Polyclonal Expansion Research Articles

Increased IgG4 responses to multiple food and animal antigens indicate a polyclonal expansion and differentiation of pre-existing B cells in IgG4-related disease

BackgroundIgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition, characterised by an elevated serum IgG4 concentration and abundant IgG4-positive plasma cells in the involved organs. An important question is whether the...

Large-scale in vitro expansion of human regulatory T cells with potent xenoantigen-specific suppression.

Xenotransplantation is a potential solution to the organ donor shortage. Immunosuppression is required for successful application of xenotransplantation but may lead to infection and cancer. Thus, strategies for immune tolerance induction need to be developed. Polyclonal regulatory T cells (Treg) play a central role in the induction and maintenance of immune tolerance and have been shown to protect against islet xenograft rejection in vivo. However, global immune suppression may be mediated by polyclonal Treg immunotherapy and a simple method for in vitro expansion of xenoantigen-specific Treg for efficient Treg application becomes necessary. Human Treg isolated from peripheral blood mononuclear cells (PBMCs) were initially cultured with anti-CD3/CD28 beads, rapamycin and IL-2 for 7days as polyclonal expansion. Expanded Treg were then cocultured with irradiated porcine PBMC as xenoantigen stimulation for three subsequent cycles with 7days for each cycle in the presence of IL-2 and anti-CD3/CD28 beads. Treg phenotype and suppressive capacity were assessed after each cycle of xenoantigen stimulation. Treg expanded with one cycle of xenoantigen stimulation retained Treg suppressive phenotype but acquired no xenoantigen specificity along with poor expansion efficiency, whereas expansion with two-cycle xenoantigen stimulation resulted in not only more than 800-fold Treg expansion but highly suppressive xenoantigen-specific Treg with effector Treg phenotype. However further increase of stimulation cycles resulted in reduced Treg suppressive potency. This study provides a simple approach to obtain high numbers of xenoantigen-specific Treg for immune tolerance induction in xenotransplantation.

Evaluation of CD69 expression as a prognosticator in chronic lymphocytic leukemia

Introduction Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the western world. It is a malignancy of mature B cells involving the blood, bone marrow (BM), and lymphoid tissues, and its cells arise from polyclonal expansion of CD5+ B lymphocytes transformed into a monoclonal population by mutational agents. CD69 is an integral membrane protein belonging to the lectin family. It is expressed after activation in all BM-derived cells except erythrocytes. CLL exhibits features of activated and antigen-experienced B lymphocytes and CD69 overexpression. CD69 is significantly correlated with poor clinical and biological prognostic factors, and this supports its introduction into routine laboratory assessment and, possibly, in a prognostic scoring system for CLL after an adequate standardization process. Objective The aim of this study was to detect CD69 expression in newly diagnosed patients with CLL by flow cytometry and correlate it with clinical and laboratory parameters to evaluate it as a prognostic factor. Patients and methods This study was conducted on 40 B-CLL patients who attended Ain Shams University Hospitals over 1 year. All patients were subjected to full medical history and clinical examination, RAI staging according to disease burden and the degree of BM involvement, abdominal ultrasonography, complete blood count with examination of peripheral blood smears, BM aspiration with morphological examination, and immunophenotyping of BM or whole peripheral blood applying monoclonal antibodies CD20, CD79b, FMC7, serum IgM, CD5/CD19, CD10, CD103, CD123, CD23, and CD38, and κ and λ light chains and CD69 expression. Results In the current study all of the studied B-CLL patients expressed CD69. Among the 40 studied patients 23 had high CD69 expression (group I) and 17 had low CD69 expression (group II). A highly significant elevation in group I patients compared with group II patients was found (P = 0.000). A highly significant reduction in hemoglobin (Hb) level (P = 0.001), elevation in lactate dehydrogenase concentration (P = 0.006), elevation in RAI staging, and elevation in CD38 expression (P = 0.005) were observed in group I. A highly significant negative correlation was found between CD69% and Hb level (P = 0.008) and platelet count (P = 0.009). A highly significant positive correlation was found between CD69% and hepatomegaly (P = 0.008) and RAI stage (P = 0.008) and significant positive correlation was found between CD69% and CD38% expression (P = 0.012). An association study was conducted between CD69% expression and all the standard prognostic factors in B-CLL patients. There was a highly significant association between CD69% expression and presence of hepatomegaly (P = 0.002) and a significant association between CD69% expression and presence of splenomegaly (P = 0.028) and low Hb levels (P = 0.013). Conclusion Several clinical and biological variables have been reported to predict the outcome of CLL patients, such as advanced patient age, male sex, higher absolute lymphocyte count, greater extent of lymphadenopathy, and raised serum β2-microglobulin levels, all of which are associated with inferior prognosis. In the current study all of the studied B-CLL patients expressed CD69. CLL patients were divided into two subsets with significantly different prognosis: those with high CD69 (group I) (≥30%) and those with low CD69 (group II) (<30%) expression; both were compared with respect to the different studied parameters. There was highly significant elevation of CD69 in group I compared with group II. There was a highly significant reduction in Hb level and elevation in lactate dehydrogenase concentration in group I patients in comparison with group II patients. A highly significant relation was found between the two groups with respect to RAI staging: an advanced RAI stage was found among group I patients in comparison with group II patients. The current study evaluated CD69 as a prognosticator and studied the significant association of its high expression with the standard prognostic factors, notably splenomegaly (P = 0.028) and hepatomegaly (P = 0.002), low Hb level, low platelet count, and advanced RAI stage. CD69 expression is associated with both immunoglobulin variable heavy chain mutation status and survival. It is recommended to perform a larger prospective study on CD69 expression in B-CLL patients, studying its relation to overall survival and progression-free survival and its re-evaluation during the course of treatment as we recommend assessment of this marker by flow cytometry among independent prognostic markers in B-CLL.

Allogeneic inducible regulatory T cells generated with retinoic acid allow the generation of mixed chimerism via nonmyeloablative conditioning in mice models

Allogeneic inducible regulatory T cells generated with retinoic acid allow the generation of mixed chimerism via nonmyeloablative conditioning in mice models

Occurrence of Non-Hodgkin Lymphoma after the Diagnosis of Persistent Polyclonal B-cell Lymphocytosis

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare indolent condition characterized by a polyclonal expansion of B cells associated with binucleated lymphocytes observable on blood smears. Though polyclonal, recurrent genetic abnormalities have been described in PPBL, supernumerary isochromosome 3q (+i(3)(q10)) being the most frequently described. We report here two clinical observations showing the occurrence of NHL after the diagnosis of PPBL. These observations lead us to recommend a closer follow-up of PPBL patients and raise the question of a relationship between PPBL and NHL.

Increasing TCR Zeta Expression and Maintaining the Clonality of T Cells from AML Patients after IL-2, IL-7 and IL-12 Induction

T-cell immunodeficiency is a common feature in patients with leukemia, lower activation of T cells was one of reasons. The TCR zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T cell activation. The proliferation and activation of T cells may be inducted by T cell related cytokines. In this study, we explored the change of TCR zeta gene expression and the clonality of T cells after induction with different immune cytokines, including IL-2, IL-7 or IL-12. CD3+ T cells sorted from peripheral blood of 4 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The expression levels of TCR zeta gene and related genes in T cells before and after induction were then analyzed by fluorescence quantitative RT-PCR. The distribution and clonality of TCR Vβ subfamily T cells were analyzed by RT-PCR and Genescan techniques. Increasing expression levels of TCR zeta gene and zap-70 (TCR zeta chain associated-protein) gene in CD3+T cells from AML patients were found after induction with single stimulating factor or the combination with different cytokines, while the expression of FcεRIγ (TCR ζ gene complementary factor) was down-regulated. We further compared the T cell clonality in CD3+T cells from AML patients after cytokine induction, eight to 22 TCR V β subfamilies could be detected in T cells from AML cases, most of them displayed polyclonal expansion. The number of expressed TCR Vβ subfamilies was increased without the change of clonality in T cells induced by CD3+CD28+IL7. In conclusion, TCR zeta gene and its related genes could be upregulated through induction with different cytokine combination such as IL-2, IL-7 and IL-12, therefore to improve the T cell activation in patients with AML. And the main effect of cytokines might to maintain the T cell clonality and nonspecific amplification of T cell clones. Further investigation can be designed to amplify the specific anti-AML TCR Vβ clones using AML associated antigens and such cytokine combination. DisclosuresShi:National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325). : Research Funding.

A Recurrent T-Cell Receptor γ Rearrangement of the Vγ9-JγP Type Represents the Major Clone of γδ T-Cells in Normal Individuals and Is Highly Suppressed in Lymphoproliferative Disorders

INTRODUCTIONγδ T-cells expressing T-cell receptor (TCR) type Vγ9Vδ2 are well-established mediators of anti-tumor immunity and have demonstrable HLA-independent cytotoxic activity against both B- and T-cell non-Hodgkin lymphoma cells. Initial clinical trials using ex vivo expanded, adoptively transferred γδ T-cells to enhance tumor immunity have shown some promise but overall results have been lackluster. This indicates that wholesale polyclonal expansion prior to transfer is not likely to be effective. Thus more refined approaches are necessary. This requires a better understanding of their TCR repertoire. We recently developed a next generation sequencing (NGS)-based method for evaluating the spectrum T-cell receptor gamma (TRG) gene rearrangements present in clinical samples. In an effort to better understand the TCR repertoire of γδ T-cells we used this strategy to evaluate a series of samples from normal individuals and from individuals with B and T-cell lymphoproliferative disorders (LPDs).METHODSDNA was isolated from samples from 11 normal individuals (all peripheral blood, PB), 11 patients with a T-cell LPD (6 PB, 3 bone marrow; BM, 2 FFPE tissues), and 5 patients with a B-cell LPD (Hairy cell leukemia; HCL, 4 PB, 1 BM). γδ T-cells were sorted from the PB of 4 of the healthy donors by FACS. TRG rearrangements were PCR amplified using consensus primers and NGS libraries were prepared and sequenced on the Ion Torrent PGM platform. The data was analyzed as follows. NGS typically yielded up to 400,000 sequencing reads which were grouped by identical V, J, and CDR3 sequences into unique rearrangements (typically 15-30,000). The prevalence of a particular TRG rearrangement (or CDR3 sequence) was determined by the number of individual NGS reads with this unique sequence per the entire data set (percent of total reads). All rearrangements were then ranked by their prevalence.RESULTSWe sequenced the TRG repertoire of isolated γδ T-cells from the peripheral blood of normal individuals (n=4). We found that a recurrent Vγ9-JγP rearrangement with the CDR3 sequence CALWEVQELGKKIKVF was always (4 of 4 samples) the most prevalent rearrangement in normal γδ T-cells (3.2-11.7-fold more prevalent than the second most common rearrangement; representing 4-11.9% of total reads). Similarly, analysis of a larger set of unsorted normal peripheral blood samples demonstrated high prevalence of the same canonical CDR3 in 5 out of 7 samples, confirming that it is very common in most individuals relative to all TRG rearrangements (among the top 10 most prevalent CDR3s in 4 of 5 samples). We also sequenced the TRG repertoire in 11 samples from patients demonstrating evidence of involvement by a T-cell LPD. Unexpectedly, all Vγ9-JγP type rearrangements were strongly suppressed in these samples including the one with the canonical CDR3. The canonical rearrangement (present in 6 /11 cases), or the most abundant Vγ9-JγP rearrangement when the canonical rearrangement was absent (absent in 5/11 cases), represented on average 0.036 ±0.024 % of all NGS reads in the samples from patients with T-cell LPDs compared to 0.72 ±0.72 % of total NGS reads in the normal controls. This represents on average a 19.9 fold reduction in the T-cell LPD samples. The median rank by abundance of the top Vγ9-JγP rearrangement dropped from 9th (normals) to 291st (T-cell LPD cases) indicating that the suppression was not simply a consequence of the presence of an abundant malignant T-cell clone in the data. The overall distribution of TRG V-segment usage in the normal and neoplastic samples was comparable (Vγ9: 9.9 ±0.55 % and 7.2 ±2.79 %, respectively). In 2 samples from patients with HCL, Vγ9-JγP rearrangements were reduced to a similar extent to that seen in the T-cell LPD cases (9.5-fold reduced; average 0.06% of NGS reads; average rank order 451st). In the other 3 cases of HCL the findings were very similar to those seen in the normal samples.CONCLUSIONSWe identified a recurrent Vγ9-JγP rearrangement by NGS representing the most abundant CDR3 in sorted γδ T-cells from normal individuals. This population, along with other clones with Vγ9 rearrangements, appeared specifically suppressed in all samples from patients with T-cell LPDs and in 2 or 5 samples from patients with B-cell LPDs (HCL), perhaps indicating a role in the disease process. Additional samples from a wider range of T- and B-cell LPDs are being analyzed. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2884: Immune-based therapy with human tumor lysate, expressing α-gal epitopes induce significant B cell response and in vivo tumor destruction against pancreatic cancer

Abstract Current strategies in the treatment of advanced pancreatic cancer (PC) offer little hope for a cure. Now treatment modalities, including immunotherapy are warranted for PC, which easily metastasizes in many organs. Although MUC1 and Mesothelin are potential targets for the immunotherapy, vaccination against a single tumor-associated antigen (TAA) seems to be insufficient. In our previous study, we showed the in vitro and in vivo effectiveness of immunotherapy though vaccination with whole PC cells engineered to express α-gal epitopes (Cancer Res; 70(13); 5259-69, 2010). It was thought that the immunogenicity of well-known as well as unknown multiple TAAs in cancer cells was upregulated by addition of α-gal epitopes and these TAAs ultimately lead to polyclonal expansion of both B and T cells. For clinical development of more effective immunotherapy, we proposed that human tumor lysate originated from resected PC is more suitable because it contains several TAAs that could elicit a marked anti-tumor response. The present study addresses the effectiveness of elicitation of both antibody production and in vivo destruction against PC by vaccination with human tumor lysate from PC patients, expressing α-gal epitopes enzymatically. Tumor specimens were obtained from 11 patients at the time of surgical resection. To express α-gal epitopes, we cloned the α1,3galactosyltransferas (α1,3GT) from a New World Monkey and expressed it in a soluble form in the yeast expression system of Pichia pastoris. For synthesis of α-gal epitopes, tumor tissues were homogenized in MES buffer, and incubated with MnCl2, UDP-Gal and α1,3GT for 4 hours at 37°C. α1,3GT KO mice were immunized with pig tissue to generate anti-Gal Ab in their sera. These mice were vaccinated intraperitoneally by either unsynthesized (control group) or α-gal tumor lysate (α-gal group). Production of anti-PC cell Ab in α-gal group was 4∼8-fold higher than that of control group. Anti-TAA Ab production was also markedly increased in comparison with that in control group (anti-MUC1 2-fold, anti-Mesothelin 4-fold). Expansion of TAAs-specific B cells was significantly higher [number of spots at 1×106 splenocytes: MUC1: α-gal vs. control; 414±26 vs. 178±46 (p=0.015), Mesothelin: 401±56 vs. 209±19 (p=0.03)]. To demonstrate in vivo tumor destruction, an animal experiment was performed. Splenocytes from vaccinated KO mice were prepared, and then transferred intraperitoneally (90×106 cells) into NOD/SCID mice. Followed by transferring, mice were challenged with 1×107 of live PANC-1 cells. In mice from α-gal group, regrowth of tumors was significantly prevented and survival period was significantly prolonged [α-gal vs. control: 91.5 vs. 43.3 days (p=0.03)]. We conclude that immunotherapy with α-gal tumor lysate obtained from PC patients with enzymatic engineering could be more effective and practicable methods for PC treatment. Citation Format: Kenta Furukawa, Masahiro Tanemura, Eiji Miyoshi, Hiroaki Nagano, Hidetoshi Eguchi, Toshimitsu Irei, Masashi Inoue, Shinya Yamashita, Yoshito Tomimaru, Naoki Hama, Hiroshi Wada, Koichi Kawamoto, Shogo Kobayashi, Katsuyoshi Matsunami, Kiyomi Taniyama, Wataru Kamiike, Masaki Mori, Yuichiro Doki. Immune-based therapy with human tumor lysate, expressing α-gal epitopes induce significant B cell response and in vivo tumor destruction against pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2884. doi:10.1158/1538-7445.AM2014-2884

Expression of the Inherently Autoreactive Idiotope 9G4 on Autoantibodies to Citrullinated Peptides and on Rheumatoid Factors in Patients with Early and Established Rheumatoid Arthritis

BackgroundThe pre-symptomatic stage of Rheumatoid arthritis (RA) is associated with pro-inflammatory cytokines and autoantibodies. High levels and epitope spread by Rheumatoid factors (RhF) and autoantibodies to citrullinated proteins signify progression towards disease expression. In established RA, the persistence of high autoantibody levels reflects production by both long-lived plasma cells and short-lived plasmablasts. Neither the relative contributions to pathogenesis by autoantibodies from either source, nor the factors responsible for deciding the fate of autoantigen specific ‘parent’ B-cells, is understood. Phenotypic markers identifying subsets of autoreactive B-cells are therefore of interest in understanding the origin and perpetuation of the autoimmune response in RA. One such phenotypic marker is the rat monoclonal antibody, 9G4, which recognises an idiotope on immunoglobuins derived from the inherently autoreactive VH-gene, VH4-34. We therefore investigated whether the 9G4 idiotope was expressed on autoantibodies in patients with RA.Methodology/Principal FindingsSera from 19 patients with established RA and those with <1year history of untreated polyarthritis either resolving into RA (n = 42) or non-RA diagnosis (n = 31) were included. Autoantibodies to cyclic citrullinated peptides (CCP), RhF and co-expression of the 9G4 idiotope were measured by ELISA. 9G4 recognised a population of anti-CCP antibodies in the majority of sera from patients with established disease and also in samples from patients with early disaese. 9G4+RhF levels were generally lower and not associated with positivity for, or levels of 9G4+CCP.Conclusions/SignificanceThe persistence of 9G4+ immunoglobulins, of any isotype, in serum is rare. We describe here the novel finding of 9G4 expression on anti-CCP antibodies in patients from the earliest symptoms of RA through to established disease. Our results suggest that 9G4 expression on anti-CCP autoantibodies was not due to polyclonal expansion of VH4-34-encoded immunoglobulins. These studies may therefore provide a new focus for investigation into the evolution of the autoimmune response in RA patients.

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV, PRRSV or PCV2.

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2—CD21+ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2+CD8α+ γδ cells and polyclonal expansion of major Vβ families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II+ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3+ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0091-x) contains supplementary material, which is available to authorized users.

Antigen-specific culture of memory-like CD8 T cells for adoptive immunotherapy.

Cytotoxic T cells typically are expanded ex vivo in culture with IL2 for adoptive immunotherapy. This culture period leads to a differentiated phenotype and acquisition of effector function, as well as a loss of in vivo proliferative capability and antitumor efficacy. Here, we report antigen-specific and polyclonal expansion of cytotoxic T cells in a cocktail of cytokines and small molecules that leads to a memory-like phenotype in mouse and human cells even during extended culture, leading to enhanced in vivo expansion and tumor control in mice.

THU0364 Autoimmune Manifestations in Patients with Multicentric Castleman's Disease

BackgroundCastleman's disease (CD) is a lymphoproliferative disorder characterized by polyclonal expansion of B lymphocytes, which features 2 clinical subtypes: unicentric and multicentric, and 3 histopathological variants: hyaline vascular, plasma cell,...

Immunological derangement in hypocellular myelodysplastic syndromes.

Hypocellular or hypoplastic myelodysplastic syndromes (HMDS) are a distinct subgroup accounting for 10-15% of all MDS patients, that are characterized by the presence of bone marrow (BM) hypocellularity, various degree of dysmyelopoiesis and sometimes abnormal karyotype. Laboratory and clinical evidence suggest that HMDS share several immune-mediated pathogenic mechanisms with acquired idiopathic aplastic anemia (AA). Different immune-mediated mechanisms have been documented in the damage of marrow hematopoietic progenitors occurring in HMDS; they include oligoclonal expansion of cytotoxic T lymphocytes (CTLs), polyclonal expansion of various subtypes of T helper lymphocytes, overexpression of FAS-L and of the TNF-related apoptosis-inducing ligand (TRAIL), underexpression of Flice-like inhibitory protein long isoform (FLIPL) in marrow cells as well as higher release of Th1 cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). It has also been documented that some HMDS patients have higher frequency of polymorphisms linked both to high production of proinflammatory cytokines such as TNF-α and transforming growth factor-β and to the inhibition of T-cell mediated immune responses such as interleukin-10, further suggesting that immune-mediated mechanisms similar to those seen in AA patients may also operate in HMDS. Clinically, the strongest evidence for immune-mediated hematopoietic suppression in some HMDS is the response to immunosuppression including mainly cyclosporine, anti-thymocyte globulin and/or cyclosporine, or alemtuzumab. Here we review all these immune mechanisms as well as the influence of this deranged cellular and humoral immunologic mileau on the initiation and possible progression of MDS. All these observations are pivotal not only for a better understanding of MDS pathophysiology, but also for their immediate clinical implications, eventually leading to the identification of MDS patients who may benefit from immunosuppression.

Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR–MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR–MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR–MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein–Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR–MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR–MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR–MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus.Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.

Enhanced Suppression of the Xenogeneic T-Cell Response In Vitro by Xenoantigen Stimulated and Expanded Regulatory T Cells

Strategies to prevent xenograft rejection while minimizing long-term immunosuppression need to be developed for successful xenotransplantaion. Polyclonally expanded human regulatory T cells (Treg) are known to suppress xenogeneic responses in vivo and in vitro. However, the risk of opportunistic infection and malignancy and the requirement for large numbers of Treg for effective suppression remain drawbacks to their clinical application. This study aimed to expand human Treg with xenoantigen stimulation and to assess their effectiveness at suppressing the xenoimmune response. Human CD4CD25CD127 Treg were stimulated with anti-CD3/CD28 beads, interleukin (IL)-2, and rapamycin for polyclonal expansion. After 7 days, Treg were further expanded with two subsequent cycles of either polyclonal stimulation or xenoantigen stimulation with irradiated porcine peripheral blood mononuclear cells with or without anti-pig SLA CII monoclonal antibody. Treg phenotype and suppressive capacity were assessed after xenoantigen stimulation. Porcine xenoantigen-stimulated Treg retained Treg phenotype but had an increased expression of human leukocyte antigen-DR, inducible costimulator, and CD45RO when compared with their polyclonally stimulated counterparts. In a pig-human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Treg demonstrated an enhanced suppressive capacity at higher ratios of responder cells:Treg and secreted higher concentrations of IL-10 and IL-35, although they were equally suppressive as polyclonally stimulated Treg in an allogeneic or polyclonal MLR. When Treg expanded in the presence of anti-pig SLA monoclonal antibody were used in the same pig-human MLR, their suppressive capacity was reduced substantially. Xenoantigen-stimulated Treg show enhanced suppressive capacity in the pig-human MLR most likely via an IL-10-mediated pathway.

SAT0205 Blys up-regulation and salivary clonal B-cell expansion characterize sjögren’s syndrome-related lymphoproliferative disorders and correlate with the new ESSDAI score

BackgroundPrimary Sjögren’s syndrome (SS) is characterized by an increased risk of lymphoma in patients with prelymphomatous manifestations (i.e., myoepithelial sialadenitis (MESA) or mixed cryoglobulinemia). Increased levels of B-lymphocyte stimulator (BLyS)...

Human T cells depend on functional calcineurin, tumour necrosis factor-α and CD80/CD86 for expansion and activation in mice

Dysregulated T cells are a hallmark of several autoimmune and inflammatory diseases; thus, models to study human T cells in vivo are advantageous, but limited by lacking insight into human T cell functionality in mice. Using non-obese diabetic (NOD), severe combined immunodeficient (SCID) or recombination activating gene-1 (RAG1)(-/-) and interleukin-2 receptor gamma-chain (IL-2Rγ)(-/-) mice reconstituted with human peripheral blood mononuclear cells (PBMCs), we have studied the mechanisms of human T cell expansion and activation in mice. Injection of human PBMCs into mice caused consistent xeno-engraftment with polyclonal expansion and activation of functional human T cells and production of human cytokines. Human T cell expansion coincided with development of a graft-versus-host disease (GVHD)-like condition observed as weight loss, multi-organ immune infiltration and liver damage. CD8(+) T cells alone were sufficient for expansion and required for disease development; in contrast, CD4(+) T cells alone expanded but did not induce acute disease and, rather, exerted regulatory capacity through CD25(+)CD4(+) T cells. Using various anti-inflammatory compounds, we demonstrated that several T cell-activation pathways controlled T cell expansion and disease development, including calcineurin-, tumour necrosis factor-α and co-stimulatory signalling via the CD80/CD86 pathway, indicating the diverse modes of action used by human T cells during expansion and activation in mice as well as the pharmacological relevance of this model. Overall, these data provide insight into the mechanisms used by human T cells during expansion and activation in mice, and we speculate that PBMC-injected mice may be useful to study intrinsic human T cell functions in vivo and to test T cell-targeting compounds.

Epstein-Barr virus-driven lymphomagenesis in the context of human immunodeficiency virus type 1 infection

Epstein–Barr virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In human immunodeficiency virus type 1 (HIV-1) infected patients, the impaired immunosurveillance against EBV may favor the development of EBV-related diseases, ranging from lymphoproliferative disorders to B cell non-Hodgkin’s lymphomas (NHL). Antiretroviral therapy (ART) has significantly modified the natural course of HIV-1 infection, resulting in decreased HIV-1 plasmaviremia, increased CD4 lymphocytes, and decreased opportunistic infections, indicating a restoration of immune functions. However, the impact of ART appears to be less favorable on EBV-related malignancies than on other AIDS-defining tumors, such as Kaposi’s sarcoma, and NHL remains the most common cancer during the ART era. EBV-driven tumors are associated with selective expression of latent oncogenic proteins, but uncontrolled lytic cycle with virus replication and/or reactivation may favor cell transformation, at least in the early phases. Several host’s factors may promote EBV reactivation and replication; besides immunodepression, inflammation/chronic immune stimulation may play an important role. Microbial pathogen-associated molecular patterns and endogenous damage-associated molecular patterns, through Toll-like receptors, activate the immune system and may promote EBV reactivation and/or polyclonal expansion of EBV-infected cells. A body of evidence suggests that chronic immune stimulation is a hallmark of HIV-1 pathogenesis and may persist even in ART-treated patients. This review focuses on lymphomagenesis driven by EBV both in the context of the natural history of HIV-1 infection and in ART-treated patients. Understanding the mechanisms involved in the expansion of EBV-infected cells is a premise for the identification of prognostic markers of EBV-associated malignancies.

In Vitro Expansion of Highly Suppressive Xenoantigen-Specific Human Regulatory T Cells for Immunotherapy in Xenotransplantation

Introduction: Lifelong immunosuression is required to prevent xenograft rejection. Strategies to modulate the xenoimmune response for clinical benefit while avoiding long-term immunosuppression need be developed. Poyclonally expanded human regulatory T cells (Treg) have shown to suppress xenogeneic response in vivo. However the risk for opportunistic infection and tumor-growth and also the requirement of large numbers of polyclonally expanded Treg for effective suppression remain drawbacks for their clinical application. These difficulties could be overcome by using antigen-specific Treg. This study aimed to expand human Treg with xenoantigen-specificity in vitro for immunomodulation in xenotransplantation. Methods: Human CD4+CD25+CD127- Treg were isolated from peripheral blood mononuclear cells (PBMC) and stimulated with anti-CD3/CD28 beads, IL-2 and rapamycin for polyclonal expansion. 7 days after polyclonal expansion, Treg were further expanded with two subsequent cycles (7 days for each cycle) of either polyclonal stimulation or xenoantigen stimulation with irradiated porcine PBMC at a 4:1 ratio of porcine PBMC:Treg. Treg phenotype and their suppressive capacity were assessed after xenoantigen stimulation. Results: Porcine xenoantigen stimulation did not affect expression of Treg markers CD25, Foxp3, CTLA-4, CD62L and GITR but slightly increased average fold expansion of Treg (807-fold with xenoantugen stimulation vs. 720-fold with polyclonal stimulation) when compared to polyclonal stimulation. In addition, Treg expanded with xenoantigen but not polyclonal stimulation revealed acquired an effector Treg phenotype with upregulated expression of HLA-DR, CD45RO, ICOS, LAG-3, IL-10 and IL-35. The acquisition of Treg effector phenotype led to enhanced Treg suppressive capacity via IL-10 in xeno-stimulated MLR, as indicated by strong suppression of xenogeneic responses (>50% suppression by xenoantigen stimulated Treg vs. 15% suppression by polyclonally stimulated Treg with Tresp:Treg ratios of 16:1). However, regardless of expansion with polyclonal or xenoantigen stimulation, Treg demonstrated a similar potency in inhibition of allo- or polyclonal-stimulated MLR. Conclusion: This study shows a feasible strategy to obtain human functional xenoantigen-specific Treg in large numbers for immunomodulation. This provides useful information for the clinical application of Treg in xenotransplantation aiming at inducing tolerance to porcine xenoantigen by minimizing general immunesuppression.

BLyS upregulation in Sjogren's syndrome associated with lymphoproliferative disorders, higher ESSDAI score and B-cell clonal expansion in the salivary glands

Primary SS is characterized by an increased risk of lymphoma in patients with prelymphomatous manifestations (i.e. myoepithelial sialadenitis or mixed cryoglobulinaemia). Serum B-lymphocyte stimulator (s-BLyS) levels in SS-related B-cell lymphoproliferative disorders were studied by integrating the results with the disease activity score and with molecular analyses of B-cell expansion in the salivary glands. Seventy-six primary SS patients (with or without lymphoma or prelymphomatous manifestations), 56 HCV-related cryoglobulinaemic vasculitis patients and 55 controls were studied. s-BLyS and molecular analyses of B-cell expansion in the salivary gland tissues were performed. Patients with SS and persistent parotid swelling underwent parotid biopsy. s-BLyS differed between SS subgroups, higher levels being documented in patients with lymphoma or prelymphomatous manifestations vs SS without [1.85 (0.45-4.12) ng/ml vs 1.12 (0.56-1.98) ng/ml; P < 0.0001]. s-BLyS levels significantly correlated with the European League Against Rheumatism (EULAR) SS disease activity index (r = 0.62, P < 0.0001, Spearman's test). Clonal B-cell expansion in the salivary glands, but not polyclonal B-cell expansion, was associated with higher s-BLyS levels [1.98 (0.45-4.12) ng/ml vs 1.15 (0.56-3.25) ng/ml; P = 0.013)]. Higher s-BLyS levels and tissue clonal B-cell expansion characterize SS with B-cell lymphoproliferative disorders, even at prelymphomatous stages. This subgroup of SS patients showed the highest EULAR SS disease activity index scores. This represents a biologic rationale for targeting both clonal B-cell expansion and s-BLyS overproduction in SS.