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Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare thromboembolic complication of adenoviral-vectored SARS-CoV2 vaccines, mediated by antibodies directed against platelet factor 4 (PF4). Given their causal role in VITT, identification of the molecular composition of anti-PF4 antibodies is crucial for developing better diagnostics and treatments. Here, we utilised a novel proteomic workflow to analyse the immunoglobulin variable (IgV) region composition of anti-PF4 antibodies at the level of the secreted proteome. Serum anti-PF4 IgG antibodies from five patients with VITT triggered by ChAdOx1 nCoV-19 vaccination were affinity purified by PF4-coupled magnetic beads and sequenced by mass spectrometry. We revealed a single IgG heavy (H)-chain species paired with a single lambda light (L)-chain species in all five unrelated patients. Remarkably, all L-chains were encoded by the identical IGLV3-21*02 gene subfamily with identical L-chain third complementarity determining region (LCDR3) lengths. Moreover, striking stereotypic features were also identified in heavy-chains anti-PF4 antibodies characterised by identical HCDR3 length and homologous sequences. In summary, we unravelled the molecular signature of highly stereotyped clonotypic anti-PF4 antibodies, indicating shared pathways of antibody production in VITT patients. These discoveries are critical to understand the molecular basis of this serious condition and develop novel therapies aimed at removing pathogenic clones.

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Reducing immunosuppression has been proposed as a means of preventing cancer in kidney transplant recipients but this can precipitate graft rejection. Here we tested whether anti-tumor natural killer (NK) cell and allo-responsive T-cell function in kidney transplant recipients may predict cancer risk and define risk of rejection. NK cell function was measured by the release of lactate dehydrogenase and T cell allo-response by interferon-γ quantification using a panel of reactive T cell Enzyme-Linked ImmunoSpot (ELISPOT) in 56 kidney transplant recipients with current or past cancer and 26 kidney transplant recipients without cancer. NK function was significantly impaired and the allo-response was significantly lower in kidney transplant recipients with cancer. With prospective follow-up, kidney transplant recipients with poor NK cell function had a hazard ratio of 2.1 [95% Confidence Interval 0.97–5.00] for the combined endpoint of metastatic cancer, cancer related death, or septic death. Kidney transplant recipients with low interferon-γ release were also more likely to reach this combined endpoint. Thus, post-transplant monitoring of allo-immunity and NK cell function is useful for assessing the risk of over immunosuppression for the development of malignancy and/or death from cancer or sepsis.

No abstract

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare thromboembolic complication of adenoviral-vectored SARS-CoV2 vaccines, mediated by antibodies directed against platelet factor 4 (PF4). Given their causal role in VITT, identification of the molecular composition of anti-PF4 antibodies is crucial for developing better diagnostics and treatments. Here, we utilised a novel proteomic workflow to analyse the immunoglobulin variable (IgV) region composition of anti-PF4 antibodies at the level of the secreted proteome. Serum anti-PF4 IgG antibodies from five patients with VITT triggered by ChAdOx1 nCoV-19 vaccination were affinity purified by PF4-coupled magnetic beads and sequenced by mass spectrometry. We revealed a single IgG heavy (H)-chain species paired with a single lambda light (L)-chain species in all five unrelated patients. Remarkably, all L-chains were encoded by the identical IGLV3-21*02 gene subfamily with identical L-chain third complementarity determining region (LCDR3) lengths. Moreover, striking stereotypic features were also identified in heavy-chains anti-PF4 antibodies characterised by identical HCDR3 length and homologous sequences. In summary, we unravelled the molecular signature of highly stereotyped clonotypic anti-PF4 antibodies, indicating shared pathways of antibody production in VITT patients. These discoveries are critical to understand the molecular basis of this serious condition and develop novel therapies aimed at removing pathogenic clones.KEY POINTSAnti-PF4 antibodies in VITT comprise highly stereotyped clonotypeA single IGLV3-21*02 encoded light chain is found in unrelated patients

The quantification of frequency of IFN-γ–producing T cells responding to donor alloantigen using the IFN-γ enzyme linked immunosorbent spot (ELISPOT) holds potential for pretransplant and posttransplant immunological risk stratification. The effectiveness of this assay, and the ability to compare results generated by different studies, is dependent on the utilization of a standardized operating procedure (SOP). Key factors in assay standardization include the identification of primary and secondary antibody pairs, and the reading of the ELISPOT plate with a standardized automated algorithm. Here, we describe in detail, an SOP that should provide low coefficient of variation results. For multicenter trials, it is recommended that groups perform the ELISPOT assays locally but use a centralized ELISPOT reading facility, as this has been shown to be beneficial in reducing coefficient of variation between laboratories even when the SOP is strictly adhered to.

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