Lymphocyte Reaction Research Articles

Immunologic reactivity and metabolic activity of blood lymphocytes associated with pathogenic form of allergic rhinosinusitis

We had studied functional and metabolic activity of blood lymphocytes in allergic rhinosinusitis (ARS) - atopic ARS and polypous rhinosinusitis (PRS). ARS is characterized by the increased level of CD25+ and HLA-DR+-lymphocytes in peripheral blood, IgA concentration in blood serum and sIgA in nasal secretion. At the same time the deviation of immune response is directed towards Th2-lymphocytes and is accompanied by high content of IL-4 and IL-6 in blood serum and nasal secretion. The activity of metabolic enzymes in blood lymphocytes in ARS is caused by exasperation of plastic processes, intensification of lipoid catabolism and increase of the speed of both anaerobic and aerobic oxidation. There are the following specific features of PRS: high level of CD19+-cells, activation of Thl-lymphocytes and high level of those cytokines, which they produce (IFN-γ and IL-2 in blood serum and nasal secretion). At the same time intensification of aerobic enzymes and lipoid metabolism and decreasing of anaerobic processes and aminoacid metabolism in blood lymphocytes are in the progress.

Metabolic Patterns in Cancer Cells and Tumor Microenvironment in Diffuse Large B-Cell Lymphoma: Tumor-Stromal Metabolic Coupling

IntroductionIt has previously been suggested that the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) has prognostic significance. Furthermore, gene expression profiling in DLBCL patients has identified separate subsets with glycolytic and mitochondrial (oxidative phosphorylative) metabolic signatures. Glycolytic metabolism forms the basis for FDG PET scans, widely used in staging and response assessment in DLBCL. While many assume that the tumor as a whole is primarily glycolytic, the metabolic patterns of cancer cells (C) and surrounding cancer-associated stromal cells (CAS) remain relatively unknown. We investigated the in situ metabolic patterns of C and CAS cells as well as tumor-associated macrophages (TAM) in DLBCL.MethodsAll patients diagnosed with DLBCL at Thomas Jefferson University from 2009-12 with available diagnostic tissue (N=28) were studied. Two immunohistochemical (IHC) biomarkers of metabolism were used as stains. Monocarboxylate transporter 4 (MCT4) shuttles glycolysis-derived lactate out of the cell and is a biomarker for glycolysis. Translocase of the outer mitochondrial membrane 20 (TOMM20) is a biomarker for mitochondrial mass, i.e. the oxidative phosphorylation phenotype. The horseradish immunoperoxidase method was used to stain 5 μm thick, paraffin-embedded tissue samples, using TOMM20 and MCT4 antibodies (Santa Cruz). Immunostains were graded on a scale of 0 to 2+ based on the intensity and percentage of immunoreactivity in C, CAS, and TAM cells. Available clinical data collected by chart review included SUVmax (SUVm) on PET/CT scan, R-IPI, treatment regimen, complete response (CR) rates and Ki-67. Fisher's exact test was used to test for association among categorical variables.ResultsCancer cells in 27/28 samples (96%) stained 2+ for TOMM20, with one sample staining 1+. MCT4 expression was completely absent in C cells in all samples. Conversely, TOMM20 was either not expressed (46%) or had 1+ expression (54%) in CAS and TAM. MCT4 expression was 2+ in 46% of CAS and 75% of TAM, and 1+ in the CAS and TAM of the remaining samples. Reactive lymphocytes (RL) had no MCT4 expression and uniform 1+ TOMM20 expression and can be regarded as internal controls. Levels of MCT4 expression in CAS correlated with MCT4 expression in TAM in the same patient (p=0.0069). 16/28 patients (57%) attained CR with anthracycline/rituximab based frontline therapy. MCT4 staining in CAS/TAM was associated with better CR rates with increasing intensity of scoring. (1+/1+ < 1+/2+ < 2+/2+; p=0.032).Table 1S.NoStageR-IPITOMM20MCT4SUVmCRCCASTAMRLCCASTAMRL14P2+1+1+1+02+2+019.82Y24*2+001+01+2+0*N34G2+1+1+1+02+2+019.00N4**2+1+1+1+02+2+0*Y54G2+001+01+2+03.50Y64P2+001+01+2+018.80N74P2+1+1+1+02+2+0*Y84P2+1+1+1+02+2+016.51N94*2+001+01+2+042.19Y10**2+1+1+1+01+1+0*N114G1+00001+1+0*Y122VG2+1+1+1+02+2+030.72Y134P2+001+01+1+0*N144P2+1+1+1+02+2+032.00N154*2+001+01+2+017.31Y164G2+001+01+2+07.90Y172*2+1+1+1+02+2+016.52Y184P2+001+02+2+038.90Y194G2+1+1+1+01+1+031.00N204G2+001+01+1+027.00N214*2+001+01+2+012.68Y224*2+1+1+1+02+2+0*Y234P2+1+1+1+01+1+06.99N244P2+1+1+1+02+2+026.91Y251*2+001+01+1+020.86N264P2+001+02+2+09.80Y274G2+1+1+1+02+2+023.50Y284P2+1+1+1+01+2+0*NP=poor G=good VG=very good *=NAConclusionsCancer cells in DLBCL do not express MCT4, but instead strongly express TOMM20, suggesting an oxidative phosphorylative rather than a glycolytic metabolic phenotype. Conversely, CAS and TAM always express MCT4, suggesting a glycolytic phenotype. Glycolysis and lactate export in CAS/TAM may be spatially related with mitochondrial metabolism in cancer cells, indicating tumor-stromal metabolic coupling. Glycolytic stroma in DLBCL may contribute to the PET-avidity in these tumors. Increasing MCT4 staining in CAS and TAM was associated with better CR rates, perhaps indicating tumors with increased chemosensitivity. Further investigation of the underlying mechanisms are needed to develop a better understanding of tumor-stromal interaction and its possible role in treatment of DLBCL. [Display omitted] [Display omitted] [Display omitted] DisclosuresPro:Seattle Genetics: Consultancy, Other: Travel expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses.

Activating Mutation of STAT3 Protects Lymphocytes from Apoptosis and Leads to a Clinical Phenotype Resembling the Autoimmune Lymphoproliferative Syndrome

(PS and UF contributed equally to this work.)Introduction:The Autoimmune Lymphoproliferative Syndrome (ALPS) is caused by inefficient clearing of T lymphocytes. Patients are thus characterized by lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias and an elevated number of double negative T cells (CD3+, TCRα/β+, CD4-, CD8-). Patients suffering from ALPS typically harbor germline or somatic mutations in genes involved in the apoptotic FAS death receptor signaling pathway (FAS, FASLG or CASP10). For 20-30% of patients, however, the genetic cause is still unknown.Methods:The objective of this study was to identify novel gene candidates underlying ALPS of unknown genetic cause. To this end, 25 patients with clinical ALPS symptoms, but without classical mutations were analyzed by whole-exome sequencing. The list of potential candidates was narrowed down using an in-house developed bioinformatic analysis pipeline for patient-based gene prioritization based on protein-protein interaction networks. Resulting candidates were validated by Sanger sequencing and their impact on Fas signaling was studied.ResultsWe identified a de novo germline mutation of the Signal Transducer And Activator Of Transcription 3 (STAT3, c.833G>A, p.R278H) in one of the analyzed patients. The patient presented at the age of nine with Coombs positive hemolytic anemia, thrombocytopenia, generalized progressive, non-infectious, non-malignant lymphadenopathy and splenomegaly. Immunophenotyping revealed increased numbers of double negative T cells (20% in peripheral blood) and over time the patient developed panhypogammaglobulinemia.We performed immunoblot analyses and could demonstrate that the level of phosphorylated STAT3 (pSTAT3-Tyr705) was elevated in the patient's lymphocytes. This finding indicated that the mutation leads to constitutive activation of STAT3. Consistently, we detected an increased expression of STAT3 target genes (including SOCS3, MMP7 and the anti-apoptotic factors BCL2 and BCL2L1) compared to wild-type controls using quantitative real-time PCR. We could also show a decreased expression of the pro-apoptotic genes BAK1 and BAX that is in accordance with the known negative regulation by STAT3. Thus, in the analyzed patient we found that the balance of pro- and anti-apoptotic factors inside the cell was skewed towards apoptosis resistance.Consistently, we could induce apoptosis in vitro applying recombinant Fas ligand, IL21 or staurosporine efficiently in cells derived from healthy controls, but only to a significantly lesser extent in cells from the patient. Moreover, in healthy cells we observed a concurrent downregulation of anti-apoptotic BCL2/BCL2L1 and an upregulation of pro-apoptotic BAX/BAK1 expression upon treatment that was completely absent in the patient's cells.Next, we tried to rescue the effect of constitutively activated STAT3 by application of a STAT3 specific inhibitor: S3I-201. When we treated the patient's lymphocytes with S3I-201 the expression levels of pro- and anti-apoptotic genes were similar to healthy controls and the sensitivity to apoptosis was restored.Conclusion:We report here a novel germline dominant STAT3 gain-of-function mutation that caused a clinical phenotype mimicking ALPS. Recent studies indicated that dominant germline STAT3 gain-of-function mutations lead to autoimmunity, hypogammaglobulinemia, and lymphoproliferation. STAT3 gain-of-function patients therefore share some clinical characteristics with ALPS patients. The clinical presentation of the patient described here differed from the phenotypes previously reported and thus extends the spectrum of STAT3 -associated diseases. The mechanism underlying the clinical symptoms of STAT3 gain-of-function patients has not yet been determined. Here, we demonstrate increased activation of STAT3 and STAT3 target genes, leading to a skewed balance of pro- and anti-apoptotic factors and apoptosis evasion as a cause for lymphocyte accumulation and resulting autoimmunity in a STAT3 gain-of-function patient. Similar to ALPS patients, diminished responsiveness of lymphocytes to apoptosis seems to be a major characteristic. The clinical phenotype may differ because mutations in STAT3 or Fas signaling genes, respectively, affect overlapping, but also distinct signaling pathways. DisclosuresNo relevant conflicts of interest to declare.

Blockade of PD-1 in Combination with Dendritic Cell/Myeloma Fusion Cell Vaccination Following Autologous Stem Cell Transplantation Is Well Tolerated, Induces Anti-Tumor Immunity and May Lead to Eradication of Measureable Disease

Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to incorporate immunotherapy in an effort to target residual disease. Our group has developed a cancer vaccine in which dendritic cells (DCs) are fused to autologous tumor cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Up-regulation of the PD-1/PDL1 pathway represents a key element contributing to tumor-mediated tolerance, and potentially muting response to vaccination. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (Pidilizumab, MDV9300) in combination with a dendritic cell/myeloma fusion cell vaccine following autologous transplantation. 22 patients have been treated with post-transplant immunotherapy. Mean age was 64. MM cells were isolated from bone marrow and were identified by expression of CD38 or CD138. Mean tumor cell yield was 118x106 cells. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 75%) and maturation markers (mean CD83 50%). DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean fusion efficiency was 41% and the mean cell dose generated was 4 x 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 92%, 89%, and 85%, respectively. As a measure of their potency as antigen presenting cells, DC/MM fusions potently stimulate allogeneic T cell proliferation ex-vivo (Mean stimulation index of 1.9, 9.2 and 7.1 for tumor, DC and DC/myeloma fusions respectively, n=21) Post-transplant immunotherapy was initiated after recovery from transplant-related toxicities. Median time from transplant to initiation of post-transplant immunotherapy was 80 days. Patients received 3 doses of Pidilizumab at 6-week intervals. DC/myeloma fusion cells vaccination is administered 1 week before each dose of Pidilizumab. To date, 22 patients have completed vaccinations and Pidilizumab. Adverse events judged to be potentially treatment related included grade 1-2 diarrhea, arthralgias, myalgias, fatigue, headache, nausea, chills, transaminitis, cytopenia, elevated TSH, and vaccine site reactions. A significant increase in circulatingtumor reactive lymphocytes was noted following post-transplant immunotherapy, as determined by T cell expressionof IFN-γ by CD8 cells following ex-vivo co-culture withautologous myeloma cell lysate. Mean percentage of tumor reactiveCD8 cells increased from 1.8% post-transplant to a peak of 9.16% following immunotherapy. In the post-transplant period, regulatory T cells fell to minimal levels and remained low throughout the period of immunotherapy. 6 patients achieved a best response of VGPR, 6 patients have achieved a nCR/CR, including 3 who converted to CR following immunotherapy. Median PFS from transplant is 19 months with ongoing follow up. In summary, DC/MM fusion cell vaccination in conjunction with PD1 blockade following ASCT was well tolerated, potently induced anti-tumor immunity, and in a subset of patients, resulted in the eradication of post-transplant measurable disease. DisclosuresRichardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

CORR Insights(®): do patients with a failed metal-on-metal hip implant with a pseudotumor present differences in their peripheral blood lymphocyte subpopulations?

T he study by Catelas and colleagues adds to the current scientific consensus that adaptive immune responses to metals occurs in a subset of all people with metal implants. The study authors also found that the incidence of pseudotumors correlates with systemic evidence of metal-related delayed-type hypersensitivity (DTH) responses and elevated metal ion levels. The current study identifies alterations in circulating adaptive immune cells in people with elevated levels of metal debris and pseudotumors. These findings support past histological and T-cell function studies, indicating pseudotumors are more prevalent in people with DTH-type immune reactivity. This highlights the growing recognition of these phenomena as central to implant performance in some individuals/implant designs. Case and group studies [4, 5] indicate that once locally activated, adaptive immune inflammation to metal contributes to (if not, mediates) the pathogenesis of poor implant performance. Metal DTH reactivity is a lymphocyte mediated response, similar to a Type IV DTH reaction. Metal-on-metal (MoM) hip failures have been associated with classic DTH responses including periimplant activated lymphocyte accumulations (aseptic lymphocytic vasculitisassociated lesions, [ALVAL]), and dermal and functional lymphocyte hypersensitivity responses to metals [2, 7, 11]. Elevated metal exposure can also cause different forms of toxicity responses in tissue, bone, and immune cells, including cobalt-induced hypoxia-like responses, postulated as causing pseudotumors [10]. Lymphocyte reactivity (metal DTH responses/ALVAL) and pseudotumors can be caused separately [6, 8]. Currently, there are no accepted thresholds for systemic metal ion levels that necessarily indicate for pseudotumor formation or immune reactions [1, 7]; it remains an increased risk association. This CORR Insights is a commentary on the article ‘‘Do Patients With a Failed Metal-onmetal Hip Implant With a Pseudotumor Present Differences in Their Peripheral Blood Lymphocyte Subpopulations?’’ by Catelas and colleagues available at: DOI: 10.1007/ s11999-015-4466-8. The author certifies that she, or any members of her immediate family, has no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research editors and board members are on file with the publication and can be viewed on request. The opinions expressed are those of the writers and do not reflect the opinion or policy of CORR or The Association of Bone and Joint Surgeons. This CORR Insights comment refers to the article available at DOI: 10.1007/s11999015-4466-8. N. J. Hallab PhD (&) Department of Orthopedic Surgery, Rush University Medical Center, 1735 W Harrison, Cohn Research Bldg, #722, Chicago, IL 60612, USA e-mail: nhallab@rush.edu CORR Insights Published online: 13 October 2015 The Association of Bone and Joint Surgeons1 2015

Simultaneous immunization of mice with Omp31 and TF provides protection against Brucella melitensis infection

Brucella vaccines consisting of live attenuated Brucella strains are currently used in livestock, but safety concerns preclude their application in humans. Subunit vaccines have recently emerged as safe and efficacious alternatives in both humans and animals. In this study, subunit vaccines were developed that consisted of a recombinant outer membrane protein (rOmp31) and the trigger factor chaperone protein (rTF) of Brucella melitensis, either alone or in combination. BALB/c mice that were immunized with rOmp31+rTF showed comparable but slightly higher TF-specific IgG1 and IgG2a antibodies as compared to mice with rTF alone. Indeed, mice given this combination had titers of rOmp31-specific antibodies similar to those immunized with rOmp31 alone. In lymphocyte reactivation experiments, the splenocytes of immunized mice, whether given either of these antigens alone or as a cocktail, exhibited a strong antigen-specific recall proliferative response and expressed high amounts of IFN-γ, IL-12, IL-10 and IL-6. Both rTF and rTF+rOmp31 vaccinated mice exhibited significantly higher CD4 and CD8 levels compared to the PBS group. The combination of rOmp31 and rTF provided protection against B. melitensis infection comparable to that of vaccine strain Rev.1. In comparison to rTF alone, combination of rTF and rOmp31 caused only a slight increase in protection level. Although combination of rTF and rOmp31 caused a non-significant increase in IFN-γ induction, antibody level, proliferation index and CD4 and CD8 frequencies compared to rTF alone, its cumulative effects on aforesaid parameters may be viewed as a better efficacy.

Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells Prolong Graft Survival in Vascularized Composite Allotransplantation.

Strategies aiming at minimization or elimination of systemic immunosuppression are key immediate goals for clinical expansion of vascularized composite allotransplantation (VCA). We compared the in vitro and in vivo immunomodulatory efficacy of adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow (BM)-derived MSCs in a rat VCA model. Both cell types were tested in vitro for suppressor function using mixed lymphocyte reactivity assays. AD-MSCs or BM-MSCs were administered intravenously (1 × 10 or 5 × 10 cells/animal) to Lewis rat recipients of mismatched Brown Norway hindlimb transplants. Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21. In vivo regulatory T-cell induction, peripheral blood chimerism, and microchimerism in lymphatic organs were analyzed. AD-MSCs and BM-MSCs exhibited strong dose-dependent suppressor function in vitro, which was significantly more pronounced for AD cells. In vivo, all animals revealed peripheral multi-lineage chimerism at four weeks (P < 0.01) independent of cell type and dosage. Regulatory T-cell levels were increased with both cell types, the most in AD-MSC groups. These immunomodulatory effects were only transient. MSC treatment resulted in long-term (>120 day) allograft survival in 47% of the animals, which correlated with durable microchimerism in BM and spleen. AD-MSCs and BM-MSCs exert immunomodulatory effects that prolong survival of immunogenic skin-bearing VCA grafts with short course (21 day) tacrolimus induction therapy. The in vivo findings in terms of allograft survival did not reflect superior immunomodulatory characteristics of AD-MSCs found in vitro.

The PD1/PDL1 axis, a promising therapeutic target in aggressive breast cancers

ABSTRACTAnalysis of PDL1 mRNA expression in ∼5,500 breast cancers showed PDL1 upregulation in 38% of basal tumors and 38% of inflammatory breast cancers (IBC). Upregulation, associated with signs of strong cytotoxic local immune response, was associated with a better survival in the basal or triple-negative subtypes, and with a better pathological response to chemotherapy in these subtypes and IBC. Reactivation of dormant tumor-infiltrating lymphocytes (TILs) by PD1/PDL1-inhibitors represents a promising strategy in these aggressive tumors.

Abstract 4974: IL-1 receptor-associated kinase-1 and -4 expression in Hodgkin lymphoma

Abstract BACKGROUND: The Toll-like receptor (TLR) mediates nuclear factor kappa B (NFkB) activation in lymphocytes using a family of intermediary proteins called IL-1 receptor associated kinase (IRAK). Mutations within and upregulation of this pathway have been described in the carcinogenesis of non-Hodgkin lymphoma (NHL), but it is not known whether IRAK proteins are present in Hodgkin lymphoma (HL). Furthermore, Epstein-Barr virus (EBV) has been shown to activate NFkB in HL but its association with proteins upstream of NFkB is not described. METHODS: 1. We performed Western blots on two Hodgkin Reed-Sternberg (HRS) cell lines to detect total IRAK-1 and -4, and phospho-IRAK-1 and -4 (the activated forms). 2. Archived lymph node biopsy samples from Hodgkin lymphoma patients diagnosed at the University of Maryland were stained for IRAK-4. RESULTS: 1. IRAK-1, IRAK-4, p-IRAK-1 and p-IRAK-4 were present in the malignant HRS cells in two HRS cell lines (HDLM-2 and KM-H2). 2. Seventeen lymph node biopsy samples showed marked positivity for IRAK-4 (mean of 2.3 on a 0-4 scale), as well as lesser positivity in surrounding reactive lymphocytes and histiocytes (mean of 1.6). The only sample to achieve a staining level of 4 was of mixed cellularity subtype, and was EBER-positive and HIV-negative. CONCLUSIONS: The IRAK proteins are present in the malignant HRS cells of Hodgkin lymphoma. Further work will examine IRAK-1, and phospho-IRAK-1 and -4 in the lymph node samples. Future studies should also examine whether IRAK causes downstream NFkB activation in HL as it does in NHL, and whether HRS cells depend on IRAK's presence for survival. A possible association with EBV infection will also be investigated. IRAK is a targetable protein with inhibitors available, so this and future studies may reveal new therapeutic strategies in relapsed Hodgkin lymphoma. Citation Format: Victoria S. Giffi, Kenisha M. Younger, Zeba Singh, Qing Chen, Eduardo Davila, Amy S. Kimball. IL-1 receptor-associated kinase-1 and -4 expression in Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4974. doi:10.1158/1538-7445.AM2015-4974

Aspiration cytology of an ectopic cervical thymoma misinterpreted as a lymphoproliferative lesion of the thyroid: A case report.

Ectopic cervical thymoma is a rare tumor that originates from ectopic thymic tissue trapped during the migration of the embryonic thymus. To the best of our knowledge, only 14 cases of ectopic cervical thymoma, which include descriptions of the cytological features based on fine-needle aspiration (FNA), have been reported thus far. The current study describes the case of a 52-year-old male presenting with an enlarging anterior neck mass that been apparent for a number of years and was now accompanied by shortness of breath. FNA cytology revealed large numbers of small lymphocytes admixed with rare groups of large, polygonal cells that were interpreted to be reactive lymphocytes or possible follicular dendritic cells. However, no definite follicular or Hürthle cells were identified. Therefore, the overall cytological features were misinterpreted as a lymphoproliferative lesion. However, subsequent histological analysis of the resected left total lobectomy specimen determined a diagnosis of thymoma, type B1. Thus, awareness of this entity combined with a careful search for thymic epithelial cells may aid in determining a correct diagnosis when FNA is performed for the evaluation of a neck mass.

Mechanisms of immunological tolerance

There is increasing interest in establishing diagnostic markers of immunological tolerance applicable to efforts to minimize drug immunosuppression in transplantation and chronic immunological diseases. It is hoped that an understanding of the diverse mechanisms that can contribute to tolerance will guide efforts to establish diagnostic tolerance biomarkers. Not only would these be valuable for management of autoimmune diseases, transplants and allergies, but they might also guide efforts to override tolerance processes in cancer and vaccine development. Where tolerance is generated by deletion or inactivation of antigen reactive lymphocytes, it is unlikely that any long-term-valid blood biomarkers might be found. Where tolerance is mediated by active regulatory mechanisms, indicators that can be usefully measured may emerge, but these would likely show significant heterogeneity reflecting the diversity of active tolerance processes operating in different individuals. Given this, the most useful “kits” might be those “smart” enough to detect this diversity of tolerance players.

Phenotypic switch of CD8(+) T cells reactivated under hypoxia toward IL-10 secreting, poorly proliferative effector cells.

CD8+ T cells controlling pathogens or tumors must function at sites where oxygen tension is frequently low, and never as high as under atmospheric culture conditions. However, T‐cell function in vivo is generally analyzed indirectly, or is extrapolated from in vitro studies under nonphysiologic oxygen tensions. In this study, we delineate the role of physiologic and pathologic oxygen tension in vitro during reactivation and differentiation of tumor‐specific CD8+ T cells. Using CD8+ T cells from pmel‐1 mice, we observed that the generation of CTLs under 5% O2, which corresponds to physioxia in lymph nodes, gave rise to a higher effector signature than those generated under atmospheric oxygen fractions (21% O2). Hypoxia (1% O2) did not modify cytotoxicity, but decreasing O2 tensions during CTL and CD8+ tumor‐infiltrating lymphocyte reactivation dose‐dependently decreased proliferation, induced secretion of the immunosuppressive cytokine IL‐10, and upregulated the expression of CD137 (4‐1BB) and CD25. Overall, our data indicate that oxygen tension is a key regulator of CD8+ T‐cell function and fate and suggest that IL‐10 release may be an unanticipated component of CD8+ T cell‐mediated immune responses in most in vivo microenvironments.

Autoantibody-positive healthy individuals display unique immune profiles that regulate autoimmunity (HUM3P.244)

Abstract Antinuclear antibodies (ANA) are detected in up to 25% of the population, yet only 5-8% develop autoimmunity. Immunological differences between ANA+ healthy individuals and SLE patients may give us insight into mechanisms by which ANA+ individuals avoid autoimmunity or transition to SLE. European-American ANA+ individuals (n=31) were matched to ANA- controls and SLE patients. Serum cytokine profiles, lymphocyte subset frequency, and lymphocyte reactivity were analyzed. Numerous pro-inflammatory cytokines including IFNγ, IL-2, IL-5 and TNFα were elevated in the serum of ANA+ healthy individuals compared to ANA- controls; however, SLE patients had even higher cytokine levels compared to ANA+ healthy individuals (p&amp;gt;0.05). Interestingly, Type I IFNs, BLyS, IL-12p40 and SCF were only increased in SLE patients (p&amp;lt;0.05). Further, IL-1RA was decreased in both ANA+ and SLE individuals (p&amp;lt;0.0001). Total monocytes and memory B cells, were elevated in ANA+ individuals compared with ANA- controls while regulatory CD4+ T cells were decreased (p&amp;lt;0.05). Further, monocytes and B cells from ANA+ individuals had increased levels of pSTAT1 and pSTAT3 following IFN-α stimulation compared to controls (p&amp;lt;0.05). These data reveal ANA+ healthy individuals exhibit similar features to SLE patients with increased inflammation and loss of immune tolerance, but differ by the absence of elevated Type I IFNs, BLyS, IL-12p40 and SCF and severely decreased IL-1RA that contribute to disease pathogenesis.

Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have been regarded as useful sources for cell-based transplantation therapy. However, immunogenicity of the cells remains the major determinant for successful clinical application. We report the examination of several hESC lines (NTU1 and H9), hiPSC lines, and their derivatives (including stem cell-derived hepatocytes) for the expression of major histocompatibility complex (MHC), natural killer (NK) cell receptor (NKp30, NKp44, NKp46) ligand, immune-related genes, human leukocyte antigen (HLA) haplotyping, and the effects in functional mixed lymphocyte reaction (MLR). Flow cytometry showed lower levels (percentages and fluorescence intensities) of MHC class I (MHC-I) molecules, β2-microglobulin, and HLA-E in undifferentiated stem cells. The levels were increased after cotreatment with interferon-γ and/or in vitro differentiation. Antigen-presenting cell markers (CD11c, CD80, and CD86) and MHC-II (HLA-DP, -DQ, and -DR) remained low throughout the treatments. Recognition of stem cells/derivatives by NK lysis receptors were lower or absent. Activation of responder lymphocytes was significantly lower by undifferentiated stem cells than by allogeneic lymphocytes in MLR, but differentiated NTU1 hESCs induced a cell number-dependent lymphocyte proliferation comparable with that by allogeneic lymphocytes. Interestingly, activation of lymphocytes by differentiated hiPSCs or H9 cells became blunted at higher cell numbers. Real-time reverse transcriptase PCR (RT-PCR) showed significant differential expression of immune privilege genes (TGF-β2, Arginase 2, Indole 1, GATA3, POMC, VIP, CALCA, CALCB, IL-1RN, CD95L, CR1L, Serpine 1, HMOX1, IL6, LGALS3, HEBP1, THBS1, CD59, and LGALS1) in pluripotent stem cells/derivatives when compared to somatic cells. It was concluded that pluripotent stem cells/derivatives are predicted to be immunogenic, though evidence suggests some level of potential immune privilege. In addition, differential immunogenicity may exist between different pluripotent stem cell lines and their derivatives.

Fungal Transverse Myelitis in a Crimson Rosella (Platycercus elegans)

This report describes a case of chronic fungal transverse myelitis in a crimson rosella (Platycercus elegans) presented for a progressive inability to stand over a 5-day period. Findings on physical examination revealed loss of neurological function to the legs, vent and tail, whereas the wings, head and neck remained neurologically normal. Radiographic imaging demonstrated a slight kyphosis in the notarial region, while findings on hematologic examination revealed nonregenerative anemia and a normal white cell count with reactive lymphocytes. A tentative diagnosis of spinal trauma was made, and supportive medical care was administered to the patient. Despite all medical treatment, the bird died 12 hours after presentation. Findings on postmortem examination revealed a fungal myelitis, meningitis and vertebral osteomyelitis associated with a healed spinal fracture in the notarial-synsacral region. No evidence of fungal infection was detected in any other location in the body. This case demonstrates how the partially segmental nature of the spinal cord blood supply, when damaged by trauma, may have led to a fungal transverse myelitis and the patient׳s subsequent death.

An examination of the utility of heterophil-lymphocyte ratios in assessing stress of caged hens

To measure stress in caged hens, differential counts of their wing vein blood were used to determine heterophil/lymphocyte (H/L) ratios and total white blood cell counts (TWBC). The H/L values of 18-wk samples from conventionally caged hens (CC) were not statistically different from hens raised in aviaries (AV) when calculated by either of 2 methods (H/L 1 and H/L 2). However, there was a high degree of variation among samples within each cage type. The TWBC data and hematology indicated leukocytosis, leukemoid reactions, and a high frequency of atypia. Reactive lymphocytes, large plasmacytoid lymphocytes, cyanophils, coccinocytes, and atypical heterophils were common. Analysis of 77-wk data indicated significant differences among 3 cage types. The H/L 1 of enriched caged (EN) hens was twice (0.91) that of either AV (0.33) or CC (0.44) hens (P < 0.01); the H/L 2 values were also highest for EN (0.46) versus AV (0.29) and CC (0.34; P <0.01). As was the case with 18-wk samples, TWBC distributions and hematological data indicated leukocytosis, leukemoid reactions, and a high frequency of atypia. Among the likely reasons for the hematological observations was the occurrence of polymicrobial bacteremia and fungemia, both of which could account for high TWBC and atypical cells. Collectively, these observations challenge the general application of the H/L ratio method when applied alone as an indicator of stress and welfare of hens caged in modern systems.

CD4+ T cell hyporesponsiveness after repeated exposure to Schistosoma mansoni larvae is dependent upon interleukin-10.

The effect that multiple percutaneous exposures to Schistosoma larvae has on the development of early CD4+ lymphocyte reactivity is unclear, yet it is important in the context of humans living in areas where schistosomiasis is endemic. In a murine model of multiple infections, we show that exposure of mice to repeated doses (4×) of Schistosoma mansoni cercariae, compared to a single dose (1×), results in CD4+ T cell hyporesponsiveness within the skin-draining lymph nodes (sdLN), manifested as reduced CD4+ cell proliferation and cytokine production. FoxP3+ CD4+ regulatory T cells were present in similar numbers in the sdLN of 4× and 1× mice and thus are unlikely to have a role in effecting hyporesponsiveness. Moreover, anergy of the CD4+ cell population from 4× mice was slight, as proliferation was only partly circumvented through the in vitro addition of exogenous interleukin-2 (IL-2), and the in vivo blockade of the regulatory molecule PD1 had a minimal effect on restoring responsiveness. In contrast, IL-10 was observed to be critical in mediating hyporesponsiveness, as CD4+ cells from the sdLN of 4× mice deficient for IL-10 were readily able to proliferate, unlike those from 4× wild-type cohorts. CD4+ cells from the sdLN of 4× mice exhibited higher levels of apoptosis and cell death, but in the absence of IL-10, there was significantly less cell death. Combined, our data show that IL-10 is a key factor in the development of CD4+ T cell hyporesponsiveness after repeated parasite exposure involving CD4+ cell apoptosis.

Cancer immunotherapy utilizing gene-modified T cells: From the bench to the clinic.

The immune system plays a critical role in the elimination and suppression of pathogens. Although the endogenous immune system is capable of immune surveillance resulting in the elimination of cancer cells, tumor cells have developed a variety of mechanisms to escape immune recognition often resulting in tumor outgrowth. The presence of immune infiltrate in tumors has been correlated with a good prognosis following treatment (Sato et al., 2005; Loi et al., 2013; Clemente et al., 1996; Galon et al., 2006). As such, immune cells such as T cells, have been harnessed in order to target cancer. Tumor reactive lymphocytes, called tumor-infiltrating lymphocytes (TILs) have been isolated and expanded from the tumor and reinfused back into patients for the treatment of melanoma. The promise of adoptive immunotherapy utilizing TILs as a robust treatment for cancer has been highlighted in patients with advanced melanoma with greater than 50% of patients responding to treatment (Dudley et al., 2005). Although TIL therapy has shown promising results in melanoma patients, it has proved difficult to translate this approach to other cancers, given that the numbers of TILs that can be isolated are generally low. To broaden this therapy for other cancers, T cells have been genetically modified to endow them with tumor reactivity using either a T cell receptor (TCR) (Parkhurst et al., 2009, 2011; Chinnasamy et al., 2011) or a chimeric antigen receptor (CAR) (Grupp et al., 2013; Park et al., 2007). This review will outline the origins and development of adoptive immunotherapy utilizing TILs leading to genetic modification strategies to redirect T cells to cancer. Potential hurdles and novel strategies will be discussed for realizing the full potential of adoptive immunotherapy becoming a standard of care treatment for cancer.

Acute abdomen in a patient with coeliac disease: a case report and literature review of enteropathy-associated T-cell lymphoma (EATl)

A 71-year-old female with a history of poorly-controlled coeliac disease presented with worsening symptoms of small bowel obstruction over 3 weeks. Jejunal, ileal and mesenteric resections were performed. The patient did not recover after surgery and died 3 weeks later. Patchy mucosal ulcerative plaques were seen, corresponding to transmural infiltration by medium-to-large atypical lymphoid cells positive with CD3 and CD56 but negative for CD5, CD8, CD4 and CD20. Similar atypical cells were identified in all regional lymph nodes, in a predominantly sinusoidal distribution. Remaining small bowel mucosa showed typical histological features of coeliac disease, comprising severe villous atrophy, intraepithelial lymphocytosis (reactive lymphocytes) and crypt hyperplasia. EATL of the small intestine with involvement of mesenteric lymph nodes, occurring in the setting of coeliac disease, was diagnosed. EATL is an uncommon lymphoma of intraepithelial T-lymphocytes mostly occurring in patients with coeliac disease. Ten to twenty percent of cases may occur sporadically without coeliac disease (type II EATL) in which monomorphic, medium-sized cells are present. Type II EATL has a distinctive immunophenotype. Both EATL types have very poor prognosis due to low tumour chemosensitivity and aggressive behaviour. Major postsurgical complications are also common in patients who are nutritionally deficient due to coeliac disease.