Chimeric antigen receptor (CAR) T cells form part of a broad wave of immunotherapies that are showing promise in early phase cancer clinical trials. This clinical delivery has been based upon preclinical efficacy testing that confirmed the proof of principle of the therapy. However, CAR T-cell therapy does not exist alone as T cells are generally given in combination with patient preconditioning, most commonly in the form of chemotherapy, and may also include systemic cytokine support, both of which are associated with toxicity. Consequently, complete CAR T-cell therapy includes elements where the toxicity profile is well known, but also includes the CAR T cell itself, for which toxicity profiles are largely unknown. With recent reports of adverse events associated with CAR T-cell therapy, there is now concern that current preclinical models may not be fit for purpose with respect to CAR T-cell toxicity profiling. Here, we explore the preclinical models used to validate CAR T-cell function and examine their potential to predict CAR T-cell driven toxicities for the future.

Interleukin 6 (IL-6) and, hence, activation of the IL-6 receptor signalling subunit glycoprotein 130 (gp130; also known as interleukin-6 receptor subunit b, IL6ST), has been linked to inflammation and tumour formation. Recently, deletion mutations in gp130 have been identified in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope and rendered gp130 constitutively active in a ligandindependent manner. Here, we show that gp130 deletion mutants, but not wild-type gp130, localise predominantly to intracellular compartments, notably the endoplasmic reticulum (ER) and early endosomes. One of the most frequent mutants, gp130 Y186-Y190del (DYY) is retained in the ER quality control system because of its association with the chaperone calnexin. Furthermore, we can show that gp130 DYY induces downstream signalling from both ER and endosomes, and that both signals contribute to ligand-independent cell proliferation. We also demonstrate that the endosomal localisation of gp130 DYY is crucial for fully fledged STAT3 activation. Therefore, aberrant signalling from intracellular compartments might explain the tumorigenic potential of naturally occurring somatic mutations of gp130.

HIT lacks a strong memory B-cell response, perhaps explaining transience and lack of anamnesis of the anti-PF4/heparin immune response. The technical modifications we describe for the ELISPOT assay, which permit detection of B-cell reactions to complex antigens, could be useful for studying other immunohematologic disorders, for example, drug-dependent thrombocytopenia and acquired hemophilia.

Activation of the IL-6 (interleukin 6) receptor subunit gp130 (glycoprotein 130) has been linked to the formation of complexes with IL-6 and the IL-6 receptor, as well as to gp130 dimerization. However, it has been shown that gp130 is present as a pre-formed dimer, indicating that its activation is not solely dependent on dimerization. Therefore the detailed mechanism of gp130 activation still remains to be deciphered. Recently, deletion mutations of gp130 have been found in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope of gp130 and resulted in a ligand-independent constitutively active gp130. We therefore hypothesized that conformational changes of this particular IL-6-binding epitope precedes gp130 activation. Using a rational structure-based approach we identified for the first time amino acids critical for gp130 activation. We can show that gp130 D2-D3 interdomain connectivity by hydrophobic residues stabilizes inactive gp130 conformation. Conformational destabilization of the EF loop present in domain D2 and disruption of D2-D3 hydrophobic interactions resulted in ligand-independent gp130 activation. Furthermore we show that the N-terminal amino acid residues of domain D1 participate in the activation of the gp130 deletion mutants. Taken together we present novel insights into the molecular basis of the activation of a cytokine receptor signalling subunit.