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Flow cytometry (FCM) and quantitative PCR (qPCR) are conventional methods for assessing CAR-T expansion, while digital droplet PCR (ddPCR) is emerging as a promising alternative. We monitored CAR-T transcript expansion in 40 B-NHL patients post-infusion of CAR-T products (axi-cel; tisa-cel; and brexu-cel) with both His-Tag FCM and ddPCR techniques. Sensitivity and predictive capacity for efficacy and safety outcomes of ddPCR were analyzed and compared with FCM. A significant correlation between CAR-T counts determined by FCM and CAR transcripts assessed by ddPCR (p < 0.001) was observed. FCM revealed median CD3+CAR+ cell counts at 7, 14, and 30 days post-infusion with no significant differences. In contrast, ddPCR-measured median copies of CAR-T transcripts demonstrated significant lower copy numbers in tisa-cel recipients compared to the other products at day 7 and day 14. Patients with a peak of CAR transcripts at day 7 exceeding 5000 copies/microg gDNA, termed “good CAR-T expanders”, were more likely to achieve a favorable response at 3 months (HR 10.79, 95% CI 1.16–100.42, p = 0.036). Good CAR-T expanders showed superior progression-free survival at 3, 6, and 12 months compared to poor CAR-T expanders (p = 0.088). Those reaching a peak higher than 5000 copies/microg gDNA were more likely to experience severe CRS and ICANS. DdPCR proves to be a practical method for monitoring CAR-T expansion, providing quantitative information that better predicts both treatment outcomes and toxicity.

CD4+ and CD8+ chimeric antigen receptor T cells (CAR-T) play different roles in the in vivo anti-tumour response, but the role of the CD4+/CD8+ ratio among infused CAR-T has not been clearly defined yet. We analysed leftovers from infused anti-CD19 CAR-T bags of 31 patients with aggressive B-cell lymphomas. The median ratio was 1.44, lower for brexu-cel compared to tisa-cel and axi-cel. The CAR+CD4+/CD8+ ratio was influenced by lactate dehydrogenase levels at apheresis, not by age, previous treatments or the CD4+/CD8+ ratio in peripheral blood. Patients with a response at 3 months after CAR-T (M3) had a lower CAR+CD4+/CD8+ ratio in the infused products compared to non-responders (ratio 0.74 vs. 2.47, p = 0.011). A CAR+CD4+/CD8+ ratio higher than the cut point of 1.12 was associated with an increased risk of treatment failure at M3 (OR 23.3, p = 0.012) and M6 (OR 10, p = 0.028). The median 6-month PFS was 76% for patients with a ratio lower than 1.12% vs. 31% for the others. The prognostic role of the CAR+CD4+/CD8+ ratio was independent of the costimulatory domain (CD28 vs. 4-1BB) of the product (OR 16.41, p = 0.041). Our data indicate a crucial role for CD8+ CAR-T and the CAR+CD4+/CD8+ ratio in predicting CAR-T efficacy.

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Observing how an action is done by others allows the observer to understand the cognitive and emotion state of the agent. This information, carried by the kinematics of the observed action, has been defined by Daniel Stern “vitality forms”. The expression and the capacity to understand the vitality forms is already present in infants, a finding indicating their importance for the development of social attunement. It has been proposed that, well before developing linguistic abilities, infants are actively engaged in non-verbal exchanges with their caregivers. This ability denotes a primordial way to relate to and understand others and presumably represents a constitutive element of interpersonal relations, namely intersubjectivity. In the present neuroimaging (fMRI) study we presented participants with videos showing hand actions performed with different velocities and asked them to judge their vitality form (gentle, neutral, rude) or their velocity (slow, medium, fast). Previous studies showed that the dorso-central insula is selectively active both during vitality form observation and execution. The aim of the present study was to assess, using multi-voxel pattern analysis (MVPA), whether in the insula there are voxels discriminating vitality form from velocity. Results showed that, consistently across subjects, in the dorso-central sector of the insula there are voxels selectively tuned to vitality forms. Supporting previous findings, these results confirm that the dorso-central insula is involved in processing the vitality forms of an action, both when carryied out in the first person and when observed in other individuals. This supports the idea that the understanding of others' behavior in terms of affective content is mediated by an automatic activation system that allows the recipient to tune in and respond to another individual's emotional state without necessarily having "formal" knowledge of what is being observed. As argued by Stern, this process would al