Delayed transfer of immune cells or the art of donor lymphocyte infusion

In the context of an allogeneic HSCT, the interplay between host and donor immune cells is considered to be the primary mechanism responsible for graft-versus-leukemia (GVL) reactivity and also able to mediate GVH

Differential effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway in acute lymphoblastic leukemia

Purpose: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established. Experimental design: We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative. Results: Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2. Conclusions: Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes

Delicate Balance Between Regulation and Stimulation at the Antigen Presenting Cell Site Determines the Likelihood of Successful In-Vitro Priming and Enrichment of Leukemia-Reactive T Cells From the NaïVe Donor Repertoire

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase

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

Influence of Polymorphisms in Innate Immunity Genes on Susceptibility to Invasive Aspergillosis after Stem Cell Transplantation

The innate immune system plays a pivotal role in the primary defence against invasive fungal infection. Genetic variation in genes that regulate this response, initiated by pulmonary macrophages, may influence susceptibility to invasive aspergillosis in patients at risk. We investigated in a clinical setting whether common polymorphisms in Toll-like receptor (TLR) and cytokine genes involved in macrophage regulation are associated with susceptibility to invasive aspergillosis. Forty-four allogeneic stem cell transplantation recipients diagnosed with probable or proven IA according to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group, were enrolled. The control group consisted of 64 allogeneic stem cell transplantation recipients without invasive aspergillosis. The TLR4 1063A>G single nucleotide polymorphism was associated with invasive aspergillosis when present in donors of allogeneic stem cell transplantation recipients (unadjusted OR 3.77 95%CI 1.08–13.2, p = 0.03). In a multivariate analysis, adjusted for occurrence of graft-versus-host-disease, Cytomegalovirus serostatus and duration of neutropenia, paired presence of the TLR4 1063A>G and IFNG 874T>A single nucleotide polymorphisms showed a trend towards increased susceptibility to invasive aspergillosis (p = 0.04). These findings point to the relevant immunological pathway involved in resistance to invasive aspergillosis and warrant further study of the effects of TLR and cytokine polymorphisms and their interaction, which may occur on different levels of the complex biological interplay between the immunocompromised host and Aspergillus sp

Identification of Clinically and Pathophysiologically Relevant Rheumatoid Factor Epitopes by Engineered IgG Targets

Objective: Rheumatoid factors (RFs), which are anti-IgG autoantibodies strongly associated with rheumatoid arthritis (RA), are also found in other diseases and in healthy individuals. RFs bind to various epitopes in the constant (Fc-) domain of IgG. Therefore, disease-specific reactivity patterns may exist. This study was undertaken in order to develop a new approach to dissecting RF epitope binding patterns across different diseases. Methods: We analyzed RF reactivity patterns in serum from patients with seropositive arthralgia, patients with RA, and patients with primary Sjögren’s syndrome (SS) using bioengineered, natively folded IgG-Fc targets that demonstrated selective RF binding toward several distinct regions of the IgG-Fc domain. Results: Rheumatoid factor responses primarily bound the Fc Elbow region, with a smaller number of RFs binding the Fc Tail region, while the Fc receptor binding region was hardly targeted. A restricted reactivity against the IgG-Fc Tail region was associated with less positivity for anti–citrullinated protein antibodies (ACPAs) and less arthritis development in arthralgia, whereas combined reactivity toward IgG-Fc Tail and Elbow regions was associated with more arthritis development. Reactivity toward the IgG-Fc Tail region was observed far more frequently in RA than in primary SS. Conclusion: Bioengineered IgG targets enable serologic characterization of RF reactivity patterns, and use of this approach appears to reveal patterns associated with ACPA detection and arthritis development in patients with arthralgia. These patterns are able to distinguish RA patients from primary SS patients. This new methodology improves the clinical value of RFs and our understanding of their pathophysiologic processes

Parametric Optimization Of Magneto-Rheological Fluid Damper Using Particle Swarm Optimization

This paper presents a parametric modeling of a magneto-rheological (MR) damper using a Particle Swarm Optimization (PSO) method. The objective of this paper is to optimize the parameter values of the MR fluid damper behavior using the Bouc-Wen model. The parametric identification was imposed beforehand in replicating the behavior of the MR fluid damper. The algebraic function from a number of hysteresis models was steered by comparing selected models: Bingham, Bouc-Wen and BoucWen by Kwok. A simulation method was operated in investigating these models by employing MATLAB reliant from the model intricacy. The experimental data was presented in terms of the time histories of the displacement, the velocity and the force parameters, measured for both constant and variable current settings and at a selected frequency applied to the damper. The model parameters were determined using a set of experimental measurements corresponding to different current constant values. It has been shown that the MR damper model’s response via the proposed approach is in good agreement with the MR damper test rig counterpar

Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT