Background

The clinical use of checkpoint inhibitor therapy, e.g., anti-CTLA-4 and anti-PD-1, has proven useful as a cancer therapy to enhance the anti-tumor immune response (Curran et al., 2010; Gubin et al., 2014). While successful in patients that have failed frontline therapy, only approximately 20% of treated patients responded to checkpoint inhibitor antibody therapy. This inactivity of treatment in a patient may be multifactorial, and may also be reflective of the specific type of cancer that a patient presents with to the clinician (Podojil et al., 2020). B7-H4 expression by tumor cells and monocyte/macrophages present within the tumor is an additional putative mechanism by which tumor cells evade anti-tumor immune responses (Salceda et al., 2005; Simon et al., 2007; Qian et al., 2011; Abadi et al., 2013). Based on these published findings, the expression of B7-H4 within the tumor microenvironment, i.e., by the tumor cells and/or by infiltrating monocyte/macrophages, is hypothesized to allow for tumor cells to evade an anti-tumor immune response via both direct inhibition of effector T cell function and increasing regulatory CD4⁺ T cell function (Wei et al., 2011; Podojil et al., 2013).

The development of in vitro methods to determine the functionality of immunological therapeutics for the treatment of human disease has relied heavily on the use of cell lines. However, while there are similarities between the signaling pathways activated within human T cells lines (Jurkat and HuT78) and primary T cells isolated from PBMCs, there are distinct differences between the level of signaling pathway intermediate activation, the timing/longevity of signaling pathway intermediate activation, and the cytokine profile expressed between human T cells lines and primary T cells (Bartelt et al., 2009). For example, besides IL-2, both Jurkat and HuT78 cells are less depenent upon costimulation and secreted significantly lower levels of T cell effector cytokines, such as IL-4, IL-5, IL-17, TNF-α, and IFN-γ upon T cell receptor stimulation (Bartelt et al., 2009). This finding limits the utility of these cell lines when studying the ability of a recombinant protein or antibody to modulate cellular activities outside of T cell proliferation and IL-2 production.

The present method can be used to determine the functionality of therapeutics utilizing primary monocytes, CD4⁺ T cells, and CD8⁺ T cells purified from healthy human donors. Several cellular considerations for determining the functionality and identifying/validating the mechanism of action for a putative checkpoint inhibitor are required for assay development. The assay must be fit-for-purpose, robust, repeatable, and transferable between collaborating laboratories. In the present example for the study of anti-B7-H4 functional activity, cells that express B7-H4 must be present within the culture system, i.e., a macrophage population that has been pre-conditioned in the presence of recombinant human cytokines to induce the expression of B7-H4. Additionally, the B7-H4 receptor must be expressed by a target immune cell population, i.e., T cells. The B7-H4 receptor is expressed by a sub-set of both CD4⁺ T cells and CD8⁺ T cells following activation (Podojil et al., 2013). Therefore, an additional consideration for the present experimental design is that since the B7-H4 receptor is not present at high levels on freshly isolated T cells, the response rate within the co-culture system will display donor-to-donor variation.

To this end, two general model are presented below. The first model utilized co-culture of B7-H4 expressing human macrophages with autologous purified CD4⁺ T cells, and the second model system utilized co-culture of B7-H4 expressing human macrophages with autologous purified CD8⁺ T cells. In brief, CD14⁺ monocytes are isolated from total PBMCs, and the CD14⁺ monocytes are cultured in the absence or presence of IL-10 and IL-6 for 3 days. Both CD4⁺ T cells and CD8⁺ T cells are isolated from the CD14- cell fraction of the healthy donor PBMC samples. The sort purified CD4⁺ T cells and CD8⁺ T cells are cryopreserved for future co-culture with the B7-H4 expressing macrophages. Figure 1 presents the number of each cell population isolated from twelve healthy donor PBMC samples. Following the initial 3 day macrophage culture in the presence of IL-10 and IL-6, the macrophages are harvested. The data show that macrophages cultured in the absence of IL-10 and IL-6 do not express B7-H4, while macrophages cultured in the presence of IL-10 and IL-6 do express B7-H4 (Podojil et al., 2020). On day 3, the cryopreserved autologous CD4⁺ T cells and CD8⁺ T cells are rapidly thawed, washed, and labeled with CFSE. The CD4⁺ T cells and CD8⁺ T cells are then co-cultured with IL-10 plus IL-6 conditioned macrophages in the presence of plate-bound anti-CD3 plus either a species and isotype matched Control antibody or anti-B7-H4. On day 6 of the assay (day 3 of macrophage-T cell co-culture) the level of CD4⁺ T cell and CD8⁺ T cell proliferation is assessed via flow cytometry (CFSE dilution), and the levels of secreted cytokine, such as IFN-γ, determined in the culture supernatants. While the use of transfected cell lines is a useful tool to quickly screen large numbers of recombinant proteins and antibodies for potential biological activity, the present protocol using primary cells allows for the validation of the initial in vitro cell line culture findings.