Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key ...requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1
03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML.
BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted ...antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, DM-sensitive antigens rely on the expression of HLA-DO-the natural inhibitor of HLA-DM. Since expression of HLA-DO is not upregulated by inflammatory cytokines, DM-sensitive antigens cannot be presented on non-hematopoietic tissues even under inflammatory conditions. Therefore, usage of CD4+ T cells directed against DM-sensitive antigens might allow induction of graft-versus-leukemia effect without GvHD. As DM-sensitivity is likely linked to low affinity peptides, it remains elusive whether DM-sensitive antigens are inferior in their immunogenicity.MethodsWe created an in vivo system using a DM-sensitive and a DM-resistant variant of the same antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM and HLA-DO) to assign the two model antigens ovalbumin (OVA) and DBY to their category. Further, we introduced mutations within the two T-cell epitopes and tested the effect on DM-sensitivity or DM-resistance. Furthermore, we vaccinated C57BL/6 mice with either variant of the epitope and measured expansion and reactivity of OVA-specific and DBY-specific CD4+ T cells.ResultsBy testing T-cell recognition of OVA and DBY on a murine B-cell line overexpressing H2-M and H2-O, respectively, we showed that OVA leads to a stronger T-cell activation in the presence of H2-O demonstrating its DM-sensitivity. In contrast, the DBY epitope does not rely on H2-O for T-cell activation indicating DM-resistance. By introducing mutations within the T-cell epitopes we could generate one further DM-sensitive variant of OVA and two DM-resistant counterparts. Likewise, we designed DM-resistant and DM-sensitive variants of DBY. On vaccination of C57BL/6 mice with either epitope variant we measured comparable expansion and reactivity of OVA-specific and DBY-specific T-cells both in vivo and ex vivo. By generating T-cell lines and clones of healthy human donors we showed that DM-sensitive antigens are targeted by the natural T-cell repertoire.ConclusionWe successfully generated DM-sensitive and DM-resistant variants for two model antigens. Thereby, we demonstrated that DM-sensitive antigens are not inferior to their DM-resistant counterpart and are therefore interesting tools for immunotherapy after allogeneic stem cell transplantation.
Systemic lupus erythematosus (SLE) is a life-threatening autoimmune disease characterized by adaptive immune system activation, formation of double-stranded DNA autoantibodies and organ inflammation. ...Five patients with SLE (four women and one man) with a median (range) age of 22 (6) years, median (range) disease duration of 4 (8) years and active disease (median (range) SLE disease activity index Systemic Lupus Erythematosus Disease Activity Index: 16 (8)) refractory to several immunosuppressive drug treatments were enrolled in a compassionate-use chimeric antigen receptor (CAR) T cell program. Autologous T cells from patients with SLE were transduced with a lentiviral anti-CD19 CAR vector, expanded and reinfused at a dose of 1 × 10
CAR T cells per kg body weight into the patients after lymphodepletion with fludarabine and cyclophosphamide. CAR T cells expanded in vivo, led to deep depletion of B cells, improvement of clinical symptoms and normalization of laboratory parameters including seroconversion of anti-double-stranded DNA antibodies. Remission of SLE according to DORIS criteria was achieved in all five patients after 3 months and the median (range) Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (2). Drug-free remission was maintained during longer follow-up (median (range) of 8 (12) months after CAR T cell administration) and even after the reappearance of B cells, which was observed after a mean (±s.d.) of 110 ± 32 d after CAR T cell treatment. Reappearing B cells were naïve and showed non-class-switched B cell receptors. CAR T cell treatment was well tolerated with only mild cytokine-release syndrome. These data suggest that CD19 CAR T cell transfer is feasible, tolerable and highly effective in SLE.
Idiopathic inflammatory myopathies are a group of rare, immune-mediated diseases that primarily affect the skeletal muscle but can also involve other organs such as the lungs, skin, and joints.1 ...Antisynthetase syndrome comprises a major cluster of such diseases and is characterised by the development of adaptive immune responses against various tRNA synthetases, including those for histidine (forming anti-Jo-1 antibodies), tyrosine (anti-PL7), alanine (anti-PL12), glycine (anti-EJ), isoleucine (anti-OJ), asparagine (anti-KS), phenylalanine (anti-Zo), and threonine (anti-HA).2 Histopathology studies of patients with antisynthetase syndrome have shown the presence of B cells and plasmablasts located adjacent to T cells in the affected muscles,3 and the condition is also associated with changes in the profile of peripheral B cells.3 In accordance with these findings, B-cell-depleting therapy with rituximab was efficacious in a subset of patients with antisynthetase syndrome, supporting the pathogenic role of autoreactive B cells.4 Antisynthetase syndrome can be refractory despite several treatment options—including glucocorticoids, intravenous immunoglobulins, T-cell-targeting drugs, and B-cell-targeting drugs—and is therefore associated with increased mortality.5 Considering the pathophysiology of antisynthetase syndrome, treatment with chimeric antigen receptor (CAR) T cells that recognise CD19⁺ B cells might be useful in refractory forms of the disease. Increased creatinine kinase concentrations and myalgia have been reported as part of cytokine release syndrome.8 After this short worsening, the patient markedly improved in physical function according to all International Myositis Assessment and Clinical Studies Group core set measures (figure F, appendix p 1). Regarding safety, the patient developed a fever (38–39°C, with no effect on blood pressure) 1–3 days after CAR T-cell treatment, which was treated with paracetamol and 3 × 720 mg tocilizumab.
Morbidity and mortality in juvenile-onset systemic lupus erythematosus are higher than adult-onset disease, and children with lupus nephritis have higher scores on the Systemic Lupus Erythematosus ...Disease Activity Index (SLEDAI).2,3 End-stage renal disease and permanent haemodialysis are severe adverse events than can occur due to juvenile-onset and adult-onset systemic lupus erythematosus, with 15% of all patients with lupus nephritis developing end-stage renal disease.4 The therapeutic goal of complete remission of lupus nephritis is only reached in 60% of patients by conventional therapies, including immunosuppressive drugs and B-cell-depleting antibodies,3 and two-thirds of adults with juvenile-onset systemic lupus erythematosus develop organ damage and impaired health-related quality of life without reaching drug-free remission.5 Due to their high B-cell depletion activity, CD19-targeted chimeric antigen receptor (CAR) T cells are a potentially powerful strategy to treat autoimmune diseases, such as systemic lupus erythematosus.6,7 In a small case series of eight patients aged 18–38 years with treatment-refractory systemic lupus erythematosus, adoptive transfer of CD19-targeted CAR T cells induced a deep reset of B cells leading to abrogation of autoreactive antibodies and durable remission, including abrogation of lupus nephritis with a follow-up time of 6–29 months.8 Herein, we report on a 15-year-old female patient with severe and rapidly progressive systemic lupus erythematosus. Low levels of complement and presence of several autoantibodies including anti-nuclear-antibody anti-double-stranded DNA (anti-dsDNA; 4545 IU/mL normal range is <100 IU/mL), anti-nucleosome, and anti-histone antibodies were evident. Because renal function further rapidly worsened under therapy escalation with high-dose methylprednisolone and cyclophosphamide, plasma separation was initiated, leading to a reduction in autoantibody levels. At our site (University Hospital Erlangen, Erlangen, Germany), we initiated CD19-targeted CAR T-cell therapy in an expanded access programme for critically ill patients according to the German Arzneimittelgesetz, §21/2 and the Arzneimittel-Härtefall-Verordnung §2 following a mandatory case review by an independent interdisciplinary board. Despite kidney failure, CAR T-cell therapy was safe and well tolerated due to appropriate dose adjustment of lymphodepletion.
Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant ...autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission.
We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded.
The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization.
In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).
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