Testing Cancer Immunotherapy in a Human Immune System Mouse Model: Correlating Treatment Responses to Human Chimerism, Therapeutic Variables and Immune Cell Phenotypes

Abstract

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2^nullIl2rγ^nullSIRPα^NOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of "responding" patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis.

Keywords: PDX (patient derived xenograft); TME (tumor microenvironment); checkpoint blockade; combination testing; humanized mice; immune correlates; immunotherapy; preclinical model.

Figure 1

Protocol for evaluation of immunotherapy treatments in HIS-BRGS mice. (A) Timeline of HIS-BRGS mouse model development. (B) Allocation of HIS-BRGS mice into experimental treatment groups A-E based on equivalent human (hCD45+), T (CD3+) and CD8+ T cell chimerism. NS, no significance [Welch’s ANOVA test]. (C) Correlation of cell counts determined by flow cytometry (Relative cell count) and hemocytometer cell count in the lymph nodes (LN, left) or tumor weight (right) [linear regression analysis, R-squared score (R2) and P value (P) in bold if statistically significant (P < 0.05)]. (D) Human (hCD45+) chimerism in the blood (PBMC), LN, spleen (SP) and tumor tissue (TIL) of female (F) vs male (M) HIS-BRGS mice [two-group t-test, two tailed].

Figure 2

The human immune system in HIS-BRGS-mice. (A) Human chimerism (%hCD45 of (h+m) CD45) among female (F) and male (M) HIS-BRGS mice at 10 weeks of age (n=35 F, n=41 M; two-group t-test p value = 0.74). (B) Percentage of human (hCD45), T cell (CD3) and B cell (CD20) subsets in PBMCs of HIS-BRGS (left panel) and BRG or NSG-A2 mice (right panel) at indicated times post engraftment. *p < 0.05, ****p < 0.0001 [paired t-test, two-tailed]. (C) Chimerism by organ for multiple HIS-BRGS mice (n=7, except for BM CD4+/CD8+,n=6) from one CB. T cell (CD3+), B cell (CD19+), myeloid population (CD11b/33/14 or 11c+), NK cell (CD56+) populations. Gate: hCD45+ or hCD45+CD3+ (CD4+, CD8+ of T). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS, no significance. [Welch’s two-group t-test, two-tailed between organs as indicated by edges of each line]. LN, lymph nodes; SP, spleen; BM, bone marrow; Thy, thymus.

Figure 3

Immunostimulatory and immunomodulating human lymphocyte subsets in the lymph nodes (LN) and spleen (SP) of HIS-BRGS mice. T cell expression of (A) activation marker, HLA-DR, and memory cell markers, CD45RO/CCR7, (B) inhibitory receptors, PD-1, TIGIT, TIM-3, and (C) regulatory T cell markers, CD25⁺FoxP3⁺. (D) CD11c⁺ B cells. Human PBMC served as a technical staining control (“PC”, top row).

Figure 4

Infiltration of human immune cells into different human tumors in untreated HIS-BRGS mice. (A) Representative flow cytometry plots showing mouse (mCD45⁺, y-axis) and human (hCD45⁺ or (^) hCD3⁺ for hematological malignancy, x-axis) leukocyte infiltration into tumors in HIS-BRGS mice: TNBC MDA-MB-231 (A1-6) and PDX (B); CRC PDX: MSI-H (A) and MSS (B-I); PDAC PDX (A, B); SCLC CDX (A, B) and PDX (C, D); ACC PDX (A, B); melanoma PDX (A) and CDX (B,C); and DLBCL CDX (A,B). Different experiments with the same tumor have the same letter but different numbers; (p) refers to a primary tumor and (m) to metastatic origin. Background staining represented by melanoma PDX A injected into non-humanized BRGS mouse (lower left). (B) Percentage of hCD45⁺ (of singlet gate, left) and CD3⁺ T cells (hCD45+ gate, right) in tumors of untreated HIS-BRGS mice. Filled symbols represent experiments in which tumors in treated HIS-BRGS mice responded to treatment and open symbols represent non-responding experiments. The data are log-transformed to approximate normal distributions. The colors represent experiments testing nothing (black), ICB monotherapy (blue), targeted therapies alone (green) or immunotherapy combinations (red). *p < 0.05, **p < 0.01, ****p < 0.0001 [The lower lines in B reflect analyses of differences in means across experiments of the same tumor type using Welch’s ANOVA (TNBC, CRC, SCLC, MEL) or two-group t-test (ACC). The upper line shows Welch’s ANOVA analysis for differences in means across all tumors, regardless of type].

Figure 5

Influence of human chimerism and timing of tumor injection on human immune infiltration in human tumors in untreated HIS-BRGS-mice. (A) Correlation of hCD45⁺ infiltration in HIS-BRGS tumors with human chimerism in lymph organs. Data from six TNBC CDX (top, A1-A6) and five CRC (bottom, CRC B2, D2, D3, E, F) independent experiments. hCD45 and hCD3 chimerism (percentage and absolute number, #) in PBMC prior to tumor injection (PBL at 15 weeks), and in the spleen (SP) and lymph nodes (LN) at end of study. (B) Positive correlation between T cell infiltration in TNBC A1-A6 and CRC tumors (B2, D2, D3, E, F) and T cell chimerism in spleens of HIS-BRGS mice. (C) Influence of timing of tumor injection on human immune cell infiltration into tumors. Human (hCD45+) or T (hCD3⁺) and mouse (mCD45⁺) immune cell infiltration into DLBCL CDXs (upper left) or PDAC PDX (lower left) with different tumor injection timepoints. Correlation of hCD45+ and hCD3⁺ infiltration into untreated TNBC CDXs (A1-A6) and the age of HIS-BRGS mice at time of tumor injection or end of study (right). *p < 0.05, ***p < 0.001 [A, B: Linear regression analysis, R-squared score (R²) and P value (P) in bold if statistically significant (P < 0.05); (C): Welch’s two-group t-test, two-tailed between organs as indicated by edges of each line].

Figure 6

Correlation of tumor growth and human chimerism in lymph organs for 5 CRC MSS PDX models (B2, D2, D3, E, F) in HIS-BRGS mice receiving either no treatment (A) or a combination immunotherapy (B). Linear regression analysis was performed between tumor specific growth rate (SGR) and immune cell populations in the blood prior to tumor injection (PBL at 15 weeks) or in the spleen (SP) and lymph nodes (LN) at end of study. [Linear regression analysis, R-squared score (R²) and P value (P) in bold if statistically significant (P < 0.05)].

Figure 7

Correlates of immune response to human tumors following combination immunotherapy treatments in HIS-BRGS mice. (A) Representative flow cytometry plots showing expression of Granzyme B (left) and TNFα and IFNγ cytokines (right) in CD4⁺ (top row) and CD8⁺ T cells (bottom row). Cells are cultured overnight with cell stimulation and blocked with Golgi Plug for final four hours to detect TNFα and IFNγ. Human PBMCs serve as technical staining control, and as stimulation controls for TNFα/IFNγ. (B) Tumor growth and immune response in HIS-BRGS mice with DLBCL CDX B tumors treated with monotherapies and combination ICI therapies. Tumor growth curves over time (left panel) and frequencies of mCD45⁺, hCD3⁺, CD4⁺ T, and CD8⁺T cell populations (left graphs), and GrB⁺CD4⁺ T cells and IFNγ⁺CD8⁺ T cells (right graphs) in indicated tissues. (C) Immune correlates of combination immunotherapy response to two CRC MSS PDXs in HIS-BRGS mice, generated from same CB. Infiltration of hCD45⁺ cells (left), and frequencies of Tregs, TNFα⁺CD4⁺ T, GrB⁺CD8⁺ and T_EM CD8⁺ cells in CRC (H, I) PDXs in HIS-BRGS mice. (D) Correlation of immunotypes and tumor growth (SGR) in a series of five independent experiments of HIS-BRGS mice bearing CRC MSS PDXs (B2, D2, D3, E, F) and treated with the same combination immunotherapy. The frequency of human (hCD45⁺), T (CD3⁺), CD4⁺ T, CD8⁺ T, B (CD19⁺), and myeloid (CD11b⁺, CD11c⁺, CD14⁺ or CD33⁺) cells (top rows), and the frequency of activated (HLA-DR⁺) T cells (2^nd row, 3^rd panel) and Tregs (2^nd row, 4^th panel) in CRC PDX relative to the specific growth rate (SGR) for that tumor. (E) Tumor growth and immune correlates in HIS-BRGS mice with DLBCL CDX A2 tumors and treated with same combination ICI therapy as in (B). Tumor growth curves over time (left panel) and correlations of tumor size with frequency of CD3, IFNγ CD4⁺ and IFNγ CD8⁺ in DLCBL A in HIS-BRGS mice. Symbols represent data from an individual tissue from tumor-bearing HIS-BRGS mouse that are untreated (black), treated with ICB (blue) alone, drug alone (green), or combination ICB (red). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 reflects significance test between two groups at edge of line. [(B,C): Two-group t-test with Welch’s correction, two-tailed; with the exception of % mCD45 and % of hCD3+ (CD4+ and CD8+) in the tumor, which was evaluated with non-parametric test due to confirmed non-normal distribution in the “combo” group. (D, E): linear regression analysis, R-squared score (R²) and P value (P) in bold if statistically significant (P<0.05)].

Figure 8

Expression of immune-related molecules on human tumors in HIS-BRGS mice. (A) Representative flow cytometry plots illustrating gating for expression on the tumors: mCD45^- hCD45^- cells are gated in the tumor cell suspension and when possible, a tumor-specific Ag (e.g. EpCAM) is included, (B) Correlation of tumor growth rates with expression of human HLA-ABC (MHC Class I), HLA-DR (MHC Class II) and PD-L1 on hCD45^-mCD45^-EpCAM⁺ CRC MSS PDX (B2, D2, D3, E, F) in HIS-BRGS mice. (C) Correlation of tumor weight (grams) and human T (hCD3) cell infiltration with expression of human HLA-ABC (Class I) and HLA-DR (Class II) on DLBCL CDX A2 in HIS-BRGS mice. Expression of HLA-ABC, HLA-DR and PD-L1 on hCD45⁺ and mCD45⁺ populations serve as positive and negative controls, respectively. [Linear regression analysis, R-squared score (R²) and P value (P) in bold if statistically significant (P < 0.05)].

Figure 9

Mouse myeloid cells in human tumors of HIS-BRGS mice. (A) Infiltration of mCD45⁺ cells into human PDX or CDX in HIS-BRGS mice, same tumors as for Figure 4B . *p < 0.05, ****p < 0.0001 [Welch’s ANOVA among means within experiments with same tumor type or across tumor types, lines indicated the tumors included in the analysis] (B) Frequencies of mouse neutrophils (PMNs) in lymph tissues and human tumors (TNBC, CRC). Normalized expression (geometric mean fluorescence intensity) of mouse MHC Class I (C) and MHC Class II (D) expression in total mCD45⁺ cells. Expression was normalized to mCD45⁺ positive control cells for each day for comparison. *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 [(B–D: Upper lines show two-group t test or Welch’s ANOVA among means of different organs in the same experiment. Two-group t-test with Welch’s correction were performed between the means of TILs (brackets)].