Utilization of the Ex Vivo LLNA

Dermal exposure to chemicals may result in allergic or irritant contact dermatitis. In this study, we performed ex vivo local lymph node assay: bromodeoxyuridine-enzyme-linked immunosorbent assay (LLNA: BrdU-ELISA) to compare the differences between irritation and sensitization potency of some chemicals in terms of the 3 end points: lymphocyte proliferation, cytokine profiles (interleukin 2 [IL-2], interferon-γ (IFN-γ), IL-4, IL-5, IL-1, and tumor necrosis factor α [TNF-α]), and ear swelling. Different concentrations of the following well-known sensitizers and irritant chemicals were applied to mice: dinitrochlorobenzene, eugenol, isoeugenol, sodium lauryl sulfate (SLS), and croton oil. According to the lymph node results; the auricular lymph node weights and lymph node cell counts increased after application of both sensitizers and irritants in high concentrations. On the other hand, according to lymph node cell proliferation results, there was a 3-fold increase in proliferation of lymph node cells (stimulation index) for sensitizer chemicals and SLS in the applied concentrations; however, there was not a 3-fold increase for croton oil and negative control. The SLS gave a false-positive response. Cytokine analysis demonstrated that 4 cytokines including IL-2, IFN-γ, IL-4, and IL-5 were released in lymph node cell cultures, with a clear dose trend for sensitizers whereas only TNF-α was released in response to irritants. Taken together, our results suggest that the ex vivo LLNA: BrdU-ELISA method can be useful for discriminating irritants and allergens.

Post-Transplant Cyclophosphamide Treatment Ameliorates Experimental Gvhd While Permitting Lymphopenic Expansion of Non-Host Reactive Donor T Cells.

Abstract 3751 Following myeloablative as well as reduced intensity conditioning regimens, recipients of allogeneic T cell replete (TCR) HLA matched sibling)or unrelated hematopoietic stem cell transplants (HSCT) experience significant acute as well as chronic graft vs. host disease (GVHD). Recent studies have reported that post-transplant cyclophosphamide (PTC) can function as an effective single agent for prophylaxis of acute and chronic GVHD (Biol. Blood Marrow Transpl. 16:482, 2010; Blood. 116:3224, 2010). To investigate the fate of non-host alloreactive donor cells, which are critical to providing immune reconstitution post-transplant, T cell replete HSCT were performed between MHC-matched allogeneic donor–recipient pairs. C3H.SW (H2b) mice were lethally conditioned 4 hrs prior to receipt of B6-CD45.1 (H2b) T cells and bone marrow. Recipients developed severe GVHD as assessed by weight loss (Fig. 1A) and clinical analyses during the first 4–5 wks post-transplant. Recipients had inverted CD4/CD8 ratios as well as an activated phenotype (CD44hiCD62Llo), severe B and T cell lymphopenia, and virtually undetectable thymic tissue. A single dose of post-transplant cyclophosphamide at day 3 (66mg/kg/body weight) blocked onset of weight loss and clinical signs of GVHD as well as lethality in C3H.SW recipients, consistent with the reduction of alloreactive anti-recipient T cells. In contrast to non-cyclophosphamide treated animals, treated recipients expressed typical CD4/CD8 ratios, significant numbers of spleen and lymph node cells (Fig. 1B) and readily detectable thymi 7–8 weeks post-HSCT. Notably, the majority of T as well as B cells in the periphery of these recipients exhibited a non-activated phenotype and were derived from donor bone marrow. Together with the observation that approximately 50% of T cells exhibited a CD44hiCD62Llo phenotype characteristic of TN, the findings are consistent with de novo derived lymphopoiesis in the thymus and marrow of post-transplant cyclophosphamide treated recipients. To assess the impact of D.3 cyclophosphamide treatment on a non-host reactive T cell population, B6 OT-I CD8 T cells (Vα2/Vβ5 TcR) were added (1-2×106) to the donor inoculum. In non-PTC treated recipients, low numbers of OT-I T cells (below input levels) were detected by 3–4 weeks post-HSCT. In contrast, in PTC-treated recipients, significant numbers of OT-I CD8 T cells were present in the recipient spleen and lymph nodes (Fig. 1C), consistent with the finding of CFSE labeled OT-I cells exhibiting division following cyclophosphamide treatment. Notably, the overall numbers of OT-I CD8 T cells in cyclophosphamide treated recipients was greater than the input levels, indicating that these cells a) survived post-cyclophosphamide treatment and b) underwent lymphopenic expansion. Consistent with these findings, CFSE studies at 72 hrs post-HSCT illustrated OT-I T cells exhibited low proliferation vs. a population of presumed B6 anti-C3H.SW alloreactive T cells. We therefore conclude that non-host reactive donor T cells undergo sufficient repair following cyclophosphamide induced alkylation to enable greater survival in contrast to rapidly dividing anti-host (GVHD) reactive populations. Vaccination studies in cyclophosphamide treated and non-treated HSCT recipients are being performed using heat shock fusion protein-transfected tumor cells to investigate the capacity to elicit anti-tumor (i.e., GVL) responses in the presence and absence of GVHD. Fig. 1: Post-transplant cyclophosphamide treated recipients demonstrate little severe weight loss vs. non-ptc recipients (panel A). Enhanced lymph node cell numbers and non-host reactive donor CD8 OT-I cell levels in ptc recipients 1 month post MHC-matched MiHA-mismatched HSCT (panels B and C). Fig. 1:. Post-transplant cyclophosphamide treated recipients demonstrate little severe weight loss vs. non-ptc recipients (panel A). Enhanced lymph node cell numbers and non-host reactive donor CD8 OT-I cell levels in ptc recipients 1 month post MHC-matched MiHA-mismatched HSCT (panels B and C). Disclosures: No relevant conflicts of interest to declare.

T-Cell Subpopulations Quantified by Flow Cytometry in Lymph Node Cell Suspensions Identify a Group of Patients with Follicular Lymphoma with Good Prognosis.

Abstract 1945 Poster Board I-968 T-cell Subpopulations Quantified by Flow Cytometry in Lymph Node Cell Suspensions Identify a Group of Patients with Follicular Lymphoma with Good Prognosis Neus Villamor, Gonzalo Gutiérrez, Joaquim Carreras, Eva Giné, Gabriela Ghita, Marta Aymerich, Montserrat Torrebadell, Antonio Martínez, Lluís Colomo, Emili Montserrat, Elías Campo, Armando López-Guillermo Hematopathology Unit and Department of Hematology, Hospital Clínic, IDIBAPS, Barcelona, Spain. Introduction: Tumor microenvironment plays an important role in the behavior of follicular lymphoma (FL), as demonstrated by gene expression profile analysis. Using this technique, an increase in macrophages has been associated with poor outcome, whilst an increase in T-cells is associated with better prognosis. Immunohistochemical analysis has been performed as alternative method to gene expression profile, but the quantification is time consuming and poorly standardized. Patients and methods: T-cell populations from lymph nodes of 68 patients (35M/33F, median age 59, range 29 to 81) with FL at diagnosis were quantified by flow cytometry in cell suspensions. The percentage of CD3, CD4, CD8, CD57, and germinal center (GC) CD4 cells (CD4+CD57+), as well as the ratio B/T, CD4/CD8, CD4/CD3, CD8/CD3 and GC-CD4/CD4 were correlated with the main initial features and the clinical outcome of the patients. The distribution according to the histological grade was: grade 1 and 2, 53 patients; grade 3a, 14; grade 3b, 1. Fifty-one percent of patients had low-risk FLIPI. 62 patients have received polychemotherapy, including rituximab in 33, whereas in 6 a watchful waiting policy was established. After a median follow-up of 4 years, 17 patients have died, with a 5-year overall survival (OS) of 77%. Results: The mean (±SD) percentage of B-cells, CD3, CD4, CD8 and GC-CD4 was 58.6% (±14.2), 36.1% (±15.2), 27.1% (±12.3), 8.7% (±5.1), and 3.4% (±2.4). No correlation was found between percentages of T cell subpopulations, B/T, CD4/CD3 and CD8/CD3 cell ratios and the clinical characteristics, failure-free survival (FFS) and OS. The median value CD4/CD8 and GC-CD4/CD4 ratios was 3.4 (range: 0.51 to 1) and 0.11 (n=56) (range: 0.02 to 0.34), respectively. Grade 3 histology was more frequently observed in patients with CD4/CD8 ratio <4.6 (percentile 0% to 75%) than in cases with CD4/CD8 ratio 34.6 (27% vs 0%; p=0.02). Advanced Ann-Arbor stage was also associated low CD4/CD8 ratio (p=0.02). Response to treatment was not related to lymphocyte subpopulations. FFS was longer in patients with CD4/CD8 34.6 (75% vs. 49% at 5 years, p=0.03), and in those with GC-CD4/CD4 30.18 (percentile 75% to 100%) (71% vs. 47% at 5 years, p=0.05). FLIPI, among other clinical variables, was able to predict OS. Patients with a CD4/CD8 ratio 34.6 had better OS than the remainder (5-year OS: 100% vs. 66%, respectively; p=0.02). Finally, patients with GC-CD4/CD4 ratio (30.18) showed a better OS than the rest (5-year OS: 100% vs. 67%, respectively; p=0.028) (figure). A multivariate analysis was performed including GC-CD4/CD4, CD4/CD8 and FLIPI, with GC-CD4/CD4 being the most important variable to predict OS in the Cox model with 53 patients (relative risk: 41; p=0.01). In conclusion, the analysis of T cell subpopulations in lymph node cell suspensions allows the identification of FL patients with a microenvironment associated with good prognosis. Flow cytometry is an easy, fast and standardized technique to assess T-cell signature in FL. Disclosures: No relevant conflicts of interest to declare.