In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Studies on the role of autoantibodies and autoantigens in rheumatoid arthritis and myositis

A major population of patients affected with rheumatoid arthritis (RA) and inflammatory idiopathic myopathies (IIM, collectively called myositis) is characterized by the presence of autoantibodies. The pathogenic impact of anti-citrullinated protein/peptide (ACPA) or anti-histidyl transfer RNA synthetase (HisRS; Jo1) autoantibodies remains largely unknown. The aims of the thesis were to understand the molecular mechanisms underlying the autoimmune component (specifically, the auto-antibody and -antigen dynamics) in RA and myositis; and to develop ACPA neutralizing compounds. Paper I: Purified anti-cyclic citrullinated peptide antibodies (aCCP2 IgG) were estimated to represent ~2% of the total RA IgG pool, and demonstrated to display distinct and individual reactivities against citrullinated epitopes from fibrinogen, collagen type II, α-enolase and vimentin. In-vivo generated autoantigens in synovial tissue and fluid were recognized by aCCP2 IgG. Paper II: Anti-CCP2 IgG purified according to the methodology described in paper I were injected in mice and shown to induce pain-like behaviour. The underlying molecular mechanism appears to be chemokine-dependent because 1) aCCP2 IgG promoted activation of murine osteoclasts with generation of CXCL1 (human IL-8); 2) aCCP2 IgG stained CD68-bearing macrophages/osteoclasts, closely localized to the calcitonin gene related peptide (CGRP)-positive sensory nerve fibers; 3) reparixin, a CXCL1/2 receptor antagonist, blocked aCCP2 IgG induced pain. Papers III and IV: Endogenously citrullinated fibrinogen peptides were found to be recognized by a large portion of sera from the Epidemiological Investigation of RA (EIRA) cohort. CCP2+ RA sera displayed 65% and 15 % immune reactivity against peptides from the fibrinogen α chain Cit573 (563-583) and Cit591 (580-600), respectively. Peptides from the β chain, Cit72 (62-81) and Cit74 (62-81), were recognized by 35% and 53% of the CCP2+ RA patients. The same fibrinogen peptides were shown to in vitro individually bind and block purified aCCP2 IgG in a dose-dependent manner, displaying a maximum of 83% blockade. Approximately 90% autoantibodies were neutralized by Cit573 and Cit591 combined, and further improvement of the blocking capacity was registered (>90%) when incubating aCCP2 IgG with a cyclic version of Cit573. Since the peptide Cit573 (also termed [Cit573]fib(563-583)) showed the best inhibition percentage, a truncated version was inserted into the stable framework sunflower trypsin inhibitor-1 (SFTI-1, and denoted s1[Cit573]fib(566-580)). This compound showed enhanced blocking capacity, 79% antibodies were neutralized with an estimated IC50 of 20 μM, in comparison to the linear counterparts (73% maximum inhibition with the IC50:s ranging from 59 to 123 μM). Stability in blood was also improved, with above 90% of s1[Cit573]fib(566-580) remaining after five hours, whereas the cyclic and linear counterparts were degraded after one hour. Using a mutant of s1, s2[Cit573,Arg575]fib(566-580), anti-[Cit573]fib(563-583) IgG (aCit573 IgG) were purified from RA plasma and estimated to comprise 0.33% of the total RA IgG pool. Binding affinity measurements demonstrated that aCit573 IgG bind the mutant scaffold peptide s3[Cit573,Arg575]fib(566-580) with high affinity (Kd = 2 nM). Thus, subsets of ACPA recognizing citrullinated fibrinogen epitopes appear to be of high affinity. Paper V: The cytoplasmic autoantigen HisRS was found in extracellular compartments (bronchoalveolar lavage, BAL, sera and plasma). High circulating levels of HisRS were found in myositis, and further increased in anti-HisRS+ patients; significant levels of HisRS were also detected in healthy individuals, whereas anti-HisRS IgG, IgA and IgM autoantibodies were only detected in sera and BAL from patients with myositis. In addition, anti-TRIM21 IgG were also identified in myositis BAL, positively correlating with the presence of anti-HisRS IgG. A so far uncharacterized factor in BAL of myositis patients was found to bind exogenous HisRS. In experiments addressing platelet activation, HisRS was found to trigger platelet activation in a dose-response dependent manner at low picomolar concentrations. In conclusion, the development of a methodology to isolate autoantibodies from RA patients’ plasma, serum and synovial fluid, provided opportunities to address the pathogenic role of ACPA. Purified aCCP2 IgG induced pain-like behaviour, raising a possible mechanism for the pain RA patients occasionally feel before clinical onset of disease or after being medicated and in remission. A stable, high affinity anti-citrullinated fibrinogen autoantibody blocking compound was designed and we propose ACPA neutralization with fibrinogen-derived peptides as a complimentary treatment strategy for ACPA+RA. ACPA pain mediated effect may also be a functional target, amenable for blocking. The presence of HisRS extracellularly suggests novel and so far undescribed functions, which merits further investigations. Finally, a possible coupled immune response among HisRS and TRIM21 in lungs of myositis patients provides new clues for the development of autoimmunity in myositis and the associated anti-synthetase syndrome

T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation

Human leukocyte antigen (HLA)-DPB1 antigens are mismatched in approximately 70% of allogeneic hematopoietic stem cell transplantations (allo-HSCT) from HLA 10/10 matched unrelated donors. HLA-DP-mismatched transplantation was shown to be associated with an increase in acute graft-versus-host disease (GVHD) and a decreased risk of leukemia relapse due to the graft-versus-leukemia (GVL) effect. Immunotherapy targeting mismatched HLA-DP is considered reasonable to treat leukemia following allo-HCT if performed under non-inflammatory conditions. Therefore, we isolated CD4^+ T cell clones that recognize mismatched HLA-DPB1 from healthy volunteer donors and generated T cell receptor (TCR)-gene-modified T cells for future clinical applications. Detailed analysis of TCR-T cells expressing TCR from candidate clone #17 demonstrated specificity to myeloid and monocytic leukemia cell lines that even expressed low levels of targeted HLA-DP. However, they did not react to non-hematopoietic cell lines with a substantial level of targeted HLA-DP expression, suggesting that the TCR recognized antigenic peptide is only present in some hematopoietic cells. This study demonstrated that induction of T cells specific for HLA-DP, consisting of hematopoietic cell lineage-derived peptide and redirection of T cells with cloned TCR cDNA by gene transfer, is feasible when using careful specificity analysis.departmental bulletin pape

Assessment of TWIST1 as an immunotherapeutic target of cancer

CD8+ T lymphocytes are key mediators of anti-tumour immunity, eliminating tumour cells through the recognition of tumour antigens. Increasing the number of characterised tumour antigens, especially those with highly specific tumour expression, may enable the development of more effective immunotherapy of cancers. TWIST1 is a basic-helix-loop-helix transcription factor (bHLB) with an important role in cell lineage determination and differentiation. It is expressed by a number of carcinomas where it functions as a pro-metastatic oncogene, but is absent or expressed at low levels in normal tissues. The main aim of this study was to investigate whether anti-TWIST1 immune responses could be generated and used to target cancer cells. Two potential HLA-A0201 restricted TWIST1-derived epitopes, SLNEAFAAL and KLAARYIDFL (referred to as SLN and KLA) were identified by in silico prediction methods and their binding to HLA-A*0201 confirmed in vitro. The peptides were assessed for their capacity to induce specific immune responses by generating cytotoxic T lymphocyte (CTL) lines from the peripheral blood of HLA-A2-positive healthy donors. SLN peptide-specific CTLs were detected in 1 out of 5 healthy donors by peptide/MHC class I pentamers and the CTL line generated showed specific cytotoxicity and the release of interferon-γ on recognition of T2 target cells pulsed with SLN. KLA peptide-specific CTLs were not detected in the four healthy HLA-A2-positive donors tested. The immunogenicity of KLA was also assessed by peptide immunisation of HLA-A*0201 transgenic mice and the in vitro stimulation of alloreactive peptide-specific CD8+ T cells from HLA-A2-negative healthy blood donors. CTLs capable of specifically killing T2 cells pulsed with KLA peptide were isolated from an alloreactive CTL bulk line using peptide/HLA-A*0201 pentamer reagents and magnetic cell sorting. The data presented here shows the existence of functional anti-TWIST1 CTL precursors within the autologous and allogeneic HLA-A*0201-restricted T cell repertoires of healthy donors, and therefore merits the further evaluation of SLN and KLA as target epitopes for the treatment of TWIST1+ tumours

Effect of Extracellular Survivin and Lymphoma Exosomes on Natural Killer Cells

Tumors alter their microenvironment to promote survival using methods such as angiogenesis promotion, growth signals, and immune suppression. The immune system becomes unresponsive to transformed neoplastic cells through a variety of methods including T cell suppression, increased myeloid-derived suppressor cells (MDSCs), and reduced natural killer (NK) cell activity. NK cells have inherent killing capabilities and thus are among the first responders in recognizing and destroying abnormal cells. However, many types of cancers inhibit the surveillance and cytotoxic abilities of NK cells by releasing exosomes, vesicles that can modulate the tumor microenvironment (TME) and intercellular communication for the purpose of enhancing tumor malignancy. These 30-150 nm sized lipid bound vesicles are secreted by many cell types, including immune cells and tumor cells, and the specific protein, lipid, mRNA and miRNA contents contribute to the complex intercellular communication occurring between malignant and normal cells. Cancer patients often have increased numbers of exosomes circulating through their body, including patients with hematological malignancies, such as lymphoma. The focus of this research was to determine the interactions between B cell lymphoma exosomes and NK cells, and characterize the resultant effects on NK cell function. A specific objective of this research was to determine whether Survivin, an Inhibitor of Apoptosis protein recently found to be localized within exosomes, has a role in modulating NK cells similar to previous findings of T cell modulations. We report that lymphoma exosomes have low levels of internalization into NK cells, and no detectable presence of immune modulating proteins MICA/B or TGF-β. Exposure of NK cells to lymphoma exosomes did not result in observable changes in degranulation or cytotoxic ability. However, treatment with recombinant Survivin protein was able to decrease NKG2D receptor levels in NK cells stimulated with target cells, and decrease protein levels of TNF-α, IFN-γ, perforin, and Granzyme B. A better understanding of the underlying processes by which Survivin or exosomes suppress immune cells in the TME may pave the way to more efficacious immunological therapies against cancer

Characterization of a stem cell population derived from human peripheral blood and its therapeutic potential in brain tumors

Adherent CD34+ cells are a stem cell enriched population with a high frequency of primitive stem cells that are genetically primed to differentiate into tissue-specific lineages. These cells make up the adherent CD34+ cells in peripheral blood which are the focus of this study. The first aim of this study was to transcriptionally characterize adherent CD34+ cells relative to nonadherent CD34+ cells using a whole human genome Affymetrix U133 plus 2 array. The subsequent analysis demonstrated transcriptional differences in genes which are involved in quiescence, cell cycle, homing and adhesion, which are likely to be relevant to the suitability of adherent CD34+ cells for clinical application. To verify the microarray analysis, a selection of representative transcripts was chosen and their relative expression was compared using real-time qPCR. To analyze further the stem cell characteristics of adherent CD34+ cells, the DNA replication timing kinetics of pluripotency-associated genes in bone marrow-derived adherent CD34+ cells, non-adherent CD34+ cells, and foetal mesenchymal stem cells were conducted and compared to those of embryonic stem (ES) cells. This showed that pluripotency-associated genes Oct4 and Nanog replicate early in the cell cycle similar to ES cells. The second aim of this study was to examine the potential use of adherent CD34+ cells in neural injury. As adherent CD34+ cells are primitive stem cells, it was reasonable to hypothesize that these cells would differentiate into neural cells in vitro, but these experiments revealed that these cells are not neurogenic and, in our hands, do not differentiate into neural cells. The experiments revealed that adherent CD34+ cells differentiate efficiently and reproducibly into microglia-like cells (CD34MG cells). CD34MG cells upregulated microglia-associated antigens and demonstrated functional characteristics of microglia cells including TNF and IL-6 release in response to lipopolysaccharide. CD34MG also stimulate responders in a mixed lymphocyte reaction, suggesting antigen presenting capabilities. To determine if these cells might be exploited for use in brain tumour therapy, it was shown that CD34MG migrate and invade in response to brain tumour glioma cells. Results also suggest that these cells are capable of presenting glioma specific antigen to immune cells in an autologous lymphocyte reaction in vitro. The combined results suggest that CD34MG cells might be exploited for therapeutic purposes in brain tumours. Stem cells are currently being explored for their therapeutic potential. This study demonstrates that adherent CD34+ cells and CD34MG cells may be useful for clinical use

HLA alloreactivity by human viral specific memory T-cells

The research described in this thesis investigates the effect of viral infection on the risk of rejection. It was previously thought that a viral specific T-cell receptor could not crossreact against allogeneic HLA molecules, however here it is proven that this is not only possible but is also common. The research performed in this thesis has important clinical implications for solid organ (kidney) and bone marrow transplantation. Viral infection commonly generates alloreactive T-cells, and may therefore be responsible for the failure of tolerance inducing regimens in primates and humans. It is shown that vaccination can also induce HLA-specific alloreactive T-cell responses and it is therefore recommended that vaccines be given to transplantation candidates at least three months prior to expected transplantation date. It is also shown that alloreactive T-cells can be tissue specific. In the final chapter of the thesis thesis it is shown that allogeneic cell therapy could conversely be useful to elicit a viral specific T-cell response in patients with infections, such as HIV.Financial support for the publication of this thesis was provided by (in alphabetical order): Astellas Pharma B.V., Beckman Coulter B.V., Europdonor, J.E. Jurriaanse Stichting, National Reference Centre for Histocompatibility, Nederlandse Transplantatie Vereniging, Novartis Pharma B.V., Pfizer B.V., Roche B.V., U-CyTech Biosciences and VPS DiagnosticsUBL - phd migration 201