Exercise as an Immune Adjuvant for Allogeneic Cell Therapies

Immune Cell Enumeration and Phenotyping

Whole blood samples will be analyzed for complete blood counts and to quantify lymphocyte and monocyte subtypes using flow cytometry and a comprehensive immunophenotyping panel. This panel is designed to identify major immune cell populations, as well as markers related to differentiation, exhaustion, migration, activation, and inhibition. Specific cell types expressing a surface protein, or combinations of surface proteins, will be reported as the percentage of cells positive for expression and/or by mean fluorescent intensity (MFI). For descriptive purposes, the cell counts of all major lymphocyte and monocyte subtypes will be expressed as cells per microliter (cells/µL) of whole blood. Additionally, isolated peripheral blood mononuclear cells (PBMCs) and expanded cell products will be quantified and phenotyped in a similar manner.

Cytolysis in vitro

We will assess whether lymphocytes collected during or after exercise, as well as cell products manufactured from these lymphocytes, are more effective at killing hematologic cancer target cells. Using in vitro assays, such as flow cytometry and bioluminescence-based assays, we will compare the cytolytic activity of both the collected lymphocytes and the manufactured cell products to those obtained under resting conditions. Results will be measured as the time required to achieve 10%, 20%, 30%, 40%, and 50% cytolysis, or as the percentage of target cells killed at specific time points (e.g., 4, 8, 24, and 48 hours). We will also evaluate the impact of combination therapies, such as monoclonal antibodies targeting the tumor model, as appropriate.

Tumor Burden and Tumor Free Survival

Tumor burden will be evaluated in immunocompromised mice engrafted with human tumors by measuring the size, number, and progression of tumors using imaging techniques such as bioluminescence, MRI, or CT scans, along with physical measurements where applicable. Overall tumor burden will be assessed through metrics like peak tumor size and photon intensity in bioluminescence imaging. Tumor-free survival will be defined as the time from treatment until either the recurrence of detectable tumors or the last follow-up without tumor recurrence. Data will be reported as overall tumor reduction (e.g., percentage decrease in tumor size or number), peak tumor burden, and photon intensity, as well as the duration of tumor-free survival in days. Additional analyses will explore the effects of treatment on delaying tumor progression and improving overall survival.

Clinical xGvHD Score

The development of xGvHD (xenogeneic graft-versus-host disease) will be assessed using a clinical scoring system with a possible aggregate score ranging from 0 to 10. Animals will be monitored regularly, and a total score of 5 or higher on two consecutive assessment days will indicate the presence of moderate xGvHD. This scoring system allows for the systematic evaluation of disease severity and progression in response to treatment.

Single-Cell Secretome

We will assess the functional phenotype of purified cell types, including CD4+ and CD8+ T cells as well as natural killer (NK) cells, isolated from blood lymphocytes or expanded cell products. This will be accomplished using the Isoplexis single-cell platform and a multiplexed analysis of over 32 cytokines. Polyfunctionality will be evaluated by quantifying the number of distinct cytokines secreted by individual cells (ranging from 0 to 32) after a culture period of 4 to 18 hours. Each assay will include controls: unstimulated cells as a negative control, tumor cells to represent a target, and phorbol myristate acetate (PMA) as a positive control.

CITE-Seq Analysis of Immune Cell Populations

We will use CITE-Seq to analyze transcriptional and surface protein differences in key human immune cell populations in response to exercise, with and without in vitro tumor challenge. Cell clusters will be annotated using the Azimuth package based on reference human PBMCs. Additionally, we will conduct differential gene expression (DE) analysis and perform Gene Set Enrichment Analysis (GSEA) using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms, applying fold-change and difference metrics for all major immune cell clusters defined by the Azimuth multimodal atlas. We will also sequence various chains of the T-cell receptor (alpha, beta, gamma and delta) to determine T-cell clonal characteristics and antigen specificity using public databases (e.g. VDJdb).

Human Cell Engraftment and Immune Reconstitution:

In xenogeneic mice, peripheral blood counts and flow cytometry will be performed on days +7, +14, +21, +28, and +35 following tail bleeding into heparinized capillary tubes. This will assess human CD45+ cell engraftment (% of total human and mouse CD45+ cells) and differences in human immune cell reconstitution using a limited panel of lineage markers due to the restricted blood volumes in mice. If necessary, selected experiments will involve euthanizing mice to evaluate differences in human CD45+ cell and lymphocyte subsets in the bone marrow and spleen.

Pathology and Immunohistochemistry

In additional experiments, tissues-including the large and small intestine, liver, lung, and skin-from xenogeneic mice will be harvested for pathological evaluation of xenogeneic graft-versus-host disease (xGvHD). Immunohistochemistry will be performed to assess the types of infiltrating human cells present in the mouse tissues. The quantification of immune cells will be conducted using image analysis software to count the number of positive-staining cells per field of view, allowing for a comparison of immune cell infiltration across different tissues and experimental conditions.