Positive T-cells Research Articles

Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium.

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an “inflammatory” monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.

Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity, immune regulation and therapy

IntroductionIn autoimmune diseases, IL-17 producing T-cells (Th17), a pro-inflammatory subset of T-cells, are pathophysiologically involved. There is little knowledge on the role of Th17 cells in granulomatosis with polyangiitis (GPA). In the present study, we investigated Th17 cells, Tregs and subsets of circulating Th17 cells in GPA and related results to disease activity.Methods42 GPA patients in remission, 18 with active disease and 14 healthy controls (HC) were enrolled. Th17 cells, their subsets and regulatory T-cells were determined by intracellular fluorescence activated cell sorter (FACS). Data are given as mean percentage ±SD of total T-helper-cells.ResultsTh17 cells are expanded in active and quiescent GPA as compared to HC (1.7±1.4% vs. 0.7 ±0.3%, P = 0.006 and 1.9 ±1.5% vs. 0.7 ±0.3%, P<0.0001). Th17 expansion is stable over time and does not decline when remission is achieved. However, a negative association of Th17 cells and steroid dosage is observed (r=-0.46, P = 0.002). The Th17 expansion was not balanced by Tregs as indicated by skewed Th17/Treg ratios in active and quiescent GPA. Th17 subsets co-producing IFNγ or IL-10 are significantly increased in GPA. GPA patients in remission not receiving maintenance therapy have significantly more IL-10/IL-17A double positive T-cells than HC (0.0501 ±0.031% vs. 0.0282 ±0.016%, P = 0.007).ConclusionsWe provide evidence for a persistent, unbalanced expansion of Th17 cells and Th17 subsets which seems to be independent of disease activity. Maintenance therapy reduces -but does not normalize- Th17 expansion.

P1-01-06: Mechanisms of Tumor Immune Escape in Triplenegative Breast Cancers (TNBC) with and without Mutated BRCA 1.

Abstract Background Triplenegative breast cancer (TNBC) is associated with a dismal prognosis, although these tumors are chemosensitive. This phenomenon could be at least in part due to tumor immune escape. The current study investigates the host's immune response to TNBC cells and explores the presence of immunesuppressive factors, such as pAKT-expression and infiltration with FoxP3 positive regulatory T-cells (Tregs), in human TNBC samples. Material and Methods: NK-cell induced lysis of tumor cells was evaluated in human breast cancer cell lines MCF-7 (ER/PR pos.), HCC 1937 (triplenegative, BRCA 1 mutated) and HCC 1806 (triplenegative). Expression of pAKT and infiltration with Tregs was determined by immunehistochemistry and evaluated semiquantatitavely (0 no expression- 3 strong expression). Control groups consisted of: Fibroadenoma (N=6), prohpyhlactic mastectomy (BRCA 1 mutated, N=3), ER/PR + breast cancer (N=13). They were compared with triplenegative breast cancers: N = 9 BRCA wildtype and N = 6 BRCA 1 mutated. Results: At an effector target/target-ratio of 10/1 NK-cell induced lysis in HCC 1937 and HCC 1806 was 2.27 and 4.45 increased, respectively, as compared to MCF 7 cells. No infiltration with Tregs was detected in fibroadenoma and prophylactic mastectomy samples. Infiltration with FoxP3-positive Tregs was 0.92 +/− 0.75 in ER/PR+ breast cancers and 2.66 +/−0.5 (p&amp;lt;0.05) in TNBC and 2.16 +/−0.98 (p&amp;lt;0.05) in TNBC with BRCA mutation, respectively. Expression of pAKT was 1.45 +/− 1.29 in ER/PR + breast cancers and 1.77 +/−1.20 in TNBC and 2.66 +/−0.51 (p&amp;lt;0.05) TNBC/with BRCA mutation. Discussion: TNBC cells stimulated the NK-cell response to a stronger extent than did ER-positive MCF 7 cells mirrored by a more pronounced NK-cell- induced lysis. Thus, the observed stronger infiltration with FoxP3-positive Tregs in TNBC tumor samples could reflect a compensatory mechanism to suppress the host's immune response. In other tumor entities, such as ovarian cancer infiltration with Tregs is associated with a worse overall survival (1). Thus, the significantly increased infiltration with Tregs, could suggest that the worse prognosis of TNBC is due to tumor immune escape and further investigation of immunemodulatory therapeutic strategies in TNBC could prove fruitful. (1) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W. Nat Med. 2004 Sep;10(9):942–9. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-06.

Lipopolysaccharide(LPS)-Induction of the HTLV-1 LTR in Monocytes

Abstract 2167The human leukemia virus type 1 (HTLV-1) gene expression is regulated by the viral proteins and various cellular transcription factors. HTLV-1 genome encodes not only structural proteins, but also non-structural proteins such as Tax, a transcriptional activator for STAT5 p12I, and HTLV-1 bZIP factor (HBZ) encoded by the minus strand of the viral genome. The functional analysis of the viral proteins such as Tax has shed light on the pathogenesis of adult T cell leukemia/lymphoma (ATL). Expression of Tax is enhanced by T-cell activation stimuli such as phorbol ester (PMA), phytohemagglutinin (PHA) or sodium butyrate in chronically HTLV-1-infected CD4+T-cells. Transgenic mouse studies with Tax expression under the control of the granzyme B promoter or the proximal Lck promoter showed that disease progression is associated with infiltration of activated T- and inflammatory cells, and dysregulated inflammatory cytokine production. More recently, a transgenic mouse model with Tax expression regulated by the LTR (LTR-Tax) showed that LTR-Tax CD4 positive T-cells are hyper-proliferative and hyper-responsive to immune stimulation and strongly produce Th1-, Th2- and Th17-associated cytokines. In addition, HTLV-1 infection causes inflammatory disease of the central nerve system, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as ATL. Aberrant cytokine gene expression is the hallmark of HTLV-1-associated diseases.HTLV-1 infection is widely distributed among mammalian cells. We previously demonstrated that Tax transactivates the promoter of human proIL-1β gene (IL1B) gene through association with two transcription factors, NF-IL6 (C/EBPβ) and Spi-1 (PU.1) in monocytes. Tax synergized with lipopolysaccharide (LPS) to induce IL1B promoter activity. Spi-1 is an Ets family protein restricted in expression to monocytes/myelocytes, B cells, mast cells and erythrocyte stem cells, while NF-IL6 is widely expressed. LPS, a component of the gram-negative bacterial cell wall involved in the activation of monocytes, binds to TLR4, leading to activation of TRAF6, IRAK and MyD88. We now extend these studies to elucidate roles of LPS and Tax on HTLV-1 LTR promoter regulation in monocytes. When HTLV-1 LTR reporter was transfected into THP-1 monocytic cells, LPS dose-dependently induced HTLV-1 LTR. The mid-LTR of the HTLV-1 gene possesses three potential Ets binding elements centered on a GGAA motif (PuB1, pets and PuB2). Elf-1 has been shown to be the predominant protein binding to the HTLV-1 Ets sites in Jurkat and peripheral blood T-cells. In the present study, mutation of the Ets sites, especially pets and PuB2 caused significant inhibition of LPS-induced LTR activity in THP-1 cells. EMSA studies using THP-1 nuclear extracts showed binding of Spi-1 to the HTLV-1 Ets sites in THP-1 cells. Anti-Spi-1 Ab, but not anti-Elf-1 Ab or anti-ets-1 Ab supershifted the complex generated by THP-1 nuclear extract and HTLV-1 LTR Ets site. However, when migration pattern of the complex was compared with recombinant Spi-1 in vitro translated in a reticulocyte lysate system, the THP-1 complex migrated slower than recombinant Spi-1 protein. In this regard, anti-IRF-8 Ab further recognized the slow complex. Several reports recently showed that IRF-8 functions as a heterodimeric complex with spi-1 for expression of relevant genes.On the other hand, when Tax expression vector was cotransfected into THP-1 cells along with HTLV-1 LTR reporter, Tax synergized with LPS to activate LTR. Spi-1 protein has three independent transcriptional activation domains (TAD); a TBP binding region, a Q domain, and a PEST region. GST pull-down studies using GST-Tax and 35S-labeled recombinant Spi-1 revealed that mutant Spi-1 lacking the TADs still retains the ability to interact with Tax. In contrast to THP-1 cells, Jurkat T-cells showed only a marginal increase in IL1B promoter activity following Tax expression. Mutations of the Spi-1 binding site in the IL1B promoter did not affect Tax-induced activity in Jurkat cells. Any factors did not bind to the IL1B Spi-1 site in Jurkat cells. Thus, our data suggest that LPS cooperates with Tax to activate the viral and various cellular genes in HTLV-1-infected monocytes. Spi-1 is a key player in the monocyte-specific gene regulation in HTLV-1 infection. Disclosures:No relevant conflicts of interest to declare.

Nuclear Factor of Activated T-Cells (NFAT1): Potential Novel Target in T-Cell Acute Lymphocytic Leukemia (T-ALL)

Abstract 4634 Background.T-cell transcription factor NFAT1 (NFATc2) has been reported to regulate cell cycle regulatory proteins including cyclins, cyclin-dependent kinases (CDKs), and p21. MicroRNA-184 (miR-184) has been noted to have close sequence homology to the 3' UTR of NFAT1 and to regulate NFAT1 mRNA translation in human adult and umbilical cord blood (UCB) CD3+CD4+ T-cells. In this study, we are characterizing the functional role of NFAT1 in T-ALL cells to determine whether its dysregulation may alter normal cell cycle regulation and thereby may contribute to T-ALL leukemogenesis. Materials/Methods.In this study we used MOLT-4 cell line to investigate the role of NFAT1 in T-ALL, since MOLT-4 represents early stage of human T-ALL (CD4+CD8+ double positive) compared with other T-cell lines including Jurkat. We developed two distinct approaches to explore NFAT1 signalling, either by manipulating the expression level of NFAT1 protein by blocking miR-184, or by overexpressing a constitutively active form of NFAT1 (caNFAT1) holding a HA-tag (Addgene plasmid 11792, A. Rao, PhD, Harvard University). caNFAT1 was introduced into gateway system expression vector pEF-DEST51. MOLT-4 cells were maintained in complete RPMI 1640 medium according to ATCC guidelines. Electroporation transfection was performed on Amaxa Nucleofector I using program C-05. 200 nM of anti-miR-184 miRNA inhibitor (Dharmacon) or 2 μ g of plasmid containing caNFAT1 was transfected into 2×10e6 MOLT-4 cells in each sample. Transfection efficiency was tested using pmaxGFP (Lonza). Proteins were extracted at various time points after transfection using RIPA buffer (PIERCE) with protease inhibitor cocktail and tested for the expression of NFAT1 (BD Biosciences), phosphorylated NFAT1 (Santa Cruz Biotechnology), HA (Roche), and beta-actin (Sigma-Aldrich). Cell cycle was measured at various time points by pelleting down 0.5–1 X10e6 cells, followed by ethanol fixation, and incubation in propidium iodide (PI) (10 μ g/ml) staining solution and flow cytometric measurement for PI incorporation. Cell apoptosis was measured by PE-conjugated annexin V staining (BD Biosciences) in transfected MOLT-4 cells including empty plasmid controls. Results/Discussion.The over-expression of caNFAT1 in MOLT-4 led to increased proportion of cells in G2 phase than in controls (10.8% vs. 7.3% on day5, and 11.8% vs. 8.1% on day6). In addition to these cell cycle changes in MOLT-4 treated with caNFAT1 plasmid, higher rates of apoptosis were noted on day 2 (28.3% vs. 15.5%), which notably leveled off from day 3 to day 6 potentially attributed to compensatory cellular responses. Previous work by this group has shown higher expression of miR-184 in MOLT-4 by 56 fold compared with normal human CD4/8 double positive T-cells. Here, miR-184 knockdown was also notable for a larger proportion of MOLT-4 cells in G2 cell cycle phase and higher apoptosis, albeit modest changes compared with that exerted by caNFAT1. Conclusion.In MOLT-4 cell line, this preliminary data suggests that increasing active non-phosphorylated NFAT1 is associated with enhanced proportion of cells in G2 phase as well as enhanced resultant apoptosis. Taken together, regulation of NFAT1 may be exploited as potential T-ALL targeted therapy. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Thalidomide in treatment of connective diseases and vasculities

Thalidomide is an immunomodulatory, anti-inflammatory and anti-angiogenic drug. Thalidomide exerts its effects by decreasing circulating CD4 positive T-cells and stimulating CD8 positive T-cells, by increasing the number of Natural Killer cells and T-helper 2 cells. Thalidomide also inhibits proliferation of stimulated T-cells and leukocyte chemotaxis. It modifies a number of integrin receptors and other leukocytic surface receptors and down-modulates cell-adhesion molecules involved in leukocyte migration. It has been demonstrated that thalidomide inhibits TNFalpha, IL-5, IL-6, IL-8, IL-12 production and increases production of IL-2, IL-10 and INFgamma. Moreover thalidomide plays an important role in inhibition of VEGF and FGF-2 mediated angiogenesis. Although the exact mechanism of action is not fully understood and only limited treatment opinions exist, thalidomide plays a role also in connective diseases and vasculities. Thalidomide has been seen efficacious in the treatment of cutaneous disorders in patients with systemic lupus erythematosus and in mucocutaneous disease in Behcet's disease with a not dose-dependent response, even if it should be restricted to selected patients because of its important side effects.

Case study interpretation—Houston: Case 1

Case study interpretation—Houston: Case 1

Flow cytometric analysis of peripheral T lymphocytes from patients with mycobacterial diseases

Flow cytometric analysis was carried out on peripheral blood cells from patients with tuberculosis (TB) (n = 84) and with mycobacteriosis other than tuberculosis (MOTT) (n = 38). A whole blood-staining-hemolysis procedure was used for the preparation of samples being analyzed, and the cells were double-stained with various combinations of fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)- labeled monoclonal antibodies. These procedures enabled us to obtain quite reproducible results. As patients of more than 70 years old showed apparently distinct T lymphocyte profiles compared with those less than 70 years of age, this investigation was carried out only on patients of less than 70 years old. 1) The proportion of total lymphocytes to total leukocytes was significantly low in TB- and MOTT- groups, when compared with that in the healthy control group, although the total peripheral leukocyte number was not significantly different from each other. Thus, absolute numbers of lymphocytes were decreased significantly in TB- and MOTT- patients. 2) The numbers of both T and B lymphocytes in peripheral blood decreased in patients of both groups, leaving the ratio of T/B relatively constant. 3) Both CD4+/CD8- and CD4-/CD8+ subsets of T lymphocytes decreased in TB- as well as MOTT- groups. However, the decrease in CD4+/CD8- subset was more manifest than that in CD4-/CD8+ subset. Among CD4+/CD8- subset the proportion of the Leu8+ subpopulation was slightly lower and among CD4-/CD8+ subset CD11b- subpopulation was slightly higher in both TB- and MOTT- groups than in healthy control group. 4) There was no significant difference in proportions of IL-2-receptor (p55 alpha chain) positive as well as HLA-DR positive T-lymphocytes between patient groups and healthy control group. 5) Both TB- and MOTT- groups were subdivided according to the extent of pulmonary lesion. Patients with the larger lesion showed remarkable decreases in the ratio of T lymphocytes to total peripheral leukocytes, the number of T lymphocytes, and the numbers of CD4+/CD8- and CD4-/CD8+ subsets, when compared with those with the smaller lesion. 6) Although the averages of absolute numbers of T lymphocytes, CD4+/CD8- and CD4-/CD8+ subsets were lower in patient groups than in the control group and the ratios of these to total lymphocyte counts and the ratio of CD4+ to CD8+ subsets were not significantly different between patient groups and control group, the distributions of each value of individual person were far broad in patient groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Elotuzumab on Circulating Lymphocytes, Chemokines, and Cytokines In Multiple Myeloma Patients

AbstractAbstract 4070Background: Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein which is highly and uniformly expressed on malignant plasma cells in multiple myeloma (MM). CS1 is also expressed at a lower level on the cell surface of natural killer (NK) cells, natural killer T-cells (NKT), and on a subset of CD8 positive T-cells, but not resting B cells, monocytes, or CD4 positive T-cells. Preclinical studies have previously demonstrated that elotuzumab kills myeloma cells via NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, in whole blood assays elotuzumab treatment resulted in the elevation of chemokines and cytokines in culture supernatants. Elotuzumab is being studied in three phase 1 clinical trials in relapsed and/or refractory MM: 1) a monotherapy dose-escalation study, 2) a combination study with bortezomib, and 3) a combination study with lenalidomide and low-dose dexamethasone. Given that NK cells express CS1, there was a concern that elotuzumab treatment could potentially lead to NK cell depletion. Therefore, the pharmacodynamic goals of these clinical studies were to examine the effects of elotuzumab treatment on lymphocyte counts (in particular NK cells), chemokine levels, and cytokine levels. Methods: Absolute lymphocyte counts were determined in peripheral blood samples using the TruCOUNT™ flow cytometry assay. Serum levels of chemokines and cytokines were measured using a multiplex, bead-based assay (Luminex®). Results: In all 3 studies, we observed no depletion of total lymphocytes or lymphocyte subsets, including CS1 positive NK cells, with elotuzumab dosing either alone or in combination with bortezomib or lenalidomide/dexamethasone. A transient decrease in the absolute number of circulating total lymphocytes (approximately 75%–90% reduction from baseline) upon first elotuzumab dose was observed, followed by a recovery of these lymphocyte counts to baseline or near baseline levels as dosing cycles continued. The transient decrease in lymphocytes included both CS1 positive and CS1 negative cell subsets. This transient decrease in lymphocyte counts was associated with increased levels of circulating chemokines and cytokines following dosing. Post-dose serum samples from study subjects had a median 16-fold (range 1.3–270-fold) elevated levels of interferon inducible protein 10 (IP-10), a chemokine well known to induce lymphocyte trafficking. Other serum analytes were elevated following the initial elotuzumab dosing, including MCP-1, IL-6, and TNF-α, also known for their role in chemotaxis and inflammatory processes. The elevated levels of chemokines and cytokines were generally not seen at subsequent doses. Conclusions: A transient decrease in both CS1 positive and CS1 negative lymphocyte counts was observed following the first dose of elotuzumab in all phase 1 studies in patients with relapsed and/or refractory MM, which resolved during subsequent dosing. This transient decrease appears to be due to lymphocyte trafficking resulting from release of chemokines and cytokines. No evidence of elotuzumab-mediated depletion of CS1 positive lymphocytes was observed. Elotuzumab treatment thus does not appear to be associated with NK cell depletion. Disclosures:Neyer:Facet Biotech: Employment. Ding:Facet Biotech: Employment. Chen:Facet Biotech: Employment. Sheridan:Facet Biotech: Employment. Rice:Facet Biotech: Employment. Balasa:Facet Biotech: Employment. Keller:Facet Biotech: Employment. Fang:Facet Biotech: Employment. Albano:Facet Biotech: Employment. Tran:Facet Biotech: Employment. Zhao:Facet Biotech: Employment. Afar:Facet Biotech: Employment.

Nuclear Factor of Activated T-Cells (NFAT1): Novel Target In T-Cell Acute Lymphocytic Leukemia (T-ALL)

AbstractAbstract 4327 Background:The well-characterized human T-cell transcription factor NFAT1 has been reported to regulate cell cycle regulatory proteins including Cyclins A, D, and p21. This group has recently identified microRNA-184 to regulate NFAT1 protein translation in human umbilical cord blood CD4 T-cells. We sought to determine if NFAT1 protein expression is dysregulated in T-ALL, and if so, its contribution, if any to T-ALL pathogenesis. Materials & Method:MOLT-4 cells were maintained according to ATCC guidelines. Normal adult and umbilical cord blood (UCB) T-cells were selected by AutoMACS (Miltenyi Biotec) and and maintained per standard protocols. Purity was confirmed in excess of 90% by flow cytometry. MOLT-4 cells were transfected with a 10 pmol:3.33 pmol ratio of anti-miR-184 miRNA inhibitor (Ambion) to siGLO non-specific RISC-free control (Dharmacon) in complete RPMI alongside corresponding controls via lipofectamine RNAi max (Invitrogen) overnight per manufacturers' instructions. Protein was isolated 48 hours after transfection and quantified by standard protein assay (BioRad). 10 μ g of each fraction was analyzed by 10% SDS-PAGE followed by Western Blotting with anti-NFAT1 (BD Biosciences) and γ-tubulin (Santa Cruz Biotechnology) antibodies. Bands were visualized using Supersignal West Pico chemiluminescent substrate (Pierce). Result:The relative levels of both NFAT1 protein and mir-184 were first compared to normal adult and UCB CD4+ T-cells. As miR-184 and NFAT1 expression levels can change over the developmental life of CD4+ T-cells, a comparison of MOLT-4 cells was made to normal UCB T-cell controls with similar surface phenotypes (CD4+/8+). NFAT1 protein expression was found to be significantly decreased in the MOLT-4 cells by Western Blot. Further, comparisons of miR-184 expression in UCB double positive T-cells to MOLT-4 by quantitative reverse transcription real-time PCR (qPCR) demonstrated a 56-fold higher expression of mir-184 in MOLT-4 compared to controls. Next, in order to test the ability of miR-184 to regulate NFAT1 levels in the MOLT-4 T-ALL cell line, lipofectamine-mediated transfections of antisense miR-184 were performed. Western Blot analysis showed the expected modest increase in the NFAT1 protein expression in MOLT-4 cells transfected with anti-miR-184. Discussion:NFAT1 protein levels are notably reduced in T-ALL cell lines and may be attributable in part to 56-fold higher expression of miR-184. Knockdown of miR-184 renders normalization of NFAT1 protein levels. Ongoing studies are designed to determine cell cycle kinetics and p21 expression in miR-184 transfected MOLT-4 cells. Dysregulation of NFAT1 may contribute to T-ALL pathogenesis, and miR-184 regulation of NFAT1 may be a target for therapeutic studies. Disclosures:No relevant conflicts of interest to declare.

Seborrheic dermatitis-like eruption following interleukin-2 administration

We describe a 72-year-old man suffering from renal cell carcinoma with lung metastases who developed scaly erythema on his face after the administration of recombinant interleukin-2 (rIL-2). A skin biopsy revealed intraepidermal and superficial perivascular infiltrate of mononuclear cells that were mainly composed of CD3 positive T-cells. There have been two cases of seborrheic dermatitis-like eruption induced by rIL-2. However, neither of them had histopathological evaluation. To our knowledge, this is the first case of seborrheic dermatitis-like eruption following rIL-2 administration whose histopathological investigations were performed.

The ITK-SYK Fusion Oncogene Induces a T-Cell Lymphoproliferative Disease in Mice Mimicking Human Disease

Peripheral T-cell lymphomas (PTCL) constitute a major treatment problem with high mortality rates due to the minimal effectiveness of conventional chemotherapy. Recent findings identified ITK-SYK as the first recurrent translocation in 17% of unspecified PTCLs and showed the overexpression of SYK in more than 90% of PTCLs. Here, we show that the expression of ITK-SYK in the bone marrow of BALB/c mice causes a T-cell lymphoproliferative disease in all transplanted mice within 8 weeks after transplantation. The disease was characterized by the infiltration of spleen, lymph nodes, bone marrow, and skin with CD3+CD4+CD8- and CD3+CD4-CD8- ITK-SYK-positive T-cells accompanied by a systemic inflammatory reaction with upregulation of interleukin 5 and INF-gamma. ITK-SYK-positive T-cells showed enhanced apoptosis resistance and INF-gamma production in vitro. The disease was serially transplantable, inducing clonal T-cell expansion in secondary recipients. The action of ITK-SYK in vivo was dependent on SYK kinase activity and disease development could be inhibited by the treatment of mice with SYK inhibitors. Interestingly, the translocation of ITK-SYK from the membrane to the cytoplasm, using a point mutation in the pleckstrin homology domain (ITK-SYK R29C), did not abolish, but rather, enhanced disease development in transplanted mice. CBL binding was strongly enhanced in membrane-associated ITK-SYK E42K and was causative for delayed disease development. Our results show that ITK-SYK causes a T-cell lymphoproliferative disease in mice, supporting its role in T-cell lymphoma development in humans. Therefore, pharmacologic inhibition of SYK in patients with U-PTCLs carrying the ITK-SYK fusion protein might be an effective treatment strategy.

Tumour-infiltrating lymphocytes and survival in patients with adenocarcinoma of the oesophagus

Introduction Tumor-infiltrating lymphocytes (TILs) and forkhead box transcription factor positive (FoxP3 +) regulatory T-lymphocytes (TREGs) have been analyzed in a variety of tumors but not in oesophageal adenocarcinoma. Patients and methods Tissue from 130 adenocarcinomas of the oesophagus was re-evaluated in the centre and periphery of tumour, respectively, using immunohistochemical staining with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-FoxP3 antibodies. Patients were stratified according neoadjuvant treatment. 106 patients proceeded directly to surgery and 24 underwent pre-operative radio-chemotherapy (RCT). Results In patients without RCT, TILs were found significantly more frequently in the periphery with the exception of CD25 + cells. Patients with centrally low counts of FoxP3 + TREGs had higher tumour stages than patients with high counts ( p < 0.011). The number of FoxP3 + TREGs was significantly associated with the number of CD8 + cells (centre: p < 0.001, periphery: p = 0.002). The multivariate regression analysis identified UICC stage (IIB/III vs. I/IIA, hazard ratio 2.6, p = 0.011) and completeness of resection (no vs. yes, hazard ratio 2.3, p = 0.015) as independent predictors of survival. RCT significantly reduced the number of TREGs in the centre ( p = 0.016) but not the number of the other TILs. Conclusion UICC stage and completeness of resection but none of the TILs were prognostic markers for long-term survival. We found no morphologic evidence that TREGs suppress immunological response, represented by the infiltration of CD8 + cells. Preoperative RCT affected the centre of tumours more than the periphery, which may indicate that it does not inhibit the host-to-tumour reaction. RCT affects TREGs more than the other TILs.

Safety and pharmacokinetic analysis of methotrexate administered directly into the fourth ventricle in a piglet model

We have developed a piglet model to assess chemotherapy administration directly into the fourth ventricle as a potential treatment for medulloblastoma and other malignant posterior fossa tumors. The objective of this study was to assess safety and pharmacokinetics after methotrexate infusions into the fourth ventricle. Catheters were inserted into the fourth ventricle and lumbar cistern in five piglets. Two milligrams of Methotrexate (MTX) was infused into the fourth ventricle on five consecutive days. Safety was assessed by neurological examination, 4.7 T MRI, and post-mortem pathological analysis. MTX levels in serum and cerebrospinal fluid (CSF) were measured, and area under the concentration-time curve (AUC) was calculated for CSF samples. No neurological deficits were caused by MTX infusions. One piglet died from complications of anesthesia induction for MRI scanning. MRI scans showed accurate catheter placement without signal changes in the brainstem or cerebellum. One piglet had asymptomatic ventriculomegaly. Pathological analysis demonstrated meningitis and choroid plexitis consisting predominantly of CD-3 positive T-lymphocytes in all piglets and a small focal area of subependymal necrosis in one. In all piglets, mean peak MTX level in fourth ventricular CSF exceeded that in lumbar CSF by greater than five-fold. Serum MTX levels were undetectable or negligible. Statistically significant differences between fourth ventricle and lumbar AUC were detected at peaks (P = 0.01) and at all collection time points (P = 0.01) but not at troughs (P = 0.36). MTX can be infused into the fourth ventricle without clinical or radiographic evidence of damage. An inflammatory response without clinical correlate is observed. Significantly higher peak MTX levels are observed in the fourth ventricle than in the lumbar cistern.

Prospective Evaluation of Proteomic Screening with An Agvhd-Specific Proteomic Pattern MS-17.

Abstract 2246Poster Board II-223Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for many hematologic malignancies or hematopoietic dysfunction syndromes in adults, but the application is still limited due to major complications, such as severe graft versus host disease (GvHD) and infectious complications. Diagnosis GvHD is based on clinical features and biopsies, a non-invasive, unbiased laboratory test using proteomics was published previously and evaluated in a multicenter setting (Weissinger et al., 2007). In order to maintain good quality data of the proteomic pattern data and due to the fact that the electrospray ionization time of flight mass spectrometer (ESI-TOF) from ABI (mariner) is no longer produced, we applied a more sensitive mass spectrometer (MS; ESI-TOF/Bruker). In order to validate the quality of the data produced with the Bruker, ESI-TOF was used since December 2007 in parallel to the mariner. Our first and previously published “aGvHD-specific pattern” (Weissinger et al., 2007) was used to evaluate and improve the pattern with the more sensitive decreased (6) or increased (11) signal intensity in patients with GvHD grade II to IV. Patients post HSCT and patients with GvHD grade I have the same pattern. In addition, the MS-17-pattern yielded at least comparable if not superior results to the previous one yielding sensitivities around 95% and specificity about 80%.To date the proteomic pattern specific for aGvHD MS17 was evaluated blindly on 400 samples collected from about 90 patients undergoing allo-HSCT at MHH since Dec. 2007.The peptide pattern MS-17 consists of 17 peptides - either absent/decreased (6) or present /increased (11) in aGvHD grade II to IV when compared to patients with clinical aGvHD grade I or without GvHD. Prospective and blinded evaluation of the patients included in this parallel diagnostic study revealed the correct classification of patients developing aGvHD about 7 days prior to the development of clinical signs and symptoms for aGVHD with a sensitivity and specificity of about 85%. Sequencing 3 of the 17 markers yielded the following results:Peptide No 5: Albumine fragment (N-terminus); Peptide No 7: ψ2 microglobulin; Peptide No 10: fragment of CD99 (MIC2; a single-chain type-1 glycoprotein). CD99 and β-2 microglobulin are particulary interesting because of their involvement in immune responses, both being expressed on leukocytes and CD 99 especially on thymocytes with involvement in apoptosis of double positive T-cells and binding cyclosporin A. Data on the pattern, signal intensity (3 dimension, mass and charge are shown in Figure 1) Disclosures:Metzger: mosaiques-diagnostics GmbH: Employment. Krons:mosaiques-diagnostics GmbH: Employment. Mischak:mosaiques-diagnostics GmbH: Patents & Royalties, coowner and founder of mosaiques-diagnostics.

The Antiproliferative Effect of Kefir Cell-Free Fraction on HuT-102 Malignant T Lymphocytes

Kefir is produced by adding kefir grains (a mass of proteins, polysaccharides, bacteria, and yeast) to pasteurized milk; it has been shown to control several cellular types of cancer, such as Sarcoma 180 in mice, Lewis lung carcinoma, and human mammary cancer. Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia, which is a fatal disease with no effective treatment. The current study aims at investigating the effect of a cell-free fraction of kefir on HuT-102 cells, which are HTLV-1–positive malignant T-lymphocytes. Cells were incubated with different kefir concentrations: the cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir cell-free fraction on the proliferation of HuT-102 cells was then assessed. The levels of transforming growth factor (TGF)-α mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction. Finally, the growth inhibitory effects of kefir on cell cycle progression and/or apoptosis were assessed by flow cytometry. The maximum cytotoxicity recorded at 80 μg/μL for 48 hours was only 43%. The percent reduction in proliferation was very significant, dose and time dependent, and reached 98% upon 60-μg/μL treatment for 24 hours. Kefir cell-free fraction caused the downregulation of TGF-α, which is a cytokine that induces the proliferation and replication of cells. Finally, a marked increase in cell cycle distribution was noted in the pre-G1 phase. In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1–positive malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.

Pathomechanism of Cellular Infiltration in the Perivascular Region of Several Organs in SAMP1/Yit Mouse

We investigated the histological changes of extra-intestinal organs, such as the liver, kidney, lung and pancreas in SAMP1/Yit mice, a human Crohn's disease model, using immunohistochemical techniques. The perivascular cellular infiltration was detected around the small vessels after 30 weeks. These infiltrating cells consisted of many CD4-positive T-lymphocytes, and small numbers of CD8- positive T-lymphocytes and IgG-positive B-lymphocytes. MAdCAM-1 and VCAM-1 were detected in vascular endothelial cells in non-affected regions of 13 and 20 week-old, as well as in the affected regions showing perivascular cellular infiltration after 30 weeks. In addition, integrin alpha4beta7 was detected on these infiltrating cells in the perivascular regions after 30 week-old. LT-beta and IL-12, cytokines of the Th-1-type immune response, were not observed in these affected regions. However, IL-4, one of the cytokines of the Th-2-type immune response, was detected on the perivascular infiltrating cells after 30 week-old. These results revealed that the changes in extra-intestinal organs were mainly caused by infiltration of CD4-positive T-lymphocytes into the perivascular regions in SAMP1/Yit mice. These cellular infiltrations were thought to be initiated by adhesion of CD4-positive T-lymphocytes to the endothelial cells mediated by MAdCAM-1 and integrin beta7. Immunohistochemistry for Th related cytokines indicated that the perivascular cellular infiltration was developed by the Th-2-type immune response in the extra-intestinal organs of SAMP1/Yit mouse.

Clinical and Immunophenotypic Differences of Patients with T/NK-Cell Large Granular Lymphocytic Leukemia

Background: According to the WHO, T/NK-cell large granular lymphocytic leukemia (T/NK-LGL) comprises 2 – 5 % of all T/NK tumors. In addition, T/NK-LGL is a very heterogeneous disease. T/NK-LGL has been divided along 2 basic types: T-cell and NKcell leukemias. We found further differences among these two basic types. Among our patients with T/NK-LGL, there were 5 immunophenotypic variants with differences in severity of their disease and other clinical parameters.Materials and Methods: Thirty five patients were diagnosed with T/NK-LGL from 01.1998 to 01.2008 (13 men and 22 women) in the age from 18 to 71 years (median age 58 years). The median survival has not been achieved. The median follow up is 72 months. Clinical common features: lymphocytosis in blood and bone marrow, cytopenia (80%), splenomegaly, rheumatoid arthritis and B-symptoms. Unusual features: lymphadenopathy, vasculitis and neuropathy. Diagnostic methods: histology, cytology and flow two-color cytometry with monoclonal antibodies (“Becton Dickinson Immunocytometry Systems” USA and “Dako” Denmark). Clonality was defined by a PCR- on T-cell receptor gene rearrangement.Results: Twenty six patients: 18 women and 8 men were accurately classified as «T/NK - cellular leukemia - LGL» according to WHO classification. The remaining 9 cases had rare immunophenotypes. All (35) patients were subdivided into 5 groups by immunophenotypic (IFT) features of leukemic lymphocytes. 1st group (4 women) had the following IFT of the leukemic lymphocytes: CD2+CD3−CD56+/−CD16+. In the 2d group, 9 patients (5 men and 4 women) had IFT of CD2+CD3+CD8+CD16+CD56+/−. There were 13 patients (3 men and 10 women) in the 3d group. Variant of IFT with leukemic T-cells was CD3+CD8+CD16−CD56−. There were 5 patients (2 men and 3 women) in the 4th group. 20–60 % peripheral blood lymphocytes simultaneous expressed CD8 and CD4 (double – positive T-cells). Others had 40–20% lymphocytes expressing only CD8+ marker. In the 5th group, there were 4 patients (2 men and 2 women). The pathologic T-cells were detected only on the bone marrow aspirate. There had heavy bone marrow aplasia or predominantly red cell aplasia. IFT of lymphocytes in the bone marrow was aberrant with presence of some markers of T-cells, including, CD8, absence of CD16 and CD56. The first three groups appeared stable and did not require chemotherapy for prolonged time (11 of 26 patients, overall 42%). A median of follow-up without treatment is 70 months. The patients of 4th and 5th group required immediate intervention and were treated with chemotherapy.Conclusions: Immunohistochemical characteristics of T-cells separate T/NK-LGL patients with stable disease from those that present with cytopenias and require immediate intervention. The molecular characteristics of these cells will be further defined.

CD11b− Donor Dendritic Cells Enhance Donor T-Cells' Th1 Polarization and Graft Versus Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

Background: Based on a clinical association of donor plasmacytoid dendritic cell (DC) content with leukemia relapses after allogeneic BMT (Waller, Blood 2001), we have previously reported that CD11b− donor DC added to a graft containing FACS-purified hematopoietic stem cells (HSC) and T-cells enhanced interferon-γ (IFN-γ) production and GvL activity in MHC-mismatched allogeneic transplant mouse models (Li, Blood 2007).Objective: In this study, we studied the mechanisms whereby donor DC in the graft modulate donor T-cell activity and the graft-versus-leukemia (GvL) effect in MiHA (C3H.SW → C57BL/6J)- and MHC (C57BL/6J → B10.BR)- mismatched models of allogeneic hematopoietic stem cell transplantation (HSCT).Methods: Mice irradiated to 11 Gy received 5 × 104 log-phase viable MMB3.19 myeloid lymphoma cells via intraperitoneal injection or intravenous injection of 1 x 105 LBRM T-cell lymphoma cells one day before transplant. Allografts consisted of 5 × 104 FACS-purified donor BM CD11b− DC or CD11b+ DC plus 3 × 103 FACS-purified c-kit+ sca-1+ lineage− hematopoietic stem cells (HSC) in combination with either 3 × 105 T-cells, 3 × 105 CD8+ T-cells or no additional T-cells transplanted via tail vein. Graft-versus-host disease (GvHD) clinical scores (based on body weight loss, posture, skin, fur texture, activity) were recorded twice weekly in non-tumor bearing recipients. In vitro proliferation and cytotoxic activity of donor-derived T-cells against tumor targets was assessed by CFSE staining and a caspase flow cytometry assay (CyToxiLux PLUS) using donor T-cells harvested from recipients on day 34 and day 82 post transplant. Serum and intracellular Th1 cytokines (IFN-γ, IL-2, and TNF-α) and Th2 cytokines (IL-4, IL-5, and IL-10) from recipients' peripheral blood and spleens day 3 and day 10 post-transplant was measured by ELISA and flow cytometry. IFN-γ direct killing of leukemia cells was tested by in vitro IFN-γ exposure.Results: In non-tumor bearing mice, recipients of all combinations of donor DC subsets, with and without donor T-cells had equivalent survival (75% – 85%) at 3 months post transplant without significant clinical signs of GvHD. Transplantation of tumor cells to recipients of HSC alone, HSC plus donor T-cells, or HSC plus T-cells and CD11b+ DC in the MiHA- and the MHC-mismatched transplant models led to 0% or 5% 3 month survival, respectively. Strikingly, tumor-bearing mice transplanted with CD11b− DC had significantly enhanced 3 month survival (35% in the MiHA-mismatched model and 45% in the MHCmismatched model) without increased GvHD (p<0.001). There was no significant difference in survival between mice that received HSC plus CD11b− DC and a mixture of CD4+ and CD8+ donor T-cells versus mice that received HSC plus CD11b− DC and only CD8+ donor T-cells. Donor T-cells harvested from recipients of CD11b− DC 34 days after transplant in the MiHA-mismatched model as well as 82 days after transplant in the MHC-mismatched model displayed increased cell proliferation following co-culture with irradiated hosttype splenocytes as a source of alloantigen compared with donor T-cells harvested from recipients of CD11b+ DC or recipients of HSC plus T-cells without donor DC. Leukemia cell killing was greater following incubation of purified donor T-cells recovered from recipients of CD11b− DC with tumor targets compared to T-cells recovered from other treatment groups. Recipients of CD11b− DC had higher serum levels of Th1 cytokines IFN-γ and IL-2 and higher number of Th1 positive donor T-cells compared with recipients of other treatment groups. In contrast, recipients of CD11b+ DC had higher serum levels of Th2 cytokines IL-4, IL-5, and IL-10 and higher number of Th2 positive donor T-cells. IFN-γ added to in vitro cultures with MMB3.19, and LBRM, had no direct cell killing effect.Conclusion: CD11b− donor DC enhanced Th1 polarization of donor T-cells and GvL without increasing GvHD. Donor CD8+ T-cells mediated tumor killing effect. CD11b+ donor DC enhanced Th2 polarization of donor CD4+ T-cells and led to limited GvHD and GvL.

Combined Treatment with Short Synthetic Anti-Lymphoma Peptide and CpG ODN; A Therapeutic Vaccine Which cures the A20 Lymphoma in Balb/C Mice.

Background. The short synthetic peptide 302 has been shown to induce rapid membrane disruption of lymphoma cells in vitro and necrosis of local tumors in the A20 lymphoma model in Balb/c mice. In order to stimulate the immune system to generate an anti-tumor response we designed a model where intra-tumor injections of peptide 302 was combined with sc injections of the Toll-like receptor 9 binding synthetic oligonucleotide CpG 1826.Methods. Balb/c mice were inoculated with A20 lymphoma cells sub-cutaneously (s.c.) on the abdomen. When the tumors reached a size of 5–7 mm, 302 peptide was administered directly into the tumors on days 1 and 6. CpG 1826 was injected s.c. on days 1–4 and 6–8. Tumor growth was measured repeatedly during follow-up. A20-specific T-cell responses were detected by culturing peripheral blood lymphocytes from treated animals for 24 hours with A20 lymphoma cells and analyzing for intracellular IFN-γ production by flow cytometry. To dissect the role of T-cell subtypes, the treatment was performed in animals depleted for CD4 or CD8 positive T-cells. In order to show A20 specific immunological memory, cured animals were re-challenged with the A20 lymphoma or the carcinoma cell line CT26.Results. Combined treatment with peptide 302 and CpG 1826 cured 8 out of 10 mice, compared to only 2 out of 10 mice who received peptide 302 alone or CpG 1826 alone. Cured mice were followed for 9 weeks without relapsing. Similar results with the combination of peptide 302 and CpG were observed in a separate experiment. The highest cure rate was achieved when injecting CpG 1826 s.c. in the tumors draining lymph node area as compared to administration of CPG 1826 in a non-draining lymph node region or intra-peritoneal. Animals treated with peptide 302 + CpG 1826 or CpG 1826 alone developed CD8-specific IFN-γ responses against A20 cells. In one separate experiment CD8 knock-out mice did not respond to the treatment, unlike animals depleted for CD4+ cells and normal mice. Only 1 out of 10 cured animals re-challenged with the A20 lymphoma developed a new tumor, a result that was reproduced in a second experiment.Conclusion: Treatment with intra-tumor injections of the anti-lymphoma peptide 302 in combination with CpG 1826 s.c. in the draining lymph node region cured established A20 tumors, induced tumor-specific CD8 positive tumor-reactive T-cells, and induced specific immunological memory. This principle represents a novel therapeutic vaccine approach.