Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model

Abstract

Objectives: Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings.

Methods: BRGSF mice reconstituted with human umbilical cord blood CD34⁺ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed.

Results: OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds.

Conclusions: These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

Keywords: BRGSF mice; cytokine release syndrome; humanized preclinical models; immunotherapy; myeloid cells; safety assessment.

Figure 1

OKT3 treatment induces cytokine release, body weight loss, and temperature drop in BRGSF-CBC mice. Presence of human proinflammatory cytokines TNF-α, IL-2, and IL-6 (A), Type II interferon IFN-ɣ (B), regulatory cytokines IL1-RA and IL-10 (C), and chemokines CCL2, CXCL10, and CXCL8 (D) in mice sera was tested at 6h, 24h, and 48h. Serum cytokine levels are shown as mean (line) and standard error (bars). Human, CCL5, G-CSF, IFN-α2, IL-7, and CCL3 were not detected in the samples. Body weight (E) and temperature (F) were measured in all mice at 6h, 24h, and 48h post-injection. Serum levels of Serum Amyloid A (SAA) was tested at 48h (G). Individual donors are identified by symbol shapes, as indicated in Supplementary Table 1 . Green and red stars correspond to statistical tests for the indicated group, at one time point compared to T0. Black stars correspond to statistical differences between hFlt3L treated and non-treated groups.

Figure 2

OKT3 treatment affects human immune cell distribution in BRGSF-CBC mice. Percentages of B (CD19⁺) and T (TCRb⁺) cells (A), pDCs, cDCs, monocytes/macrophages, and neutrophils (B) in total human immune cells (CD45⁺) were analyzed from all mice spleens 48h after OKT3 treatment. Percentages of activated pDCs (C), cDCs (D), monocytes/macrophages (E), and neutrophils (F) were determined as single CD86⁺, single CD80⁺, and double CD80⁺/CD86⁺ cells in each population. Individual donors are identified by symbol shapes, as indicated in Supplementary Table 1 . Gating strategy is shown in Supplementary Figure 2 . P value < 0.05 was considered statistically significant (*). **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3

OKT3 treatment induces cytokine release, body weight loss and immune cell distribution alteration in BRGSF-PBMC and hFlt3L-boosted BRGSF-CBC mice. Presence of human TNF-α, IL-2, IFN-ɣ, IL-10, IL-6, IL1-RA, CCL2, and CXCL10 in mice sera was tested at 2h, 6h, 24h, and 48h (A). Serum cytokine levels are shown as mean (line) and standard error (bars). Body weight was measured in all mice at 2h, 6h, 24h, and 48h post-injection (B). Humanization rate in splenocytes of all Vehicle-injected mice were determined by flow cytometry (C). T cells (TCRb⁺) in total human immune cells (CD45⁺) were analyzed from all mice spleens, 24h or 48h after OKT3 treatment (D). Individual donors are identified by symbol shapes, as indicated in Supplementary Table 3 . Green and red stars correspond to statistical tests for the indicated group, at one time point compared to T0. Black stars correspond to statistical differences between groups. P value < 0.05 was considered statistically significant (*). **P<0.01, ***P<0.001, ****P<0.0001.

Figure 4

Anti-VISTA therapeutic antibody JNJ treatment induces cytokine release and myeloid cells depletion in hFlt3L-boosted BRGSF-CBC mice. Cytokine serum levels (A) were analyzed in all mice upon JNJ treatment at indicated time points. Percentages of monocytes/macrophages in total human immune cells (B), CD86⁺ activated monocytes/macrophages (C), cDCs in total human immune cells (D), and CD86⁺ activated cDCs (E) were analyzed from mice spleens at 24h and 48h by flow cytometry. Individual donors are identified by symbol shapes, as indicated in Supplementary Table 5 . Purple stars correspond to statistical tests for the indicated group, at one time point compared to T0. Black stars correspond to statistical differences between groups. P value < 0.05 was considered statistically significant (*). **P<0.01, ***P<0.001, ****P<0.0001.