Hypoxia Selectively Impairs CAR-T Cells In Vitro

Abstract

Hypoxia is a major characteristic of the solid tumor microenvironment. To understand how chimeric antigen receptor-T cells (CAR-T cells) function in hypoxic conditions, we characterized CD19-specific and BCMA-specific human CAR-T cells generated in atmospheric (18% oxygen) and hypoxic (1% oxygen) culture for expansion, differentiation status, and CD4:CD8 ratio. CAR-T cells expanded to a much lower extent in 1% oxygen than in 18% oxygen. Hypoxic CAR-T cells also had a less differentiated phenotype and a higher CD4:CD8 ratio than atmospheric CAR-T cells. CAR-T cells were then added to antigen-positive and antigen-negative tumor cell lines at the same or lower oxygen level and characterized for cytotoxicity, cytokine and granzyme B secretion, and PD-1 upregulation. Atmospheric and hypoxic CAR-T cells exhibited comparable cytolytic activity and PD-1 upregulation; however, cytokine production and granzyme B release were greatly decreased in 1% oxygen, even when the CAR-T cells were generated in atmospheric culture. Together, these data show that at solid tumor oxygen levels, CAR-T cells are impaired in expansion, differentiation and cytokine production. These effects may contribute to the inability of CAR-T cells to eradicate solid tumors seen in many patients.

Keywords: BCMA; CAR-T; CD19; hypoxia; immunotherapy; microenvironment; tumor.

Figure 1

Hypoxia decreases chimeric antigen receptor-T cell (CAR-T cell) expansion. CD19 CAR-T cells (A) and B cell maturation antigen (BCMA) CAR-T cells (B), along with control T cells, were cultured in an 18% oxygen incubator for the entire 13-day expansion period (red lines), or were cultured in the 18% oxygen incubator for the first 5 days and then in a hypoxia chamber for the remaining 8 days (blue lines). Data-points represent the average and standard error of 4 separate experiments. * p = 0.02 (day 12) and ** p < 0.001 (day 13) for hypoxic vs. atmospheric CD19 CAR-T cells. ** p < 0.001 for hypoxic vs. atmospheric BCMA CAR-T cells.

Figure 2

Hypoxia does not affect CAR-T cell frequency. CD19 CAR-T cells (A) and BCMA CAR-T cells (B) were stained with an anti-FLAG antibody or BCMA protein, respectively. Representative flow cytometry plots showing CAR expression on the X-axis (the Y-axis is an empty channel) are on the left. Charts showing the average and standard error of 4 separate experiments are shown on the right.

Figure 3

Hypoxia inhibits CAR-T cell differentiation. PBMC (A), CD19 CAR-T cells (B) and BCMA CAR-T cells (C) were stained with antibodies for CD27 and CD45RO. CAR-T cells were first gated using the anti-FLAG antibody or BCMA protein. Representative flow cytometry plots showing CD27 and CD45RO expression are on the left; the CAR-T plots show only the gated CAR-T cells. Charts showing the average and standard error of 4 separate experiments are shown on the right. * p < 0.05 and ** p < 0.005.

Figure 4

Hypoxia increases the CD4:CD8 ratio. CD19 CAR-T cells (A) and BCMA CAR-T cells (B) were stained with antibodies for CD27 and CD45RO, along with the anti-FLAG antibody or BCMA protein. Representative flow cytometry plots showing CD27 and CD45RO expression are on the left; the CAR-T plots show only the gated CAR-T cells. Charts showing the average and standard error of 4 separate experiments are shown on the right. * p = < 0.05.

Figure 5

Hypoxia does not affect CAR-T cell cytotoxicity. (A) CD19 CAR-T cell RTCA assay. (B) BCMA CAR-T cell RTCA assay. Left: HeLa, HeLa-CD19, CHO, and CHO-BCMA cells were monitored overnight as they adhered to the plate and formed a monolayer. The next day, atmospheric CD19 CAR-T cells, BCMA CAR-T cells or control T cells were added to the monolayers at an E:T ratio of 10:1 (vertical bars). The cultures were monitored for approximately 24 more hours. Traces show the average of 3 wells. Right: Cytotoxicity in the RTCA assays was calculated at the end of the assays. Data-points represent the average and standard error of 4 separate experiments.

Figure 6

Hypoxia decreases CAR-T cell granzyme B and cytokine production. The media from the CD19 RTCA assay (A) and BCMA RTCA assay (B) was analyzed by ELISA for the levels of granzyme B, IFN-γ, IL-2 and IL-6. Data-points represent the average and standard error of 2–4 separate experiments. * p < 0.05 and ** p < 0.005.

Figure 7

Hypoxia decreases CAR-T cell granzyme B and cytokine production in response to tumor cells. CD19 CAR-T cells or control T cells (A) were co-cultured with CD19⁺ Raji cells or CD19⁻ K562 cells. BCMA CAR-T cells or control T cells (B) were cultured with BCMA⁺ RPMI8226 cells, BCMA⁺ MM1S cells or BCMA^– K562 cells. The medium from the co-cultures was analyzed by ELISA for the levels of granzyme B, IFN-γ, IL-2, and IL-6. Data-points represent the average and standard error of 2-4 separate experiments. * p < 0.05 and ** p < 0.005.

Figure 8

Hypoxia does not affect CAR-T cell PD-1 upregulation. Top: the cells from the CD19 CAR-T cell co-cultures were analyzed by flow cytometry for FLAG staining (i.e., CD19 CAR expression) vs. PD-1 expression. Bottom: the percentages of CD19 CAR-T cells, BCMA CAR-T cells, or control T cells expressing PD-1 were plotted; data-points represent the average and standard error of 2–4 separate experiments.

Figure 9

CAR-T cell expansion in 5% oxygen results in greater cytotoxicity and decreased IFN-γ/IL-2 production. CD19 CAR-T cells and control T cells expanded in 18% oxygen or 5% oxygen were analyzed for cell expansion (A), differentiation (B), cytotoxicity (C), and cytokine production during the RTCA assay (D). Data points indicate averages of 2–3 replicates; * p < 0.05 and ** p < 0.005.