The presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal... Show moreThe presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal long-term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA-producing B cells. We have genetically engineered an HLA-A2-specific CHAR (A2-CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti-HLA-A2 antibody-expressing target cells. In addition, we have performed A2-CHAR-Tc cytotoxic assays in an immunodeficient mouse model. A2-CHAR expressing T cells could selectively eliminate HLA-A2 antibody-producing B cells in vitro. The cytotoxic capacity of A2-CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2-CHAR-T cells could identify and eradicate HLA-A2 antibody-producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody-producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation. Show less
Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in... Show moreLuminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r(2): 0.946, class II r(2): 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r(2): 0.952, class II r(2): 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended. Show less
Jong, E. de; Quint, K.D.; Ghalbzouri, A. el; Verdijk, R.M.; Goeman, J.J.; Heidt, S.; ... ; Bavinck, J.N.B. 2023
Background: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC... Show moreBackground: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC, downregulation of HLA class I is associated with poor prognosis. Since the degree of HLA expression on tumor cells could play a role in immunogenicity and pathophysiology of cSCC metastasis, we hypothesized that decreased HLA expression is associated with an increased risk of metastasis.Methods: We compared HLA expression between primary metastasized cSCCs, their metastases, and nonmetastasized cSCCs from the same patients. Samples were stained for HLA-A, HLA-B/-C and quantified by calculating the difference in immunoreactivity score (IRS) of the primary cSCC compared with all nonmetastasized cSCCs. Results: The mean IRS score for HLA-B/C expression was 2.07 point higher in metastasized compared to nonmetastasized cSCCs (p = 0.065, 95 % CI -0.18-4.32). 83.3 % of the primary metastasized cSCCs had an IRS score of 4 or higher, compared to 42.9 % in non-metastasized cSCCs. Moderately to poorly differentiated cSCCs had more HLA class I expression compared to well-differentiated cSCCs. Conclusion: Contrary to immunocompetent patients, HLA-B/C expression tends to be upregulated in metastasized cSCC compared to non-metastasized cSCC in SOTR, suggesting that different tumor escape mechanisms play a role in SOTR compared to immunocompetent patients. Show less
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options... Show moreHLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity. Show less
