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Cell-based therapies using chimeric antigen receptor (CAR-T) cells have shown great promise for treating cancer. Applying this approach to regulatory T cells (Tregs) may provide treatments applicable to other disorders, including tissue rejection and autoimmune diseases, such as Type 1 Diabetes and multiple sclerosis, where a defect in Tregs allows the immune system to mediate the destruction of self-tissues.

A subset of T lymphocytes, versatile and multifaceted Tregs interact with multiple cell types to provide suppressive mechanisms to keep the immune system in check. Over 50 registered clinical trials are utilizing polyclonal Tregs; however, the use of Tregs with uncharacterized antigen specificities has led to undesirable effects. The accurate profiling and characterization of these immune cells are critical.

To counter the undesirable effects of polyclonal cells, CAR Tregs can be targeted to specific antigens to create immune tolerance to protect particular tissues from destruction.

In a January 2020 Genetic Engineering & Biotechnology News webinar, Leonardo Ferreira, PhD, Postdoctoral Scholar and Jeffrey G. Klein Family Diabetes Fellow at the University of California, San Francisco, and Jonathan Chen, technology co-inventor at IsoPlexis, discussed CAR Tregs and how the IsoPlexis IsoLight single-cell functional proteomics system is crucial in the informed design and development of this next generation of cell therapies. To view the on demand webinar, visit here.

In the webinar Ferreira discussed his work focused on Type 1 Diabetes, on designing pancreas-specific second-generation CAR Tregs to suppress effector T cells. In this model, the CAR is one single polypeptide chain that contains a receptor recognition portion, the zeta chain of the T-cell receptor and a co-stimulatory receptor, 28z or 41BBz.

Isolated human Tregs from peripheral blood were stimulated, transduced, and then expanded in culture. In vitro experiments determined CAR-mediated activation, specificity, proliferation, and stability using TSDR methylation, an epigenetic mark. Mass cytometry (CyTOF) analysis determined that CAR-mediated 28z signaling induced stronger activation than 41BBz.

For the in vivo work, NALM6 cells, a B-cell derived leukemia, were injected into NSG mice. NALM6 cells cause demise in three weeks; survival if CD19 CAR-T effector cells are added. Theoretically, CAR Tregs should revert this protection but, in this experiment, the CAR Tregs failed to suppress the effect of the effector cells, an unexpected finding. The finding that the CAR Tregs could control the growth of the CD19 tumor in vivo was intriguing and warranted further investigation.

Additional assessment of in vivo suppression of allo T cell-mediated tumor killing showed no difference between Tregs with a mock or experimental CAR. It was hypothesized that cytokines could be responsible as research into previously published work showed that TNF-α suppresses NALM6 tumor growth in mice.  Flow cytometry analysis of intracellular cytokine production (IFN-γ, IL-10 and TNF-α) showed little or no cytokine expression with mock Tregs; the 28Z Tregs seemed to be hot-wired to produce IFN-γ, and TNF-α.

For a more in-depth analysis, the IsoPlexis IsoLight system was used to quantitate the cytokine production at the single-cell level to determine the signaling of the Treg subsets. The 32-plex panel single-cell analysis divides the cytokines into effector, stimulatory, chemoattractive, regulatory and inflammatory categories. Polyfunctionality also was compared between Tregs and effector cells.

Results showed that the CAR seemed to be partially reprogramming the Tregs to have an effector function, even though initial tests confirmed their identity as Tregs. Even with the same CAR, Tregs and T effectors behaved differently; the CAR signaling appeared to lead to production of more effector cytokines impacting CAR Treg suppression in vitro.

To summarize Ferreira’s results, CAR Tregs maintained Treg phenotype and function upon in vitro activation and effector cell proliferation in vitro and in vivo, yet also controlled the growth of target-expressing cells. In addition, CAR signaling had a profound impact on CAR Treg-mediated suppression and cytokine production.

The IsoPlexis system fills a missing gap in defining the functional phenotyping of each immune cell by analyzing extracellular function. The technology complements flow based systems used to define surface markers, and genomics or single cell transcriptomics used to define the complete cellular transcript.

The single-cell phenotyping platform differentiates immune functional biomarkers by defining extracellular cytokine production of each T-cell, monocyte and NK cell to achieve unique correlative clinical immune biomarkers, and differentiating mechanistic information.

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