Monocyte lineage–derived IL-6 does not affect chimeric antigen receptor T-cell function
Introduction
The primary toxicity associated with highly active cellular therapy using CD19 chimeric antigen receptor (CD19 CAR) T cells is the hyper-inflammatory state known as cytokine release syndrome (CRS) [1]. This toxicity is characterized by clinical symptoms ranging from a mild influenza-like syndrome to extreme elevations in core body temperature and life-threatening multi-organ failure. In our original report of CD19 CAR T cells for acute lymphoblastic leukemia (ALL), we described significant elevations in several serum cytokines, most notably interleukin (IL)-2, IL-6 and interferon-gamma (INF-γ) in patients with CRS [1]. The first patient treated in our phase 1 study experienced life-threatening toxicity in the form of distributive shock requiring multiple vasopressors and respiratory failure requiring prolonged mechanical ventilation. Administration of the anti–IL-6 receptor agent tocilizumab several days into this critical illness resulted in prompt hemodynamic stabilization, pointing to the central role of IL-6 in causing these symptoms. Other groups have observed a similar IL-6–driven syndrome with CD19 CAR T cells, and treatment with anti-IL-6 therapy has become standard practice for the management of severe CRS [2], [3]. Use of this agent has been cautious, however, as we have a limited understanding of the cellular source of IL-6 during CRS, whether IL-6 is secreted by CAR T cells themselves as a means of homeostatic support in a rapidly dividing cell population, or if IL-6 promotes CAR T-cell efficacy. Several cellular sources of IL-6 have been identified, including macrophages, dendritic cells and B and T lymphocytes [4], [5], [6], [7]. T cells have been identified as the primary source of pathologic IL-6 in models of multiple sclerosis [7], and T-cell–derived IL-6 has been implicated in mediating a positive feedback loop driving TH17 cell differentiation [8]. Innate cells are often the primary sources of IL-6; however, these studies permit that T cells themselves may be the source of IL-6 observed in CRS. Although classical T-cell activation in response to infection and auto-antigens has been studied for decades, the effect of CAR-mediated T-cell activation is poorly understood, and CAR-driven activation may generate distinct cytokine support needs and have a distinct effect on T-cell–mediated IL-6 production. In the absence of a deeper understanding of the role of IL-6 in CAR T-cell function and in CRS, balancing the management of severe toxicity with optimization of anti-tumor activity has been driven by empirical trial and error.
A recent report from our group has provided initial insight into the physiology of CRS. In the first prospective examination of a large panel of serum cytokines in pediatric and adult patients receiving CD19 CAR T-cell therapy for ALL, we observed that elevations in IFN-γ, IL-6, IL-8, soluble IL-2 receptor-α (sIL-2Rα), soluble IL-6 receptor (sIL-6R), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1β) and granulocyte-macrophage colony stimulating factor (GM-CSF) were associated with the development of severe CRS [9]. Early elevations in IFN-γ, serum glycoprotein 130 (sgp130), a component of sIL-6R, and sIL-1RA were predictive markers of development of severe CRS. Both T-cell expansion and baseline disease burden have been speculated to be primary determinants of severity of CRS; however, expansion itself was not associated with development of CRS. Similarly, disease burden alone did not provide any further predictive modeling over serum cytokine levels. Examination of all patients who received tocilizumab therapy upon manifestation of severe CRS demonstrated a consistent and rapid resolution of toxicity after administration, with discontinuation of vasopressors within 24–36 h, confirming the clinical significance of IL-6 in mediating toxicity.
The immunologic cascade that results from CAR-mediated T-cell activation, as opposed to native T-cell receptor (TCR)-mediated activation, and the resulting cellular events that lead to the biochemical derangements of CRS have clear clinical relevance; two adult patients treated at the University of Pennsylvania have died while experiencing CRS, and many more have experienced significant morbidity. Other institutions have experienced similar morbidity and mortality as a result of CRS [3], [10]. The study discussed above [9] provided a detailed cytokine profile of patients who experienced CRS, and we observed that cytokine dynamics in CRS are almost identical to those in hemotophagocytic lymphohistiocytosis (HLH). This inflammatory syndrome is driven by macrophage activation, suggesting that CAR T cells are unlikely to be lone actors in CRS, and other immune cells may be key players. Our clinical understanding thus far has hinged on elevations in IL-6, and although IL-6 seems to drive clinical symptoms, it is clear that it is one of several cytokines that becomes up-regulated during CAR T-cell activation in vivo, suggesting that a network of cytokine signaling contributes to CRS.
To further elucidate the cellular drivers of CRS, we isolated antigen-presenting cells (APCs) derived from the monocyte lineage and performed in vivo and in vitro co-culture experiments to identify the cellular events leading to the cytokine elevations associated with this clinical syndrome. Here, we observe that although T cells alone are sufficient for the production of some CRS-associated cytokines, both activated T cells and APCs are necessary for production of IL-6, and this dependence is not reliant on cell-to-cell contact. We further identify that monocyte-derived cells are responsible for IL-6 secretion that results in response to CAR-mediated T-cell activation. Finally, we demonstrate that CAR-activated T-cell function is not affected by the presence of IL-6-secreting APCs and confirm that CAR T cells from patients experiencing CRS do not produce IL-6. These details of the CRS cascade provide not only a deeper immunologic understanding of this syndrome but also further opportunity for safe management of CRS without compromising CAR T-cell efficacy.
Section snippets
Xenograft studies and patient samples
Six to 10-week old NOD-SCID-γc−/− (NSG) mice were obtained from the Jackson Laboratory or bred in-house under an approved Institutional Animal Care and Use Committee protocol and maintained in pathogen-free conditions. Patient leukemia and T cells were obtained under a Children's Hospital of Philadelphia Institutional Review Board–approved protocol (CHP959 and CHP784, respectively). T-cell engineering for this study has been described previously [1]. Animals were given 106 primary human ALL
Combining CD19 CAR T cells and targets fails to mimic clinically observed CRS in xenograft mice
To evaluate the role of CAR-activated T cells in CRS, we created a patient-derived xenograft model of an aggressive and multiply refractory pediatric ALL. The malignant cells used to establish this xenograft were derived from the first patient with ALL treated at our institution (patient 1 or CHP959-100) [1]. As reported, this patient experienced grade 4 toxicity, including need for prolonged vasopressor support and mechanical ventilation. Clinical CRS was accompanied by a significant elevation
Discussion
Although engineered T-cell therapies have been tested in clinical trials for 2 decades, only the past 5 years have witnessed significant efficacy of this therapy [15], [16], [17], [18]. In a classic example of an unexpected toxicity of a first-in-human therapy, highly active CD19 CAR T-cell therapy has been complicated by the development of CRS in a significant percentage of patients treated across several institutions, with 25–30% requiring intensive care unit support. The clinical description
Acknowledgments
We thank A. Seif and H. Bassiri for review of this manuscript, and J. Perazelli, J. Storm, L. Winestone and B. Moghimi for their technical assistance.
This work was supported in part by grants from Stand Up To Cancer–St. Baldrick's Pediatric Dream Team Translational Research grant (SU2C-AACR-DT1113) and Pennsylvania Department of Health (to SAG), as well as the St. Baldrick's Foundation and Gabrielle's Angel Foundation for Cancer Research (to DMB). Additional funds provided by the First Annual
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