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. 2017 Jul;19(7):867-880.
doi: 10.1016/j.jcyt.2017.04.001. Epub 2017 May 11.

Monocyte lineage-derived IL-6 does not affect chimeric antigen receptor T-cell function

Nathan Singh  1 , Ted J Hofmann  2 , Zachary Gershenson  3 , Bruce L Levine  4 , Stephan A Grupp  5 , David T Teachey  2 , David M Barrett  2
Affiliations

Monocyte lineage-derived IL-6 does not affect chimeric antigen receptor T-cell function

Nathan Singh et al. Cytotherapy. 2017 Jul.

Abstract

Background aims: Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has demonstrated remarkable success in targeting B-cell malignancies but is often complicated by serious systemic toxicity in the form of cytokine release syndrome (CRS). CRS symptoms are primarily mediated by interleukin 6 (IL-6), and clinical management has focused on inhibition of IL-6 signaling. The cellular source and function of IL-6 in CRS remain unknown.

Methods: Using co-culture assays and data from patients on our clinical CAR T-cell trials, we investigated the cellular source of IL-6, as well as other CRS-associated cytokines, during CAR T-cell activation. We also explored the effect that IL-6 has on T-cell function.

Results: We demonstrated that IL-6 is secreted by monocyte-lineage cells in response to CAR T-cell activation in a contact-independent mechanism upon T-cell engagement of target leukemia. We observed that the presence of antigen-presenting cell-derived IL-6 has no impact on CAR T-cell transcriptional profiles or cytotoxicity. Finally, we confirm that CAR T cells do not secrete IL-6 in vivo during clinical CRS.

Discussion: These findings suggest that IL-6 blockade will not affect CD19 CAR T-cell-driven anti-leukemic cytotoxicity, permitting enhanced control of CRS while maintaining CAR T-cell efficacy.

Keywords: chimeric antigen receptor; cytokine release syndrome; interleukin 6.

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Figures

Figure 1.
Figure 1.
Serum cytokine concentrations in xenograft mice bearing primary pediatric ALL treated with CD19 CART cells. NSG mice were given 106 primary ALL cells and 5 × 106 autologous CD19 CART cells 7 days later. Serum was collected 3 days after T-cell delivery, and a subgroup of animals was given tocilizumab on days 1 and 3 after T cells. Human cytokine concentrations are measured in picograms per milliliter.
Figure 2.
Figure 2.
Cytokine expression after cellular co-culture. T cells, targets and APCs were combined at a ratio of 10:50:1, respectively. Supernatants were collected after 18 h of co-culture. Cytokine levels are measured in picograms per milliliter. *Statistical difference across the group; P < 0.05. **Statistical difference between the two groups; P < 0.05.
Figure 3.
Figure 3.
Cytokine secretion from co-culture experiments combining monocyte-lineage cells with T cells and targets. Monocyte-lineage cells were differentiated in vitro, and T cells, targets and APCs were combined at a ratio of 10:50:1, respectively. Supernatants were collected at 18 and 48 h and analyzed for cytokine concentrations, measured in picograms per milliliter.
Figure 4.
Figure 4.
Transcriptional analysis of isolated cell populations. T cells and targets were isolated from APCs using trans-well inserts and co-cultured for 18 h. Six hundred and ninety-seven RNA transcripts were quantified from each cell population, and log counts of each are displayed. Transcriptional profiles of (A) CD19 CART cells when combined with targets and when combined with targets and pooled monocytes, (B) APCs when combined with targets and with targets and untargeted T cells and (C) APCs when combined with targets and untargeted T cells, and when combined with targets and targeted T cells are presented as linear regression plots.
Figure 5.
Figure 5.
Transcript profile of activated CD19 CART cells and monocyte-lineage APCs. Cells were harvested from trans-well co-culture of CD19 CART cells, Nalm-6 leukemia and pooled monocytes after 18 h. Transcript counts from T cells are displayed in blue and counts from APCs in red. Full quantification of each transcript is presented in Supplementary Table S1.
Figure 6.
Figure 6.
T-cell degranulation in the presence of APCs. T cells expressing (A) no CAR molecule, (B) GD2-targeted CAR or (C) CD19-targeted CAR were combined with CD19+ target ALL cell line Nalm-6. Degranulation was measured by quantification of CD107a surface expression.
Figure 7.
Figure 7.
Transcriptional profile of peripheral blood mononuclear cells collected from patients with ALL treated with CD19 CART cells. Peripheral blood was collected on first day of fever after engineered T-cell infusion. Seven patients had T cells detectable in peripheral blood with no detectable ALL, and three patients had only ALL cells and no detectable T cells.
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