T-cell reconstitution can restrict tumor growth of HLA-A2 matched A375 melanoma cells. Hu-mice, as described in Fig. 1 that have 90 to 120 cells/μl circulating CD45+/CD8+ cells (closed circle, blue line) when challenged with melanoma cells (10⁵; s.c.), can restrict tumor growth significantly (*p = 0.0281) when compared to non-reconstituted NSG mice (n = 3; closed circles, brown line) and Hu-mice with high circulating B-cells (n = 4; >65% [550 to 600 cells/μl] CD20⁺; open circles, magenta line) have unrestricted tumor growth. Tumor growth measurements are recorded using a digital caliper by an independent researcher. f–h Treatment with anti-PD-1 antibody can restrict tumor growth of melanoma cells. f Schema for anti-PD-1 therapy. Hu-mice with CD45⁺/CD8⁺ (90 to 120 cells/μl) and cells were randomized, and they receive melanoma cells (10⁵; s.c.). When tumors are palpable, mice receive anti-PD-1 (10 mg/kg; i.p. injections) every week for four injections and tumor growth measurements are recorded. g, h Anti-PD-1 therapy restrict melanoma growth. Hu-mice (n = 10) treated with anti-PD-1 antibody can restrict tumor growth of two different melanoma cells [(WM3629 [HLA-A3]; g] and [A375 [HLA-A2]; h]) significantly (blue line, closed circles; *p = 0.0437 for WM3629 and p = 0.0547 for A375) when compared to Hu-mice treated with control IgG (n = 5; magenta; open circles) or non-reconstituted age and sex-matched NSG mice (n = 10; brown; closed circles) treated with the ant-PD-1 antibody. Two different batches of Hu-mice were used for this experiment and they had comparable CD45⁺/CD8⁺ T-cell counts. Unrestricted tumor growth in presence of anti-PD-1 antibody was observed when Hu-mice were challenged with an aggressive phenotype of the tumor (Supplementary Fig. 8). Data are presented as mean values ± SEM for e–h. A one-sided paired t-test was used for analysis when p-values are provided. Source data are provided as a Source Data file.