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FACS results for autologous PD-L1 CAR-T cells. (a) Non-transduced T cells were used as control. (b) Transduced T cells. Both a and b were stained with biotinylated PD-L1::human Fc fusion protein and APC-labelled streptavidin. Approximately 20% of the transduced cells were CAR-positive T cells.
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Cytotoxic effects of PD-L1 CAR transduced T cells on lung carcinoma cells, as compared to the effects of PD-1 fusion receptor T cells. The cell-killing effects of PD-1 fusion receptor (PD1-CD28Z-CD3zeta) T cells and PD-L1 CAR-T cells were investigated after co-incubation with lung cancer cells in different effector to target ratios. CAR-T cell killing by CD38 CAR cells, PD-1 fusion receptor T cells and PD-L1 CAR-T cells were investigated after co-incubation with lung cancer cells in different effector to target ratios (E:T = 3:1, 10:1 and 30:1, respectively). n = 3. (a-c) H1975 cells; (d-f) H3122 cells. CD38-specific CAR-T cells were included as the control. PD-L1 CAR-T cells were significantly more potent than PD-1 fusion receptor T cells. One-way ANOVA and the Student's t-test were used. *P-value < 0.05;**P-value < 0.01.
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The patient was discharged from hospital as per schedule (Supplementary figure 2).
CT imaging of the mediastinal window of the patient. The results obtained on four representative days are shown. (a) Day -12 before the patient was administered CAR-T cells: maximum tumor diameter, 78.6 mm; (b) Day +29, that is when the patient came for the first follow-up: the maximum tumor diameter, 73.3 mm (indicating stable disease [SD]); (c) Day +49, that is 2 days after the patient was admitted because of ALL/ARDS and respiratory failure: maximum tumor diameter, 70.2 mm (SD); (d) Day +64, that is when the patient recovered from ALI/ARDS: maximum tumor diameter, 72.8 mm.
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On Day -12, the maximum tumor diameter was found to be 78.6 mm on performing computed tomography (CT) imaging of the mediastinal window (Figure 3a), and the Eastern Cooperative Oncology Group (ECOG) performance status was estimated as 1. Figure 3b). FACS analysis detected PD-L1-specific CAR-T cells in peripheral blood, which accounted for 3.30% of his peripheral blood T cells (Figure 5c and d), looks odd on its own on this line indicating high CAR-T cell proliferation after infusion in relation to his absolute lymphocyte counts (Supplementary table 1). The pulmonary infiltrates gradually resolved (Figure 6b and c), and chest CT imaging showed that the tumors shrank further (70.2 mm; Day +49, 2 days after being transferred to the ICU), which indicated SD (Figure 3c).
Pathological examination of the patient's lung tumor via core needle biopsy. (a) Representative section of H.E. staining showing adenocarcinoma. Scale bar: 100 microm. (b, c) High-power observations of tumor and non-tumor regions in a. Scale bar: 50 mum. (d) Corresponding section of IHC staining. (e, f) High-power observations of PD-L1 expression in tumor and non-tumor regions in d. Tumor cells were positive for PD-L1 expression, whereas the adjacent non-malignant lung tissue was negative. Scale bar: 50 microm.
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Pathological analysis confirmed the diagnosis of NSCLC; the tumor was a poorly differentiated adenocarcinoma (Figure 4a) with a proliferative rate of 40% (percentage of Ki67-positive area). The PD-L1 expression levels, as determined by immunohistochemistry (IHC) with the Ventana rabbit monoclonal antibody SP142, were heterogeneous; approximately 20% of the tumor tissue was positive for PD-L1 (Figure 4b and e), whereas the surrounding normal lung tissues were negative (Figure 4c and f).
PD-L1 CAR-T cells proliferated greatly after transfusion in the patient's circulation.(upper panels) No PD-L1 CAR-T cells were detected on Day +11, when the patient was discharged from the hospital after CAR-T cell infusions. (a) Patient's PBMCs stained with APC-labelled streptavidin alone. (b) Patient's PBMCs stained with both biotinylated PD-L1::human Fc fusion protein and APC-labelled streptavidin. (middle panels) Approximately 3.30% of the total T cells were CAR positive on Day +29. (c) Patient's PBMCs stained with FITC CD3 and APC-labelled streptavidin. (d) Patient's PBMCs stained with FITC CD3 and both biotinylated PD-L1::human Fc fusion protein and APC-labelled streptavidin. (lower panels) PD-L1 CAR-T cells were undetectable after the patient developed ALI/ARDS on Day +48. (e, f) The staining patterns were the same as those in a and b, respectively.
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Figure 3b). FACS analysis detected PD-L1-specific CAR-T cells in peripheral blood, which accounted for 3.30% of his peripheral blood T cells (Figure 5c and d), looks odd on its own on this line indicating high CAR-T cell proliferation after infusion in relation to his absolute lymphocyte counts (Supplementary table 1). Table 1), while CAR-engineered T cells could not be detected in the peripheral blood after tocilizumab and corticosteroid administration (Figure 5e and f). To confirm the cytokine levels, the patient's sample was also sent to another third-party laboratory for further analysis, which showed consistent results (Supplementary table 2).
Bilateral extensive pulmonary infiltrates evolved with treatment. Representative chest X-ray images obtained at the following time points have been provided: (a) when the patient developed ARDS and was admitted to the ICU on Day +47; (b) at 1 week after administration of corticosteroids and the IL-6 receptor blocker on Day +53; (c) after transfer out of the ICU on Day +57. At the onset of ALI/ARDS, extensive bilateral pulmonary infiltration was noted, in addition to the tumor and atelectasis in the left lung. The infiltrates rapidly resolved on treatment with the tumor and atelectasis present.
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The chest X-ray showed extensive bilateral pulmonary infiltration in addition to the silhouette of the upper left lung tumors (Figure 6a). The pulmonary infiltrates gradually resolved (Figure 6b and c), and chest CT imaging showed that the tumors shrank further (70.2 mm; Day +49, 2 days after being transferred to the ICU), which indicated SD (Figure 3c).
The patient's condition rapidly improved after treatment. (a) The patient's body temperature started decreasing, and his blood oxygen saturation also immediately increased. (b) The patient's serum cytokine IL-6 and CRP levels immediately decreased.
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The dyspnoea and pyrexia rapidly decreased, blood oxygen saturation increased (Figure 7a), and IL-6 levels showed a steep decline (Figure 7b).