Gideon Hönger

IMPORTANCE: Immunotherapy with checkpoint inhibitors targeting the PD-1 (programmed cell death 1) axis has brought notable progress in patients with non-small cell lung cancer (NSCLC) and other cancers. However, autoimmune toxic effects are frequent and poorly understood, making it important to understand the pathophysiologic processes of autoimmune adverse effects induced by checkpoint inhibitor therapy. OBJECTIVE: To gain mechanistic insight into autoimmune skin toxic effects induced by anti-PD-1 treatment in patients with non-small cell lung cancer. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study was conducted from July 1, 2016, to December 31, 2018. Patients (n = 73) with non-small cell lung cancer who received anti-PD-1 therapy (nivolumab or pembrolizumab) were recruited from 4 different centers in Switzerland (Kantonsspital St Gallen, Spital Grabs, Spital Wil, and Spital Flawil). Peripheral blood mononuclear cells, tumor biopsy specimens and biopsies from sites of autoimmune skin toxic effects were collected over a 2-year period, with patient follow-up after 1 year. MAIN OUTCOMES AND MEASURES: Response to treatment, overall survival, progression-free survival, and development of autoimmune toxic effects (based on standard laboratory values and clinical examinations). RESULTS: Of the cohort of 73 patients with NSCLC (mean [SD] age, 68.1 [8.9] years; 44 [60%] men), 25 (34.2% [95% CI, 24.4%-45.7%]) developed autoimmune skin toxic effects, which were more frequent in patients with complete remission or partial remission (68.2% [95% CI, 47.3%-83.6%]) than those with progressive or stable disease (19.6% [95% CI, 11.0%-32.5%]) (χ2 = 14.02, P < .001). Nine T-cell antigens shared between tumor tissue and skin were identified. These antigens were able to stimulate CD8+ and CD4+ T cells in vitro. Several of the antigen-specific T cells found in blood samples were also present in autoimmune skin lesions and lung tumors of patients who responded to anti-PD-1 therapy. CONCLUSIONS AND RELEVANCE: These findings highlight a potential mechanism of checkpoint inhibitor-mediated autoimmune toxic effects and describe the association between toxic effects and response to therapy; such an understanding will help in controlling adverse effects, deciphering new cancer antigens, and further improving immunotherapy.

BACKGROUND: Defining the clinical relevance of donor-specific HLA-antibodies detected by single-antigen flow-beads (SAFB) is important because these assays are increasingly used for pretransplant risk assessment and organ allocation. The aims of this study were to investigate to which extent HLA-DSA detected by SAFB represent a risk for antibody-mediated rejection (AMR) and diminished allograft survival, and to define HLA-DSA characteristics predictive for AMR. METHODS: In this retrospective study of 334 patients with negative complement-dependent cytotoxicity crossmatches, day-of-transplant sera were analyzed by SAFB, HLA-DSA determined by virtual crossmatching, and the results correlated with the occurrence of AMR and allograft survival. RESULTS: Sixty-seven of 334 patients (20%) had HLA-DSA. The incidence of clinical/subclinical AMR at day 200 posttransplant was significantly higher in patients with HLA-DSA than in patients without HLA-DSA (55% vs. 6%; P<0.0001). Notably, 30/67 patients with HLA-DSA (45%) did not experience clinical/subclinical AMR. Death-censored 5-year allograft survival was equal in patient without HLA-DSA and patients with HLA-DSA but no AMR (89% vs. 87%; P=0.95), whereas it was 20% lower in patients with HLA-DSA and AMR (68%; P=0.002). The number, class, and cumulative strength of HLA-DSA determined by SAFB, and prior sensitizing events were not predictive for the occurrence of AMR. CONCLUSIONS: These results support the utility of SAFB for pretransplant risk assessment and organ allocation, and suggest that improvement of the positive predictive value of HLA-DSA defined by SAFB will require an enhanced definition of pathogenic factors of HLA-DSA.

Preformed donor-specific HLA-antibodies antibodies (DSA) are a major risk for early antibody-mediated rejection (AMR). This prospective study evaluated the accuracy of pretransplant risk assessment using virtual crossmatching (virtualXM) (i.e. comparing HLA-typing of the donor with the recipient’s HLA-antibody specificities determined by flow-beads). Sixty-five consecutive patients were stratified according to virtualXM results: patients without DSA (n = 56) were considered low risk and received standard immunosuppression; patients with DSA (n = 9) were considered high risk and received additional induction with anti-T-lymphocyte-globulin (ATG) and intravenous immunoglobulins. Despite induction therapy 4 of 9 patients with DSA (44%) had clinical/subclinical AMR, whereas only 2 of 56 patients without DSA (4%) (p = 0.002). Notably, one of these two patients had early AMR likely induced by non-HLA-antibodies; the other had subclinical AMR at month 6 consistent with de novo DSA. The results of virtualXM and retrospectively obtained flow-cytometric crossmatches (FCXM) (n = 59) were concordant in 51 patients (86%), four patients (7%) were virtualXM−/FCXM+ and none had AMR, four patients (7%) were virtualXM+/FCXM− and one had AMR. VirtualXM can accurately define absence or presence of DSA and may become an invaluable tool for organ allocation and pretransplant risk assessment. However, further studies need to address whether all HLA-antibodies detected by flow-beads are clinically relevant. Preformed donor-specific HLA-antibodies antibodies (DSA) are a major risk for early antibody-mediated rejection (AMR). This prospective study evaluated the accuracy of pretransplant risk assessment using virtual crossmatching (virtualXM) (i.e. comparing HLA-typing of the donor with the recipient’s HLA-antibody specificities determined by flow-beads). Sixty-five consecutive patients were stratified according to virtualXM results: patients without DSA (n = 56) were considered low risk and received standard immunosuppression; patients with DSA (n = 9) were considered high risk and received additional induction with anti-T-lymphocyte-globulin (ATG) and intravenous immunoglobulins. Despite induction therapy 4 of 9 patients with DSA (44%) had clinical/subclinical AMR, whereas only 2 of 56 patients without DSA (4%) (p = 0.002). Notably, one of these two patients had early AMR likely induced by non-HLA-antibodies; the other had subclinical AMR at month 6 consistent with de novo DSA. The results of virtualXM and retrospectively obtained flow-cytometric crossmatches (FCXM) (n = 59) were concordant in 51 patients (86%), four patients (7%) were virtualXM−/FCXM+ and none had AMR, four patients (7%) were virtualXM+/FCXM− and one had AMR. VirtualXM can accurately define absence or presence of DSA and may become an invaluable tool for organ allocation and pretransplant risk assessment. However, further studies need to address whether all HLA-antibodies detected by flow-beads are clinically relevant.

BACKGROUND: A modified single antigen bead (SAB) assay measuring C1q binding to human leukocyte antigen antibodies has recently been introduced. The aim of this study was to investigate the determinants of C1q binding on SAB. METHODS: Sera from 73 sensitized patients were analyzed by the generic IgGpan as well as IgG subclass specific SAB assays and correlated with the standard and an anti-human globulin (AHG) enhanced C1q test. RESULTS: Among 2,665 SABs with positive IgGpan results (mean fluorescence intensity [MFI]>500), strong complement-binding IgG1 and IgG3 subclasses accounted for a median of 99% (interquartile range, 76%-100%) of the total IgG amount. IgGpan MFI alone showed a very strong association with standard C1q positivity (r=0.72), which was superior to a model including all IgG subclass MFI (r=0.62). Combining all IgG subclass MFI and IgGpan MFI only marginally improved the prediction of standard C1q positivity compared with IgGpan MFI alone (Δr=0.02). IgGpan MFI greater than 14,154 predicted standard C1q positivity, with 92% sensitivity and 96% specificity. Notably, 1,840 (93%) of the 1,974 C1q-negative SABs contained human leukocyte antigen antibodies with strong complement-binding IgG1 and IgG3 subclasses. Anti-human globulin significantly enhanced the signal in the C1q assay, but the association of AHG C1q positivity with IgGpan MFI was less strong (r=0.51). CONCLUSION: C1q binding on SAB is strongly associated with IgGpan MFI. IgG subclass information only marginally improves prediction of C1q binding likely because complement-binding IgG1 and IgG3 subclasses dominate regarding frequency and relative amounts. A negative C1q assay result does not indicate the absence of strong complement-binding IgG subclasses.