Provided methods of obtaining a plurality of T cell receptors specifically recognizing a target tumor antigen peptide from an individual that has clinically benefitted from an immunotherapy, such as Multiple Antigen Specific Cell Therapy. Also provided tumor-specific TCRs, engineered immune cells expressing the TCRs and methods of treating a disease using the engineered immune cells.

Expansion of cytotoxic T lymphocytes (CTLs) is a crucial step in almost all cancer immunotherapeutic methods. Current techniques for expansion of tumor-reactive CTLs present major limitations. The present invention comprises a novel method to effectively produce and expand tumor-activated CTLs using high-voltage pulsed electric fields. Tumor cells were subjected to high-frequency irreversible electroporation (HFIRE) with various electric field magnitudes and pulse widths, or irreversible electroporation (IRE). The treated tumor cells were subsequently cocultured with CD8+ cytotoxic T cells along with antigen-presenting cells. Tumor-activated CTLs can be produced and expanded when exposed to treated tumor cells. The activated CTLs produced with the present invention could be used for clinical applications with the goal of targeting and eliminating tumors.

Provided methods of obtaining a plurality of T cell receptors specifically recognizing a target tumor antigen peptide from an individual that has clinically benefitted from an immunotherapy, such as Multiple Antigen Specific Cell Therapy. Also provided tumor-specific TCRs, engineered immune cells expressing the TCRs and methods of treating a disease using the engineered immune cells.

The present disclosure provides compositions and methods for monitoring cell to cell (“cell-cell”) interactions in vitro, ex vivo, and in vivo. In some embodiments, the present disclosure provides for the use of these compositions and methods (i) to identify and enrich for tumor-specific antigen (TSA) reactive T cells from tumor infiltrating lymphocytes (TILs) or circulating T cells; (ii) to identify and enrich for T cells that recognize autoantigens in a particular autoimmune disease, and/or (iii) to identify and enrich for antigen specific regulatory T cells that have the potential to be exploited to treat autoimmune disease. In some embodiments, the present disclosure also provides for methods of treating diseases, e.g., cancer with such TSA reactive T cells isolated via the present methods.

Cell culture systems and methods provide improved immunotherapeutic product manufacturing with greater scalability, flexibility, and automation. Cell culture systems are configured with interchangeable cartridges, allowing versatility and scalability. Systems are configured to have multiple connected cell culture chambers, which allows parallel processing of different types of cells. Gas-impermeable cell culture chambers and methods for generating cells in closed systems prevent contamination and user error. Methods for recycling cell culture medium provide additional efficiencies.

Disclosed are compositions and methods for suppressing without killing alloreactive and/or autoreactive lymphocytes. The methods can be used for preventing graft versus host disease (GVHD) in subjects receiving donor cells or treating autoimmunity. In particular, chimeric antigen receptor (CAR) polypeptides are disclosed that can be used with adoptive cell transfer to suppress alloreactive or autoreactive lymphocytes. Also disclosed are regulatory T cells that are engineered to express these CARs. Therefore, also disclosed are methods of suppressing alloreactive or autoreactive lymphocytes in a subject in need thereof that involves adoptive transfer of the disclosed regulatory T cells engineered to express the disclosed CARs.

The present invention provides a method of treating a cancer in an individual using activated T cells or PBMCs induced by antigen presenting cells (such as dendritic cells) loaded with a plurality of tumor antigen peptides. The method may further comprise administration of the antigen presenting cells loaded with the plurality of tumor antigen peptides to the individual. The methods may be used singly or in combination with an immune checkpoint inhibitor. Precision therapy methods customized for the individual using neoantigen peptides or based on the mutation load in the tumor of the individual are provided. Methods of preparing the activated T cells, methods of monitoring the treatment, and methods of cloning tumor-specific T cell receptors are further disclosed. An isolated population of cells comprising the activated T cells, as well as compositions and kits useful for cancer immunotherapy are also provided.

FOXP3.sup.+ regulatory T cells (Tregs) can represent powerful adoptive immunotherapies for autoimmune diseases, metabolic diseases, and other chronic inflammatory diseases. The present invention is related to the ability to maintain and expand stable Treg lines and can provide insight into FOXP3.sup.+ Treg physiology and can enable feasible strategies of Treg-based immunotherapy.

Provided methods of obtaining a plurality of T cell receptors specifically recognizing a target tumor antigen peptide from an individual that has clinically benefitted from an immunotherapy, such as Multiple Antigen Specific Cell Therapy. Also provided tumor-specific TCRs, engineered immune cells expressing the TCRs and methods of treating a disease using the engineered immune cells.

Cell culture systems and methods provide improved immunotherapeutic product manufacturing with greater scalability, flexibility, and automation. Cell culture systems are configured with interchangeable cartridges, allowing versatility and scalability. Systems are configured to have multiple connected cell culture chambers, which allows parallel processing of different types of cells. Gas-impermeable cell culture chambers and methods for generating cells in closed systems prevent contamination and user error. Methods for recycling cell culture medium provide additional efficiencies.