Preferential HLA-DRB1*11 Dependent Presentation of CUB2 Derived Peptides by ADAMTS13 Pulsed Dendritic Cells

Abstract Abstract 489 Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura (TTP). Recently, HLA-DRB1*11 has been identified as a risk factor for the development of acquired TTP. Here, we identified ADAMTS13-derived peptides presented on MHC class II alleles. Dendritic cells from a panel of both HLA-DRB1*11 positive and negative donors were pulsed with ADAMTS13 and the HLA-DR-presented peptide repertoire was analyzed by mass spectrometry. Interestingly, pulsing of dendritic cells of HLA-DRB1*11 positive donors with 100 nM ADAMTS13 resulted in presentation of a single CUB2 derived ADAMTS13 peptide. This peptide was not presented by HLA-DRB1*11 negative donors. Pulsing of HLA-DRB1*0301 positive cells with 100 nM ADAMTS13 resulted in presentation of a different CUB2 domain derived peptide in 2 out of 3 donors analyzed. Pulsing of dendritic cells employing higher concentrations of ADAMTS13 (500 nM) resulted in increased presentation of ADAMTS13 derived peptides by both HLA-DRB1*11 positive and negative donors. In DRB1*11 negative donors peptides derived of multiple domains were presented which included spacer, TSR2-8, metalloprotease and disintegrin domains. Interestingly, the CUB2 domain peptide (specific for DRB1*11 when dendritic cells were pulsed with 100 nM ADAMTS13) was also presented under these conditions. Apparently, this peptide can be presented by multiple MHC class II alleles although higher concentrations of ADAMTS13 are required for its presentation on DCs derived of non-DRB1*11 positive donors. Interestingly, the diversity of ADAMTS13 derived peptides presented by iDCs of donors HLA-DRB1*11 was not affected by pulsing of iDCs with higher concentration of ADAMTS13. Also, under these conditions the only peptides that were presented were derivatives of the CUB2 domain derived peptide that was also presented at lower concentrations of ADAMTS13. Our results clearly demonstrate that this peptide is efficiently presented when compared to other ADAMTS13-derived peptides. Therefore, we hypothesize that efficient presentation of this CUB2 domain derived peptide on DRB1*11 may provoke proliferation of low affinity self-reactive CD4+ T cells that have escaped negative selection in the thymus and contribute to the onset of acquired TTP. Together these findings may provide further insight in the initiation of the autoimmune reactivity against ADAMTS13 in patients affected by TTP. Disclosures: No relevant conflicts of interest to declare.

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Generation Of Anti-Tumor CD4+ T Helper Cells Using Genetically-Engineered Dendritic Cells Expressing Leukemia-Associated Antigens

Blood ◽

10.1182/blood.v122.21.2027.2027 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2027-2027

Author(s):

Dhanalakshmi Chinnasamy ◽

Pawel Muranski ◽

Manuel Franco-Colon ◽

Sawa Ito ◽

Nancy F. Hensel ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Adoptive Immunotherapy ◽

Cd4 T Cells ◽

Conflicts Of Interest ◽

Class Ii ◽

High Avidity ◽

Th Cells ◽

Antigenic Targets

Abstract Adoptive transfer of antigen-specific T cells is a potentially curative strategy for patients with solid tumors and leukemia. Most clinical trials of adoptive T cell therapy have used cytotoxic CD8+ T cells recognizing MHC class I-restricted tumor antigens. Despite overwhelming evidence suggesting the fundamental influence of CD4+ T cells on the immune system, clinical experience with tumor-specific CD4+ Th cells is almost non-existent. Unlike most other tissues, bone marrow-derived cells constitutively express MHC class II and CD4+ T cells play crucial role in mediating the curative GVL effect after allogeneic SCT and donor lymphocyte infusion (DLI). Furthermore, experimental evidences suggest that MHC class II-restricted antigenic targets recognized by CD4+ T cells exist in both solid cancers and in hematological malignancies. Therefore adoptive immunotherapy using CD4+ T cells in the setting of leukemia might be especially relevant. The goal of this study is to establish a simplified non-individualized protocol of generating LAA-reactive CD4+ T cells from patients and normal donors for adoptive immunotherapy directed against common leukemia-associated antigens (LAA) expressed in acute myeloid leukemias (AML) and myelodysplastic syndrome (MDS). We isolated naïve and memory CD4+ T cells from 3 normal donors and stimulated with twice at weekly interval with autologous monocytes pulsed with libraries of overlapping 15-amino acid length peptides (pepmixes) derived from WT-1, MAGE A3 and A4, PRAME and SSX2 antigens. At the end of the experiment CD4+ T cells were evaluated for reactivity against each LAA by analyzing their ability to specifically release cytokines (IL-2, TNF-α, and IFNγ) using flow cytometry. LAA-specific cells were found in either naïve or memory-derived CD4+ T cells upon stimulation with relevant pepmixes in all donors tested. However specific cytokine production could not be demonstrated when the same T cells were exposed to LAA-transduced autologous targets (LCL and T cells), raising the possibility that the majority of pepmix-reactive cells recognized epitopes that were not naturally processed. Therefore, as an alternative strategy to induce LAA-specific cells capable of targeting only therapeutically-relevant epitopes, we used autologous dendritic cells (DCs) transduced with a lentiviral vector encoding MAGE A3 antigen. Autologous CD4+ T cells were stimulated with MAGE A3 or mock-transduced DCs at an interval of 7-10 days and tested for their antigen-specific cytokine secretion. At the end of the culture we observed that Th cells expanded in presence of MAGE A3-expressing DCs and contained a significant number of cells possessing specific reactive against MAGE A3 pepmix (Figure), but not to unrelated antigenic targets, suggesting induction of LAA-reactivity against naturally-processed MAGE A3 epitopes. In summary, we demonstrate the feasibility of generating specific anti-tumor CD4+ T cells using autologous DCs engineered to express a full-length tumor antigen. This approach allows for selective expansion of polyclonal Th cells recognizing only naturally processed MHC class II-restricted epitopes. Therefore, this strategy circumvents the limitation inherent to usage of overlapping peptide libraries that might induce the expansion of high-avidity T cells specific to epitopes that are irrelevant to in vivo recognition of tumor targets. Furthermore, this approach does not rely on a particular pre-defined MHC class II restriction element, thus it is applicable to majority of donors or patients irrespective of their MHC haplotype. Disclosures: No relevant conflicts of interest to declare.

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