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. 2018 Apr 7;26(6):1423–1434. doi: 10.1016/j.ymthe.2018.04.006

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice

Christopher A Pennell 1,∗, Jessie L Barnum 2, Cameron S McDonald-Hyman 2, Angela Panoskaltsis-Mortari 2, Megan J Riddle 3, Zhengming Xiong 4, Michael Loschi 2, Govindarajan Thangavelu 2, Heather M Campbell 1, Meghan D Storlie 1, Yosef Refaeli 5, Scott N Furlan 6, Michael C Jensen 6, Leslie S Kean 6, Jeffrey S Miller 7, Jakub Tolar 2,3, Mark J Osborn 2, Bruce R Blazar 2
PMCID: PMC5986973  PMID: 29735365

Abstract

The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.

Keywords: B cell aplasia, CAR T cell, CD19, cytokine release syndrome, mouse model, neurologic adverse events, toxicity

Pennell et al. describe a human CD19 transgenic mouse model that mirrors the tumor efficacy and morbidities associated with CAR T cell therapy for human B cell malignancies. This model should allow approaches designed to reduce toxicity while preserving anti-tumor effects to be explored prior to testing in the clinic.

Introduction

CD19-specific chimeric antigen receptor (CAR) T cell therapy is revolutionizing the treatment of B cell malignancies.1 Up to 90% remission rates have been reported in patients with relapsed/refractory B cell acute lymphoblastic leukemia following adoptive transfer of autologous T cells genetically engineered to express CD19-specific CARs.2, 3 Other trials confirm the efficacy of CAR-T-cell-based cancer therapies, particularly for hematologic malignancies.4, 5, 6, 7, 8, 9, 10 Collectively, these studies suggest durable remissions induced by CAR T cells reflect the cells’ specificity, potency, and persistence.

CD19-specific CAR T cell therapy for B cell malignancies also has limitations. The most immediate are life-threatening acute toxicities followed by immunodeficiency due to B cell aplasia.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 Chronic B cell aplasia can result from the continued depletion of healthy CD19+ B cells by persistent CAR T cells, necessitating long-term immunoglobulin replacement therapy. Acute toxicity is thought to result from the systemic activation of CAR T cells following engagement of CD19+ malignant (on target/on tumor) and healthy (on target/off tumor) cells. Such T cell activation often produces a cytokine release syndrome (CRS), which can range from mild and self-limited to severe and fatal if left untreated. CRS is characterized by high fever, hypotension, edema, myalgia, and respiratory distress and appears related to the action of proinflammatory cytokines, including interleukin-6 (IL-6), interferon γ (IFN-γ), and tumor necrosis factor alpha (TNF-α).14, 15 Indeed, a common management option for anti-CD19-CAR-T-cell-induced CRS is treatment with the IL-6R antagonist tocilizumab.16, 17, 18 CRS in anti-CD19-CAR-T-cell-treated patients is often accompanied by neurologic adverse events (AEs), which, in rare cases, are fatal.3, 9, 10, 13, 19, 20, 21 Emerging data suggest activation of endothelial cells in the CNS leads to increased blood-brain barrier (BBB) permeability and subsequent high levels of proinflammatory cytokines, vascular disruption, and neurologic AEs.13 However, the exact pathogenesis of AEs, and whether it is causally related to CRS, remain to be determined.

To obtain mechanistic insights into the biology of CRS and AEs, and as a foundation for future interventions designed to minimize CD19-specific CAR T cell toxicities while preserving anti-tumor efficacy, we sought to develop an animal model that combined anti-human CD19 (hCD19)-CAR-T-cell-induced morbidities and tumor-induced mortality. We reasoned such a model minimally required healthy and malignant B-cell-specific expression of hCD19 at sufficient levels to drive CAR T cell responses and murine T cells (CART-19 T cells) that expressed a clinically validated hCD19-specific CAR construct.22, 23 This combination would permit us to study CART-19-T-cell-induced on-target/off-tumor toxicity and on-target/on tumor-efficacy. Zhou et al.24 developed and characterized C57BL/6 (B6) mice that expressed a hCD19 transgene (hCD19TG) exclusively on B cells. We hypothesized adoptive transfer of CART-19 T cells into hCD19TG mice would cause morbidities similar to those associated with the clinical use of hCD19-specific CAR T cell therapy. To test this hypothesis, we considered the optimal level of hCD19 expression and B cell frequencies that might best simulate the patient setting. We selected for study hCD19TG mice that were hemizygous for hCD19 expression, overcoming the low B cell frequency and correspondingly lower antigen load in hCD19TG+/+ mice and matching more closely hCD19 antigen density on healthy and malignant B cells.24 hCD19TG+/− mice with or without hCD19-transduced murine B-lymphoma cells were given congenic T cells transduced with a clinically validated hCD19-specific CAR.22, 23 We now report these recipients experience morbidities and mortality similar to those occurring clinically.

Results

Choice of hCD19TG Hemizygous Recipients

Rather than using homozygous hCD19TG mice, we generated hCD19TG hemizygous mice as recipients of CART-19 T cells because hCD19TG expression decreases mature B cell frequency in a dose-dependent manner.24 Relative to B6 wild-type (WT) mice, peripheral blood B cell frequencies were ∼50% and ∼75% reduced in hCD19TG hemizygous and homozygous mice, respectively (Figures 1A and 1B). As expected, peripheral blood B cells from B6 WT mice did not express hCD19 (Figure 1C). CD45R (B220) expression did not differ significantly between mature B cells from WT and hCD19TG+/− mice, although it was significantly lower on hCD19TG+/+ B cells -(Figure 1D). Cell surface expression of endogenous mouse CD19 (mCD19) and hCD19TG were inversely related (Figures 1E and 1F), perhaps due to competition for transcription factors or cofactors required for CD19 cell surface expression.25, 26, 27 Consistent with gene dosage, hCD19 expression on mature B cells in hemizygous mice was half that of the homozygotes (Figure 1F). As expected, hCD19 tissue expression in the hemizygotes was the same as reported in the homozygotes,24 in that it was restricted to B cells (as evidenced by the lack of detectable hCD19+CD45R− cells in all organs examined, including brain, duodenum, ileum, and colon [ascending and descending]; C.A.P., unpublished data). Together, the higher frequency of peripheral B cells and expression of WT B cell levels of CD45R led to the choice of hCD19TG+/− mice as CART-19 T cell recipients.

Figure 1.

Figure 1

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Frequencies of Peripheral Blood B Cells and hCD19TG Expression

Blood was collected from 6- to 16-week-old mice of the indicated hCD19TG genotypes. After incubating with fluorophore-conjugated mAbs specific for human or mouse CD19 (hCD19 or mCD19, respectively) or CD45R, leukocytes were analyzed by flow cytometry. Lymphocytes were gated on their forward and side scatter profiles and then gated for single cells based on forward scatter height by width. Top row: the y axes display percentages of lymphocytes expressing (A) CD45R, (B) mCD19, or (C) hCD19 from mice with the hCD19TG genotypes shown on the x axes. Bottom row: the y axes represent the median fluorescence intensities (MFIs) of (D) CD45R, (E) mCD19, or (F) hCD19 expression on peripheral blood lymphocytes from mice with the hCD19TG genotypes shown on the x axes. Each symbol represents an individual mouse. The means ± SEM for each population are indicated. **p < 0.01; ***p < 0.001; ns (not significant); one-way ANOVA with Tukey’s multiple comparison test.

CAR Retroviral Construct and Generation of Mouse CART-19 T Cells

CART-19 T cells were generated with a construct (Figure S1) used in clinical trials; autologous T cells genetically modified with this CAR construct design are currently being evaluated in a phase 1/2 clinical study of CD19+ relapsed/refractory leukemia (ClinicalTrials.gov Identifier NCT02028455).22, 23 The construct encodes a single-chain Fv (sFv) derived from the hCD19-specific mouse monoclonal antibody (mAb) FMC63 joined via hCD8α-derived hinge and transmembrane regions to a cytoplasmic signaling module. The signaling module encodes a membrane proximal hCD137 (4-1BB) costimulatory domain upstream of an hCD3ζ domain, followed by a gene encoding the viral T2A peptide to permit “ribosomal skipping” and translation of truncated, membrane-bound human epidermal growth factor receptor (hEGFR) as a separate protein.23, 28 The truncated hEGFR is functionally inert and serves as a selection and tracking marker as well as a target for depletion of transduced cells with cetuximab, if indicated.29 Controls included T cells transduced with just the hEGFR portion of the CAR construct (hEGFR T cells) or GFP (GFP T cells).

Transduction efficiencies depended primarily on titer and storage duration of the retroviral preparation and ranged from 26% to 90% with a median of 71% (Figure S2). To model most CAR clinical trials, transduced cells were not enriched prior to use but were normalized for transduction efficiencies. Accordingly, mice in different experiments receiving the same number of transduced cells typically received different numbers of total cells due to variability in transduction efficiencies. This variability did not measurably affect function on a per CART-19 T cell basis (see below).

CART-19 T Cells Are Activated by hCD19TG+/− B Cells In Vitro

To determine whether B cells from hCD19TG+/− mice could activate CART-19 T cells, CART-19 T cells were cultured overnight without the anti-CD3/-CD28 mAb-coated beads and exogenous IL-2 added for T cell activation and expansion in vitro. These CART-19 T cells were then cocultured for 36 hr with freshly isolated hCD19TG+/− splenic B cells at 1:10 ratios, respectively, before assessing CART-19 T cell activation phenotypically and functionally. In all cases, the same numbers of transduced T cells were compared. CART-19 T cells cocultured with hCD19TG+/− B cells expressed significantly higher levels of CD25, CD44, CD69, CD107a, and CD279 (PD-1) than controls, consistent with antigen-specific activation (Figure 2A). CD62L and CD197 (CCR7) were both significantly elevated on CART-19 T cells incubated with hCD19TG+/− B cells, although to lower levels relative to the other measured activation markers. Enhanced expression of CD62L and CD197 was unexpected because their levels typically drop upon T cell activation.30, 31 This may be a consequence of the 4-1BB signaling domain in the CAR, as 4-1BB ligation can drive the formation of central memory-like cells.32

Figure 2.

Figure 2

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Activation of CART-19 T Cells by hCD19TG+/− B Cells

(A) CART-19 T cells or control hEGFR T cells were cocultured with B cells at 1:10 (T:B) ratios for 36 hr and then analyzed by flow cytometry for the indicated CD markers. CD expression levels were measured by MFI. (B) Supernatants were harvested from each culture in (A) to measure soluble cytokines using a cytometric bead array. The black solid lines indicate the limits of detection. (C) CART-19 T cells or control hEGFR T cells were cultured for 4 hr with 50,000 B cells from hCD19TG+/− or B6 (−/−) mice at the effector:target (T:B) ratios indicated on the x axis. The numbers of viable B cells were then quantitated by flow cytometry. (D–F) 5E+06 CART-19 or hEGFR CD45.1+ T cells were injected intravenously (i.v.) into hCD19TG+/− recipients treated the day before with 300 mg/kg CY. Three days after cell transfer, splenocytes were harvested, reacted with fluorophore-conjugated mAbs, and analyzed by flow cytometry. Lymphocytes were gated on their forward and side scatter characteristics and then gated for single cells based on forward scatter height by width. These data are representative of 2 or 3 experiments. (D) Expression levels of the indicated CD antigens on CD8α+hEGFR+ gated lymphocytes were measured by MFI. (E) The absolute numbers of transduced cells (CD45.1+hEGFR+) per spleen were quantitated by flow cytometry. (F) The absolute numbers of B cells per spleen were quantitated by flow cytometry. (G) Unmanipulated hCD19TG+/− mice and 300 mg/kg CY-pretreated hCD19TG+/− recipients of 1E+06 hEGFR or CART-19 T cells were bled 55 days post-ACT, and the frequencies of peripheral blood B cell lymphocytes (mCD19+/mCD45+) were determined by flow cytometry. All data are presented as the mean ± SEM (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; ns (not significant); two-tailed unpaired t test (D), Mann Whitney U test (E and F) or one-way ANOVA with Tukey’s multiple comparison test.

Cytokine production was used as a functional measure of antigen-specific CART-19 T cell activation. Soluble IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α from the cocultures described above were quantified by flow cytometry using cytokine bead arrays. Compared to controls, CART-19 T cells cocultured with hCD19TG+/− B cells produced significant amounts of IL-2, IL-4, IL-6, IFN-γ, and TNF-α; IL-10 and IL-17A were not detectable (Figure 2B). This cytokine pattern is consistent with antigen-specific T effector cell activation.33, 34

To measure CART-19 T cell cytolytic function, varying numbers of transduced cells were cultured with fixed numbers of B cell targets; absolute numbers of viable B cells were determined 4 hr later. As expected, CART-19 T cells lysed hCD19TG+/− B cells at significantly higher levels than control hEGFR T cells; lysis of B6 WT B cells was not significantly different between CART-19 or hEGFR-T effectors (Figure 2C). Together, the enhanced expression of activation markers and cytokines, and specific cytolysis of hCD19TG+/− B cells, argue for hCD19-specific activation of mouse CART-19 T cells.

CART-19 T Cells Are Activated following Transfer into hCD19TG+/− Recipients

To determine whether CART-19 T cells differentiated and proliferated in vivo upon antigen stimulation, 5E+06 CART-19 T cells were adoptively transferred into hCD19TG+/− mice. Recipients were given a lymphodepleting dose (300 mg/kg) of cyclophosphamide (CY) prior to adoptive cell transfer (ACT) to enhance CART-19 T cell engraftment; this approach models current CAR clinical trials, which employ lymphodepletion to maximize the therapeutic effect of adoptively transferred T cells.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 17, 35 Because clinical data suggested T effector cells infused within two days of conditioning were more efficacious than T cells infused at later time points, recipients received CY the day before ACT.35, 36 Transduced cells were recovered from spleens three days after cell transfer. As compared to control hEGFR T cells, CART-19 T cells expressed significantly higher levels of the activation markers CD44, CD107a, and CD279 (PD-1), but not CD69 (Figure 2D). CART-19 T cells also expanded significantly in hCD19TG+/ recipients; mean splenic CART-19 T cell numbers were 130-fold higher than the control splenic hEGFR T cells (Figure 2E). This corresponded with an almost complete loss of splenic B cells in the CART-19 T cell recipients; the mean splenic B cell frequency in CART-19 cell recipients was >1,000-fold lower than that of hEGFR T cell controls (Figure 2F).

To assess longer-term B cell aplasia, peripheral blood B cell frequencies in hCD19TG+/− mice were measured 55 days after ACT. Peripheral blood B cell frequencies in hEGFR T cell recipients (18.4% ± 4.1%) were not significantly different from unmanipulated hCD19TG+/ mice (24.8% ± 1.6%), but the B cell frequencies in the CART-19 T cell recipients (1.9% ± 0.6%) were significantly reduced (Figure 2G). CART-19 T cell frequencies in peripheral blood were not significantly elevated (not shown) as compared to hEGFR T cell frequencies in respective ACT recipients on day 55, suggesting the CART-19 T cells mediating B cell depletion persisted in other organs (e.g., bone marrow).

CART-19 T Cells Eliminate Malignant hCD19+ B Cells In Vivo

To assess on-target/on-tumor activity, 1E+06 CART-19 T cells were adoptively transferred into hCD19TG+/− mice bearing GFP+ TBL12 lymphoma cells that we modified to express hCD19 and luciferase (TBL12.hCD19; Figure S3).37 hCD19TG+/− mice were injected intraperitoneally (i.p.) with 1E+06 TBL12.hCD19 cells followed 8 days later by 300 mg/kg of CY (to model the clinical situation in which tumor burden is reduced and patients are lymphodepleted to facilitate CAR T cell engraftment).35, 36 Without CY, this tumor cell dose was 100% lethal within 11–13 days (not shown). With CY alone or in combination with 1E+06 control hEGFR T cells, tumor burden was reduced >1,000-fold (Figure 3A), but eventually all mice succumbed to tumor within a month (Figure 3B). In contrast, treatment with CY followed 1 day later by 1E+06 CART-19 T cells reduced tumor burden to undetectable levels and resulted in 80% survival at 55 days post-tumor challenge. These data reveal CART-19 T cells are potently cytotoxic and can rescue mice from an otherwise uniformly lethal tumor challenge.

Figure 3.

Figure 3

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CART-19 T Cell Anti-tumor Effects

hCD19TG+/− mice (n = 5 per group) were injected i.p. with 1E+06 TBL12.hCD19 cells on day 0. On day 8, each mouse was injected i.p. with 300 mg/kg CY. On day 9, mice in the indicated groups were injected with 1E+06 (1M) CART-19 or hEGFR T cells or no cells. (A) Tumor growth, as measured by bioluminescence intensity, was determined on the indicated days. (B) Mice were monitored regularly for survival. Values in (A) represent the mean ± SEM per group. *p < 0.05; ***p < 0.001; (A) one-way ANOVA with Tukey’s multiple comparison test or (B) log rank (Mantel-Cox).

CART-19 T Cells Cause Dose- and Antigen-Dependent On-Target/Off-Tumor Toxicity In Vivo

The relatively early death in some hCD19TG+/− recipients given CY and CART-19 T cells led us to speculate that CART-19 T cells may mediate acute on-target/off-tumor toxicity. To test this, non-tumor-bearing hCD19TG+/− mice were treated with CY and the next day adoptively transferred with CART-19 T cells. Two lymphodepleting CY (200 and 300 mg/kg) and two CART-19 T cell (1E+06 and 3E+06) doses were compared to determine whether toxicity was dose dependent. The treated mice were monitored daily for survival, body weight, appearance, and activity. Appearance and activity were recorded as one clinical score, with summed scores of 0 and 8 indicating healthy and moribund mice, respectively.38

CART-19 T cells caused dose-dependent acute toxicity and death in hCD19TG+/− recipients. These effects correlated with both the number of transferred CART-19 T cells and the CY dose. A dose of 3E+06 CART-19 T cells in hCD19TG+/− recipients pretreated with 300 mg/kg CY caused rapid and significant weight loss with a mean clinical score of 8 on a 0–8 scale as early as day 5 and uniform mortality typically 5–8 days after transfer; a three-fold lower CART-19 T cell dose caused slower weight loss, peak clinical scores exceeding 7, and a 40% death rate (Figures 4A–4C). In hCD19TG+/− recipients pretreated with 200 mg/kg CY, the effects of 3E+06 and 1E+06 CART-19 T cells were slightly less severe and acute, with peak clinical scores of ∼7 on day 5 and day 7, along with 80% and 20% mortality, respectively (Figures 4D–4F).

Figure 4.

Figure 4

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CART-19 T-Cell-Induced Toxicity and Lethality in hCD19TG+/− Recipients

hCD19TG+/− mice were injected i.p. with either 300 (A–C and G) or 200 (D–F) mg/kg of CY. 24 hr later (day 0), mice were injected i.v. with 1E+06 (1M) or 3E+06 (3M) CART-19 T cells. Controls included uninjected mice (unmanipulated; A–C), mice injected with just 300 mg/kg CY (CY only; A–C), and mice injected with 300 mg/kg CY followed the next day by 3M GFP-T cells. Mice were weighed and scored on the indicated days and euthanized when moribund. These cell and CY dose response results are representative of five experiments. Values represent the mean ± SEM per group (n = 5–8 per group). (G) Six days after adoptive transfer of 3E+06 CART-19 T cells or hEGFR T cells, mice were gavaged with 800 mg/kg FITC-dextran, rested for 4 hr, and then bled. Relative fluorescence values of diluted plasma were obtained. The y axis shows the interpolated concentrations of plasma FITC-dextran whereas the x axis lists the treatment groups (n = 7; CART-19 or 12; hEGFR T and CY only). These data are pooled from two similar experiments. Asterisks denote statistical comparisons at the same time points among the following groups: 1M or 3M CART-19 versus 3M GFP-T (A–C and F); 3M versus 1M CART-19 (D and E). *p < 0.05; **p < 0.01; ***p < 0.001; ns (not significant); (A, B, D, and E) one-way ANOVA with Tukey’s multiple comparison test, (C and F) log rank (Mantel-Cox), or (G) two-way unpaired t test.

The rapid weight loss in hCD19TG+/− recipients following CART-19 T cell transfer indicated systemic manifestations. Because B cells are present in the gastrointestinal track in gut-associated lymphoid tissue and regional lymph nodes, we considered the possibility that CAR-19 T cells caused gastrointestinal track damage by lysis of gut B cells in situ or by activated T cells that triggered a proinflammatory cytokine response causing epithelial cell injury, similar to that seen in donor anti-host T-cell-mediated graft-versus-host disease.39, 40, 41 To determine whether the intestinal epithelial barrier was compromised following CART-19 T cell transfer, hCD19TG+/− mice were pretreated with 300 mg/kg CY and 24 hr later were adoptively transferred with 3E+06 CART-19 or control hEGFR T cells. Six days after ACT, the mice were gavaged with dextran conjugated to fluorescein isothiocyanate (FITC) and bled 4 hr later. There were significantly higher levels (11-fold) of FITC in the plasma of the CART-19 cell recipients relative to the hEGFR T cell control recipients (Figure 4G). Consistent with the loss of epithelial cell integrity in CY-treated recipients of CART-19, but not control transduced T cells, histopathological analysis of the colon on day 2 showed readily detectable ulcerations and lymphocyte infiltrates (Figure S4).

hCD19TG+/− mice treated with high cell doses (≥3E+06) of CART-19 T cells often displayed behavioral anomalies suggestive of neurologic AEs. These anomalies manifested as movement disorders, such as ataxia (unpublished data). To gain insight into the biological nature of these behaviors, hCD19TG+/− mice were pretreated with 300 mg/kg CY and 24 hr later were adoptively transferred with 3E+06 CART-19 or hEGFR T cells. Five days after ACT, the mice were sacrificed and their brains removed, flash frozen, and sectioned. The sections were reacted with a mAb specific for the microglial cell marker Tmem119 and the DNA dye DAPI.42 Three fields per brain were imaged, and the frequencies of Tmem119+ cells were determined as percentages of DAPI+ cells. Representative images of stained brain sections show fewer Tmem119+DAPI+ cells in recipients of CART-19 versus control hEGFR T cells (Figures 5A and 5B, respectively). Quantification revealed the frequencies of Tmem119+DAPI+ cells were significantly lower in the brains of CART-19-treated mice as compared to control mice (Figure 5E). Allelically marked (CD45.1+) T cells were detectable in the brains of CART-19 T cell recipients, but not in control mice (Figure S5). These findings are consistent with reports of anti-CD19 CAR T cells being present in the CNS of patients following intravenous cell infusion.13

Figure 5.

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Loss of Tmem119+ CNS Cells following CART-19 ACT

(A–D) Tmem119 (A and B; green) or rabbit isotype (C and D; green) and nuclei (DAPI; blue) stained cerebral cortex sections (40× magnification) from hCD19TG+/− mice injected 5 days previously with 3E+06 (A and C) CART-19 or (B and D) hEGFR T cells. (E) Percentage of Tmem119+ cells divided by the number of DAPI+ cells is shown. Each column represents data pooled from 3 sections per mouse and 4 mice per group. *p < 0.05; unpaired t test.

The toxicity and ultimate lethality caused by CART-19 T cells in hCD19TG+/− recipients were antigen specific. hCD19TG+/− and B6 WT mice were treated with CY (300 mg/kg) followed the next day by CART-19 T cells (5E+06). These doses were lethal to hCD19TG+/− recipients, as all such treated mice died within 7 days post-ACT (Figures 6A–6C). In contrast, B6 WT recipients were unaffected as compared to the B6 WT CY-treated controls (Figures 6D–6F). These data, combined with the lack of in vitro cytotoxicity of CART-19 T cells toward B6 WT B cells (Figure 2C), show that CART-19 T-cell-mediated cell death is antigen specific.

Figure 6.

Figure 6

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Antigen-Specific Toxicity of CART-19 Cells

(A–C) With the exception of the unmanipulated controls, all hCD19TG+/− mice were injected i.p. with 300 mg/kg of CY. These mice were injected i.v. with 5E+06 CART-19 T or GFP T cells 24 hr later; mice in the CY only group were not adoptively transferred. (D and E) Wild-type B6 mice were injected i.p. with 300 mg/kg of CY. 24 hr later, mice in the CART-19 T cell group received i.v. injections of 5E+06 CART-19 T cells from the same lots injected into the hCD19TG+/− recipients (A–C). Mice were weighed and scored on the indicated days and euthanized when moribund. Values represent the mean ± SEM per group (n = 5 per group). The results shown in (A)–(C) are representative of over 10 experiments; the results shown in (D)–(F) are representative of three experiments. Asterisks denote statistical comparisons at the same time points between the CART-19 and GFP T groups. *p < 0.05; ***p < 0.001; (A and B) one-way ANOVA with Tukey’s multiple comparison test or (C) log rank (Mantel-Cox).

Transcriptomic Analyses Demonstrate Upregulation of Innate and Adaptive Immune Responses

To obtain mechanistic insights into acute toxicity, NanoString transcriptomic analyses were performed on colons and spleens of hCD19TG+/− mice treated with CY (300 mg/kg) the day before ACT of 3E+06 CART-19 or hEGFR T cells (accession numbers for the transcriptomic data are provided in Table S1). Principal-component analysis (Figure 7A) documented global differences in transcript expression in both organs on days 2 and 5 post-ACT, influenced by CART-19 T cells. To determine the immune pathways modulated after CART-19 infusion, pathway analysis was performed using the “HALLMARK” gene sets from the Molecular Signatures Database.43, 44, 45 Pathways associated with both innate (e.g., IL-6, IFN-α, complement, and inflammatory response) and adaptive (e.g., IFN-γ and allograft rejection) immunity were upregulated in the colon and spleen (Figure 7B). These data document significant immune activation, specific for the CART-19 recipients, including the upregulation of IL-6 signatures previously observed in patients receiving CAR T cell therapy.3, 8, 14, 16 The most significant changes in immune-response gene expression occur in the spleen on day 2, followed by the colon on day 2 and spleen on day 5 post-ACT.

Figure 7.

Figure 7

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Transcriptomic Analyses of Affected Tissues and IL-6 Neutralization

hCD19TG+/− mice were adoptively transferred with 3M CART-19 or hEGFR T cells and RNA was isolated from spleens and colons and interrogated by NanoString transcriptomic analyses. (A) Principal-component analysis reveals differences in transcript count variance between animal cohorts in both spleen and colon. The variances explained by PC1 and PC2 are 44.6% and 18.4%, respectively. (B) Pathway analysis shows IL-6-, IFN-α- and IFN-γ-related signatures are overrepresented in differentially expressed transcripts. (C) 4 hr and 24 hr after adoptive transfer of 3E+06 transduced T cells, recipients were injected intraperitoneally with 400 μg of the indicated antibody. Weight losses relative to pretreatment weights are shown on the y axis. Values represent the mean ± SEM per group (n = 5 per group). *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA with Tukey’s multiple comparison test.

Neutralizing IL-6 Blunts CART-19 T-Cell-Induced Weight Loss

We sought to determine whether antagonizing IL-6 signaling blunted CART-19 T-cell-induced toxicity in our model, as in patients treated with CD19-specific CAR T cells.3, 8, 14, 16 hCD19TG+/− recipients were treated with 300 mg/kg CY one day prior to ACT of 3.0E+06 CART-19 T cells or control hEGFR-T cells. Four hours before and 24 hr after ACT, recipients were injected i.p. with 400 μg of control or cytokine-neutralizing mAbs. The IL-6-specific mAb significantly prevented CAR-19 T-cell-induced weight loss early (day 2) and later (day 6) after infusion (Figure 7C). The IFN-γ-specific mAb significantly blunted weight loss early (day 2), but this effect waned by day 6, consistent with the early (day 2) IFN-γ-response gene signatures in the colons and spleens of CART-19 recipients. Taken together, these data point to tissue injury induced by IL-6 and IFN-γ proinflammatory cytokines.

Discussion

This preclinical model using anti-hCD19-CAR mouse T cells, hCD19+ mouse B lymphoma cells, and hCD19 hemizygous transgenic recipients replicates the clinical advantages and limitations of hCD19-specific CAR T cell therapy: tumor eradication; systemic toxicity; and B cell aplasia. Upon adoptive transfer into hCD19TG+/− recipients, CART-19 T cells differentiate into cytolytic effectors that reduce splenic B cell frequencies >1,000-fold in three days, replicating clinical B cell aplasia. This is accompanied by acute toxicity manifested by significant weight loss, worsening clinical scores, neurologic AEs, and death of the recipients within a week if the CART-19 T cell dose equals or exceeds three million.

The toxicity cascade initiated by CART-19 cells in hCD19TG+/− mice is driven, at least in part, by the proinflammatory cytokines IFN-γ and IL-6. This is most evident in the early post-ACT period based on the transcriptomic analyses of colons and spleens from hCD19TG+/− recipients two days post-CART-19 T cell transfer and the transient prevention of weight loss by an IFN-γ-neutralizing mAb. Like IFN-γ, mAb-mediated neutralization of IL-6 also significantly blunts weight loss. Unlike IFN-γ, however, IL-6 neutralization has a more durable effect, as weight loss is still significantly blunted six days after ACT.

Although CART-19 T cells secrete IL-6 in vitro when cultured with B cells from hCD19TG+/− mice, it is not yet clear whether IL-6 derived from CART-19 cells drives pathology in vivo in our model. Indeed, emerging data suggest the source of IL-6 that drives CRS is not CAR T cells but rather myeloid cells or vascular endothelial cells or both. In a preclinical mouse model of CRS induced by Erb-B-specific human CAR T cells, macrophage depletion prior to ACT ameliorates both toxicity and IL-6 release, implicating macrophages, possibly following CAR T-cell-derived IFN-γ-induced activation, as the pathogenic IL-6 source.46 In vitro coculture assays of patient-derived CAR T cells, leukemic cells, and peripheral blood mononuclear cells demonstrate that IL-6 is secreted by monocyte-lineage cells in a contact-independent mechanism following CAR T cell activation by leukemic cells; additional data show CAR T cells do not secrete IL-6 in vivo during clinical CRS.47 These observations are all consistent with the significant elevation we find in the IL-6/JAK/STAT3 signaling pathway in macrophage-rich spleens. Another potential source of IL-6 that drives clinical CRS is vascular endothelial cells. Dual RNA in situ hybridization of lymph node sections from a patient who succumbed to CAR T-cell-induced CRS showed IL-6 was not expressed by CD3+ (i.e., CAR T cells) but rather by von Willebrand factor+ vascular endothelial cells and, to a lesser extent, by LYVE1+ lymphatic endothelial cells.48

These data and ours suggest a biphasic process in which IFN-γ, released by CAR T cells upon ligand engagement, induces IL-6 production by non-CART-19 cells; more intense T cell activation with a higher total body B cell mass, made possible by use of hCD19TG+/− recipients, would be predicted to lead to more severe systemic toxicity. This process is in agreement with other preclinical models of T-cell-activation-induced toxicity and with clinical trials in which patients experiencing CRS have elevated serum levels of IFN-γ and IL-6.2, 46, 49, 50 Data from CAR T clinical trials, however, suggest IFN-γ can also drive CRS via IL-6-independent mechanisms, as some CRS patients given the IL-6R antagonist tocilizumab are non-responsive.51 Regardless, our data are consistent with IL-6 overexpression in patients experiencing CRS subsequent to CAR T cell therapy and the corresponding ability to blunt CRS with tocilizumab.3, 8, 14, 16

The gastrointestinal track epithelial injury that occurs in hCD19TG+/− mice following CART-19 ACT may be due to lysis of B cells in gut-associated lymphoid tissue or gut regional lymph nodes. Another possibility is that activated CART-19 cells may lead to bystander, pathogenic T cell responses to gut flora antigens after intestinal injury, similar to those reported in adoptive T cell transfer models of inflammatory bowel disease, amplifying local gut injury and their systemic manifestations.52 Regardless of the underlying cause, loss of epithelial cell integrity may cause death by serving as a portal for systemic circulation of endotoxin and microbes present in the intestine. This is consistent with a report from Brentjens et al.53 that implicated sepsis in the death of a patient with chronic lymphocytic leukemia 40 hr after infusion of CD19-specific CAR T cells. Similarly, Hill et al.12 recently reported that the risk of infection after CD19 CAR T cell therapy was associated with higher doses of CAR T cells and severity of CRS and neurotoxicity. As opposed to fungi and viruses, bacteria were the most common cause of infections in the first 28 days after CAR T cell infusion, with bacteremia occurring in 50% of these cases. Neurotoxicity has also caused fatalities in patients receiving CD19 CAR T cell therapy.13, 17, 18, 53 Gust et al.13 reported that severe neurotoxicity was associated with increased BBB permeability, high concentrations of proinflammatory cytokines (including IFN-γ) in cerebrospinal fluid, and vascular endothelial cell dysfunction.

In our model, neurotoxicity appears to occur in hCD19TG+/− recipients of high-dose (≥3E+06) CART-19 cells, as evidenced by behavioral anomalies and significant decreases in Tmem119+ cells.42, 54 In the CNS, Tmem119 is expressed exclusively on microglial cells, the resident parenchymal macrophage lineage cells, and distinguishes them from blood-derived macrophages. Tmem119 is a transmembrane protein that induces the development of myoblasts into osteoblasts by upregulating expression of the transcription factor ATF4; Tmem119 is also required for spermatogenesis and testicular differentiation.55, 56, 57, 58, 59 However, its function in microglial cells is unknown. Costaining of brain sections for Tmem119 and another microglial cell marker, AIF-1/Iba-1, shows the frequencies of Tmem119+/AIF-1+ cells were reduced in the CART-19 recipients relative to controls (C.A.P., unpublished data). These data suggest that Tmem119 expression is not downregulated but that microglial cells are lost in CART-19 recipients. It is not yet clear whether CNS resident CART-19 cells cause microglial cell loss non-specifically following activation by hCD19+ B cells or whether it is driven by systemic cytokines that cross the blood-brain barrier. Nonetheless, loss of Tmem119-expressing microglial cells following CART-19 ACT most likely disrupts normal CNS homeostasis and function and could contribute to death in severe cases.

Despite these many similarities in tumor efficacy and toxicities between our model and clinical findings, there are differences. For example, serum levels of IL-6, C-reactive protein, and ferritin were not detectably elevated in hCD19TG+/− recipients experiencing weight loss and worsening clinical scores following CART-19 ACT (C.A.P., unpublished data), in contrast to CAR-treated patients experiencing CRS.3, 8, 14, 16 Patients with macrophage activation syndrome (MAS)/hemophagocytic lymphohistiocytosis may also experience CRS, and the pathophysiology of the two conditions may overlap.49 Immunohistochemistry of liver biopsies from children with MAS revealed massive mononuclear cell infiltrates that included numerous IL-6- and TNF-α-expressing macrophages plus IFN-γ-expressing CD8+ lymphocytes.49 In contrast, there were no significant differences in liver pathology between CART-19 and hEGFR T control recipients (C.A.P., unpublished data), consistent with the lack of elevated CRP and ferritin. The reasons for the lack of detectable liver damage in CART-19 T cell recipients are not yet clear. These data, together with the transcriptomic and cytokine neutralization results, suggest IL-6 levels are elevated locally in tissue microenvironments and not systemically.

The immunocompetent animal model we describe employs a construct tested in the clinic and captures both the acute toxicity and B cell aplasia currently limiting CD19-specific CAR T cell therapy for CD19+ malignancies. Other immunocompetent animal models replicate tumor efficacy and B cell aplasia, but not acute toxicity. For example, Brentjens et al.60 reported an immunocompetent B6 mouse model in which B cell aplasia occurred following ACT of syngeneic T cells transduced with a CAR construct encoding a sFv derived from the 1D3 rat anti-mCD19 mAb linked to mCD28/mCD3ζ signaling domains. Although their model provided insight into how a small population of bone marrow progenitor B cells could drive CAR T cell persistence, the model did not replicate the acute toxicities observed clinically nor did it permit the testing of human CD19-specific therapeutic platforms. Similarly, Kochenderfer et al.61 reported a single infusion of B6 CAR T cells expressing a 1D3-derived anti-mCD19 sFv linked to mCD28/mCD3ζ signaling domains into tumor-bearing syngeneic recipients eradicated both B lymphoma and normal B cells; normal B cell aplasia was long lasting, presumably due to CAR T cell persistence.

A model reported by Pegram et al.62 was more similar to ours in that mouse lymphomas transfected with hCD19 were used as tumor targets and CAR T cell recipients were hCD19TG+/− B6 mice; however, these mice lacked endogenous mCD19 and had one hCD19TG copy (versus 9–14 copies in the hCD19TG+/− mice we used). T cells from hCD19TG+/− B6 mice were transduced with a CAR construct encoding a hCD19-specific sFv derived from the SJ25C1 mAb linked to a mCD3ζ signaling domain. Despite eradicating tumors and inducing long-lasting B cell aplasia, these CAR T cells did not induce acute toxicity.62 It is unclear whether the lack of toxicity reflects the lack of endogenous mCD19 or different levels of hCD19TG expression on B cells in the hCD19TG+/−/mCD19−/− mice or differences in the CAR construct relative to our model. Indeed, a key difference between our model and these three is the inclusion of the 4-1BB signaling domain in our CAR construct. The second-generation CAR constructs employed by the Davila and Kochenderfer groups encoded CD3ζ- and CD28-derived signaling domains, whereas the Pegram CAR construct contained just a CD3ζ-derived signaling domain.60, 61, 62 Inclusion of 4-1BB in many CAR constructs prevents T cell exhaustion that results from CAR-mediated tonic signaling and concomitant CD3ζ phosphorylation and also may permit more robust and sustained responses.63 Alternatively, 4-1BB-derived signals may induce a qualitatively or quantitatively different proinflammatory cytokine microenvironment than CD28-derived signals, favoring the toxicity observed in our model.64

Because our preclinical small animal model of hCD19 CAR T cell therapy replicates clinical observations, it should be a highly useful platform for further mechanistic studies exploring toxicities and related B cell aplasia reflective of hCD19-CAR T cell anti-tumor potency. Approaches to reduce toxicity while preserving anti-tumor effects can be readily explored prior to testing in the clinic.

Materials and Methods

Mice

B6, B6.PL-Thy1a/CyJ, and B6.SJL-PtprcaPepcb/BoyJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). Dr. Thomas Tedder (Duke University) kindly provided the B6 TG-1 line that contains 9–14 copies of a bacterial artificial chromosome hCD19 transgene.24 We bred TG-1 mice to WT B6 mice to generate hCD19TG+/− hemizygotes. All mice were housed in a specific pathogen-free facility and used with University of Minnesota Institutional Animal Care and Use Committee approval.

Tumor Model

TBL12 is a B lymphoma from a B6 mouse expressing Eμ-MYC, anti-hen egg lysozyme antibody, and soluble hen egg lysozyme transgenes.37 We generated TBL12.hCD19 cells by retrovirally transducing TBL12 cells with a construct (Figure S4) encoding hCD19, GFP, and luciferase and flow sorting GFP-expressing cells. hCD19TG+/− mice were injected i.p. with 1E+06 TBL12.hCD19 cells 8 days before treatment with 300 mg/kg of CY (Sigma-Aldrich, St. Louis, MO) in PBS. Tumor growth was measured by bioluminescence using an IVIS Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA) 6–8 min after injection of 3 mg D-luciferin potassium salt (Gold Biotechnology, St. Louis, MO) in PBS.

Retroviral Constructs and Transduction of Primary T Cells

The CD19-specific CAR and truncated hEGFR coding regions were subcloned from a lentiviral construct into the MP71 retroviral vector optimized for T cell expression.22, 23, 65 Negatively enriched splenic T cells were retrovirally transduced as described in the Supplemental Materials and Methods.

ACT

One day prior to ACT, randomized male and female recipients were injected i.p. with 100–300 mg/kg body weight of CY. Transduced T cells were washed and adjusted to the desired number prior to tail vein injection. To neutralize cytokines, mice were injected i.p. 4 hr before and 24 hr after ACT with 400 μg of monoclonal rat immunoglobulin G1 (IgG1) specific for mouse IL-6 (MP5-20F3), mouse IFN-γ (RA-6A2), or horseradish peroxidase (isotype control; BE0088; Bio X Cell, West Lebanon, NH).

Toxicity Measurements

Appearance and activity were recorded as one clinical score based upon the most prominent features observed. Scores of 0–2 were assigned to each of four criteria: activity; fur texture; posture; and weight.38 Summed scores of 0 and 8 indicate healthy and moribund mice, respectively. To assess intestinal barrier function, mice were orally gavaged with 800 mg/kg with FITC-dextran (Sigma-Aldrich) and FITC-serum levels measured 4 hr later (see Supplemental Materials and Methods).40 The FITC-dextran assay, in vitro functional assays, flow cytometry, histopathology, RNA isolation, and expression analyses are described in Supplemental Materials and Methods.

Statistical Analyses

All experiments were replicated at least twice. Prism v6.0f (GraphPad Software, La Jolla, CA) was used for all statistical analyses, except for the noted transcriptomic analyses. The statistical tests used for each analysis are provided in the appropriate figure legends.

Author Contributions

Conceptualization, C.A.P., J.S.M., M.J.O., and B.R.B.; Methodology, C.A.P., J.S.M., and B.R.B.; Formal Analysis, C.A.P., A.P.-M., S.N.F., L.S.K., and B.R.B.; Investigation, C.A.P., J.L.B., C.S.M.-H., M.J.R., Z.X., M.L., G.T., H.M.C., and M.D.S.; Resources, C.A.P., Y.R., M.C.J., M.J.O., and B.R.B.; Writing – Original Draft, C.A.P. and B.R.B.; Writing – Reviewing and Editing, all authors; Funding Acquisition, C.A.P., J.S.M., and B.R.B.

Conflicts of Interest

M.C.J. holds a patent on the truncated EGFR (US 8802374 B2) construct used in this study and is a cofounder of Juno Therapeutics.

Acknowledgments

The authors acknowledge the kind gifts of TG-1 mice from Dr. Thomas Tedder (Duke University) and pCL Eco DNA from Dr. Inder Verma (Salk Institute for Biological Studies) and appreciate the technical assistance provided by Anthony DeFranco, Amber McElroy, Drs. Ryan Flynn and Patricia Taylor, and the staffs of the University of Minnesota Genomics Core and University Flow Cytometry Resource. The study was supported by gifts from the Minnesota Masonic Charities and the Atwater family to the Masonic Cancer Center and grants from the Randy Shaver Cancer Research and Community Fund and the National Cancer Institute of the NIH under award numbers R01 CA72669 and 2P01 CA 065493.

Footnotes

Supplemental Information includes Supplemental Materials and Methods, five figures, and one table and can be found with this article online at https://doi.org/10.1016/j.ymthe.2018.04.006.

Supplemental Information

Document S1. Supplemental Materials and Methods and Figures S1–S5
mmc1.pdf (2.9MB, pdf)
Table S1. Accession Numbers for Transcriptomic Data
mmc2.xlsx (14.1KB, xlsx)
Document S2. Article plus Supplemental Information
mmc3.pdf (4.7MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Materials and Methods and Figures S1–S5
mmc1.pdf (2.9MB, pdf)
Table S1. Accession Numbers for Transcriptomic Data
mmc2.xlsx (14.1KB, xlsx)
Document S2. Article plus Supplemental Information
mmc3.pdf (4.7MB, pdf)

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