Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape.

“Humanized” mice have been widely used for the characterization of human cancer progression and as a powerful preclinical model. Standardization of multicolor phenotyping could help to identify immune cell patterns involved in checkpoint-related complications. Therefore, we applied established protocols for immune cell profiling to our humanized Patient-Derived Xenograft (hPDX) model. hPDX are characterized by the co-existence of a human immune system and a patient-derived tumor transplant. These mice possess a human-like immune system after CD34+ stem cell transplantation while the reconstitution level of the immune system was not related to the quantity of transplanted CD34+ cells. Contamination ≤ 1.2% by CD3+ cells in the hematopoietic stem cell (HSC) transplant did not trigger abnormal T cell maturation. Different B and T cell differentiation stages were identified, as well as regulatory T cells (Tregs) and exhausted T cells that expressed TIGIT, PD-1, or KLRG1. Overall, the application of standardized protocols for the characterization of immune cells using flow cytometry will contribute to a better understanding of immune-oncologic processes.