Immunological Intervention Research Articles

Role of innate cytokines in mycobacterial infection.

Cells of the innate immune system produce cytokines and lipid mediators that strongly influence the outcome of mycobacterial infection. In the case of Mycobacterium tuberculosis, the lung is a critical site for this interaction. Here, we review current information on the role of the major innate cytokine pathways both in controlling initial infection as well as in promoting and maintaining adaptive T-cell responses that mediate host resistance or immunopathology. Understanding this important feature of the host-pathogen interaction can provide major insights into the mechanisms of virulence and can lead to new approaches for immunological intervention in tuberculosis and other mycobacterial diseases.

Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that emerged in the late 1980s in both Europe and North America as the causative agent of porcine reproductive and respiratory syndrome (PRRS), now the most important disease of swine worldwide. Despite extensive characterization of PRRSV proteins by direct analysis and comparison with other arteriviruses, determinants of virulence, pathogenesis and protective immune recognition remain poorly understood. Thus, we hypothesized that additional ORFs are present in the PRRSV genome that may contribute to its biological properties, and so we screened highly purified virions of strain VR2332, the prototype type 2 PRRSV, for evidence of novel polypeptides. A 51 aa polypeptide was discovered that is encoded by an alternative ORF of the subgenomic mRNA encoding the major envelope glycoprotein, GP5, and which is incorporated into virions. The protein, referred to as ORF5a protein, is expressed in infected cells, and pigs infected with PRRSV express anti-ORF5a protein antibodies. A similar ORF is present as an alternative reading frame in all PRRSV subgenomic RNA5 genes and in all other arteriviruses, suggesting that this ORF5a protein plays a significant role in arterivirology. Its discovery also provides a new potential target for immunological and pharmacological intervention in PRRS.

Multi-Dimensional Resistance Phenotype Allows Quiescent Leukemic Stem Cells Residing After Tyrosine Kinase Inhibitor Treatment to Escape From Immunological Interventions With Alloreactive T Cells and NK Cells

Chronic myeloid leukemia (CML) is well responsive to various therapeutic strategies, including tyrosine kinase inhibitors (TKI) and allogeneic stem cell transplantation combined with donor lymphocyte infusion (DLI). Although initial complete responses are frequently achieved, recurrence of the disease is a common phenomenon occurring sometimes years after the therapeutic intervention(s). This suggests that an initially undetectable subpopulation of leukemic precursor cells is capable of escaping therapy.

Influence of Interferon-γ Deficiency in Immune Tolerance Induced by Male Islet Transplantation

BackgroundTraditionally, interferon-γ (IFN-γ) was regarded as a pro-inflammatory cytokine, however, recent reports suggested role of IFN-γ in immune tolerance. In our previous report, we could induce tolerance to male antigen (HY) just by male islet transplantation in wild type C57BL/6 mice without any immunological intervention. We tried to investigate the influence of IFN-γ deficiency on tolerance induction by male islet transplantation.MethodsTo examine the immunogenicity of male tissue in the absence of IFN-γ, we transplanted male IFN-γ knock-out (KO) skin to female IFN-γ KO mice. Next, we analyzed male IFN-γ KO islet to streptozotocin-induced diabetic female IFN-γ KO mice. And, we checked the functionality of grafted islet by graft removal and insulin staining.ResultsAs our previous results in wild type C57BL/6 mice, female IFN-γ KO mice rejected male IFN-γ KO skin within 29 days, and did not reject male IFN-γ KO islet. The maintenance of normal blood glucose level was dependent on the presence of grafted male islet. And the male islet recipient did not reject 2nd challenge of male islet graft also.ConclusionDeficiency of IFN-γ does not have influence on the result of male skin graft and male islet transplantation. Conclusively, male islet transplantation induced T cell tolerance is not dependent on the presence of IFN-γ.

Frontiers in targeting glioma stem cells

Patients with glioblastoma multiforme (GBM – WHO grade IV) seldom recover. This is due to the infiltrative nature of these tumours and the presence of cellular populations with ability to escape therapies and drive tumour recurrence and progression. In some cases, these resistant cells exhibit stem properties [glioma stem cells (GSC)]. This article aims at discussing relevant issues on GSC resistance to current therapies and outlines possible and promising avenues in regard to novel therapeutic strategies, such as pharmacological, immunological and viral interventions.

Multi-Dimensional Resistance Phenotype Allows Subpopulation of Quiescent Chronic Myeloid Leukemia Stem Cells to Universally Escape From Therapeutic Attack

AbstractAbstract 203Chronic myeloid leukemia (CML) is well responsive to various therapeutic strategies, including conventional chemotherapy, tyrosine kinase inhibitors (TKI), but also allogeneic stem cell transplantation combined with donor lymphocyte infusions (DLI). Although initial complete responses are frequently achieved, recurrence of the disease is a common phenomenon occurring sometimes years after the therapeutic intervention(s). Although acquired resistance is sometimes observed, the majority of relapses can be controlled with re-introduction of therapeutic pressure. This indicates that an initially undetectable subpopulation of leukemic precursor cells is capable of escaping the therapy and may give rise to a relapse of the disease after discontinuation of the therapeutic pressure. Since the different therapeutic strategies are based on different anti-tumor effector mechanisms, different escape variants of leukemic precursor cells may reside. To test this hypothesis, we investigated in detail the phenotype and apoptosis gene expression fingerprint of the subpopulations of CML precursor cells residing after conventional chemotherapy (Ara-C, daunorubicin, and camptothecin), TKI treatment (imatinib, dasatinib), and immunological interventions with high affinity antigen-specific cytotoxic donor T cells or NK cells. CD34+ CML precursor cells were isolated from patients and labeled with the green or red flurorescent dyes PKH67 or 26 to allow visualization of individual cell divisions. Quantitative flowcytometric analysis applying counterstaining with antibodies for different cell surface molecules allows a detailed analysis of the phenotype of the precursor cells capable of escaping the different treatment modalities. Although as expected the majority of CML precursor cells and their malignant progeny were efficiently targeted, a small subpopulation of non-dividing CD34 bright cells, comprising on average 0.05–0.2% of the initial CD34+ population, resided after the different strategies. In the absence of therapeutic pressure these cells were capable of producing malignant progeny, illustrating their proliferative capacity. In contrast to the quiescent population persisting during therapeutic pressure, the malignant progeny developing from this population after discontinuation of the treatment was again normally responsive to re-introduction of the therapeutic intervention. Next, we performed cross-resistance analysis by combining the different strategies in sequential order. We demonstrated that despite the different anti-tumor effector mechanisms, the population of quiescent leukemic precursor cells residing after each individual therapeutic intervention shows cross-resistance to the other treatment modalities. To investigate the escape mechanisms underlying this general resistance phenotype, we performed detailed analysis of the cell surface expression of molecules involved in the interaction with immune effector cells like T and NK cells. In addition, we isolated the population of quiescent CML precursor cells residing after therapeutic intervention and compared the apoptosis gene expression fingerprint of these cells with the profile of total CD34+ CML precursor cells, their proliferating malignant offspring and CD34+ precursor cells isolated from peripheral blood of healthy stem cell donors after G-CSF induced stem cell mobilization. This fingerprint was made using a quantitative array PCR containing probes for 86 apoptosis-related genes (SABiosciences). The phenotype analysis showed inferior expression of HLA-class I and several adhesion molecules crucial for the formation of a high avidity interaction with immune effector cells, including CD54, CD58 and CD49d. Moreover, the genetic profiling revealed significant downregulation (4–182 fold) of crucial players of the different main apoptosis pathways, including caspase-3 and -8, Fas, TNFR, and different association molecules like FADD, TRADD, and TRAFs, and a 4.8–6.2 fold upregulation of the most important inhibitor of the death receptor pathway c-FLIP (CFLAR). Interestingly, we observed a 55 fold downregulation of the TKI-targeted fusion gene partner Abl1. These data show that a subpopulation of CML precursor cells with potential self-renewal capacity harbors a multi-dimensional resistance phenotype allowing them to escape from different therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Chimerism Analysis Is Predictive of Relapse-Free Survival After Allogeneic Transplantation for MDS

AbstractAbstract 1332Chimerism analysis permits evaluation of the extent of donor engraftment following allogeneic hematopoietic cell transplantation (HCT) via differentiation between donor- and recipient-derived cells. Though relapse remains a major cause of treatment failure post-HCT for hematologic malignancies, the clinical utility of chimerism analysis for the early detection of morphologic relapse varies between different diseases and remains controversial. The predictive value of chimerism analysis for relapse rates and mortality in myelodysplastic syndromes (MDS) is not well-described. We reviewed serial chimerism results for 72 consecutive patients (pts) who underwent allogeneic HCT for MDS between 1999 and 2009; 9 pts were excluded from analysis due to lack of appropriately timed chimerism studies. Donor engraftment was initially assessed 28 days post-HCT and then at 2-week intervals through day 100. Chimerism studies were performed with peripheral blood using a short tandem repeat assay by PCR-based analysis. Acute and chronic GVHD rate, relapse-free survival (RFS), and overall survival (OS) were assessed for patients with donor leukocyte chimerism and T-cell chimerism ≥95% and <95%. The median age was 51 years (range 20–70) and 52% were male. The median time from MDS diagnosis to HCT was 5.4 months; 9 pts (14%) were in complete remission at the time of HCT, 11 (18%) were in partial remission, 17 (27%) had relapsed or refractory disease, and 26 (41%) were untreated. HCT-comorbidity index was low in 25 pts (40%), intermediate in 18 (29%), and high in 20 (32%). 31 pts (49%) received sibling donor HCT and the remainder received an unrelated donor graft. 40 (63%) received bone marrow and 23 (37%) received peripheral stem cells. Myeloablative (MA) busulfan- or cyclophosphamide-based preparative regimens were used in 47 pts (75%) and a non-myeloablative (NMA) regimen with fludarabine and TBI was given to the remaining pts. 61 pts achieved ≥95% donor leukocyte chimerism at a median of 29 days and 39 evaluable pts achieved ≥95% donor T-cell chimerism at a median of 42 days. Two pts did not achieve donor leukocyte chimerism ≥95% and 9 did not achieve donor T-cell chimerism ≥95% at any timepoint. Univariable analysis of prognostic factors for relapse showed that donor leukocyte chimerism ≥95% was significantly associated with lower probability of relapse (hazard ratio [HR] 0.11, 95% confidence interval [CI] 0.02–0.51, p=.005), whereas prior exposure to radiation therapy (excluding exposure during HCT preparative regimen) was associated with increased probability of relapse (HR 3.48, 95% CI 1.14–10.60, p=.028). Multivariable analysis implicated donor leukocyte chimerism <95% as the only independent risk factor for relapse. Transplant type (MA vs NMA) and cell source did not significantly impact the likelihood of relapse. Donor leukocyte chimerism ≥95% was not associated with acute or chronic GVHD. Univariable analysis of risk factors for survival showed that donor leukocyte chimerism ≥95% was associated with improved RFS (HR 0.29, 95% CI 0.09–0.97, p=.045) but not OS, both findings of which were confirmed on multivariable analysis. Donor T-cell chimerism ≥95% was not significant in univariable analysis; however, in multivariable analysis, controlling for comorbidity scores, donor T-cell chimerism <95% was associated with lower risk of chronic GVHD (HR 0.18, 95% CI 0.04–0.88, p=.034) but did not significantly impact relapse, RFS, or OS. In conclusion, achievement of a high donor leukocyte chimerism post-HCT for MDS is associated with improved RFS. Donor T-cell chimerism, however, is not predictive of outcome and its routine use for MDS pts should be reevaluated. Pts with donor leukocyte chimerism <95% might be considered for immunologic interventions such as withdrawal of immunosuppression or donor lymphocyte infusion. Disclosures:No relevant conflicts of interest to declare.

Characterization of age-related changes in bovine CD8 + T-cells

Evaluation of the changes induced by immunological interventions requires a baseline against which to compare those changes. The age-related changes in the CD8 + T-cell population of cattle were studied. The results indicate that CD8 + T-cells could be divided into γ/δ TCR1 + and γ/δ TCR1 − according to their expression of the γ/δ T-cell receptor. As a proportion, the CD8 + γ/δ TCR1 + population appears to increase with age. Within the CD8 +γ/δ TCR1 − a population of cells expressing a profile of surface molecules previously associated with effector memory T cells (CD45RO +, CD62L −, CD27 −, CD45RA − and CD28 −) increases with age. Furthermore, a parallel increase with age in the proportion of CD8 +CD45RO + T cells that express the cytotoxic granule protein perforin was observed. In peripheral tissues, namely lungs, it was found that the majority of CD8 + T cells present expressed a phenotype indicative of previously primed T cells (high expression of CD45RO and perforin). In contrast, only a small population of memory CD8 + T cells was present in lymphoid tissue where most of the CD8 + T cells expressed a naïve phenotype. In conclusion, in cattle, like in human, CD8 + T cells that express a phenotype associated with antigen experience accumulate with age that may play a role in immunocompetence as the individual ages.

Immunological intervention strategies aimed at regulatory T cells (LL1-4)

Immunological intervention strategies aimed at regulatory T cells (LL1-4)

A Tractable Experimental Model for Study of Human and Animal Scabies

BackgroundScabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australia's Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica.Methodology/Principal FindingsOver five years and involving ten independent cohorts, we have developed a protocol for continuous passage of Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort.Conclusions/SignificanceA reliable pig/scabies animal model will facilitate in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaption. It is a most needed tool for the further investigation of this important and widespread parasitic disease.

Second-line options for refractory steroid-sensitive and -resistant nephrotic syndrome

Although initially, many children with idiopathic nephrotic syndrome respond to steroid therapy, a repeated course for patients with relapses often causes significant steroid toxicity. Patients with frequent relapses or steroid dependency thus require alternative treatment, and so far, cyclophosphamide or levamisole have been regarded as first-choice options, although the latter is no longer available in many countries. Data are accumulating that mycophenolic acid may be an alternative for these patients. Calcineurin inhibitors (cyclosporine A or tacrolimus) are usually effective and are often used after cytotoxic treatment, but long-term treatment is necessary, raising concerns regarding the accumulation of side effects. Still, some patients show a tendency to relapse even on this maintenance regimen and some even have a refractory course that creates a medical dilemma. For this situation, recent data have demonstrated an effect of monoclonal antibodies directed to B cells – rituximab, a drug that may also prove to be a therapeutic option in less complicated cases. Patients that do not respond to initial steroid treatment need genetic testing and a renal biopsy, since focal segmental glomerulosclerosis may be present. Treatment options include pulse methylprednisolone, often in addition to calcineurin inhibitors (mainly cyclosporine but also, recently, tacrolimus). Cyctotoxic treatment, especially intravenous cyclophosphamide, has been found to be effective in steroid-resistant nephrotic syndrome by some studies but is inferior to calcineurin inhibitors. In addition, mycophenolic acid and rituximab have been used in children with primary focal segmental glomerulosclerosis; however, response seems to be inferior in comparison with patients with steroid-sensitive nephrotic syndrome. Taken together, idiopathic nephrotic syndrome, including steroid-sensitive as well as steroid-resistant patients, is a potentially serious disorder. Although much progress has been made in recent years and a wide arsenal of immunological interventions is available, some patients have a treatment refractory course. Prospective studies or at least standardization of treatment for complicated cases is urgently needed.

MVA-nef induces HIV-1-specific polyfunctional and proliferative T-cell responses revealed by the combination of short- and long-term immune assays

Several vaccination trials are evaluating the modified vaccinia virus Ankara (MVA) as a delivery vector in various clinical settings. In this paper, we present the reevaluation of a therapeutic vaccination trial in human immunodeficiency virus (HIV)-1-infected individuals treated with highly active antiretroviral therapy using MVA-expressing HIV-1 nef. Immunogenicity of MVA-nef was assessed using multicolor flow cytometry. Vaccine-induced polyfunctionality and proliferative capacity, which are associated with nonprogressive HIV-1 infection, were detectable by combining two immune assays. By means of short-term polychromatic intracellular cytokine staining, we observed a significant increase in polyfunctional Nef-specific CD4 T cells expressing interferon-γ, interleukin (IL)-2 and CD154 after vaccination, whereas changes in the quality of CD8 T-cell response could not be observed. Only the additional use of a long-term polychromatic Carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay revealed vaccine-induced Nef-specific CD8, as well as CD4 T cells with proliferative capacity. The correlation between vaccine-induced IL-2 production by CD4 T cells and the increase in proliferating Nef-specific CD8 T cells suggests a causal link between these two functions. These results highlight the importance of combining sophisticated immunomonitoring tools to unravel concealed effects of immunological interventions and support the use of the poxvirus-derived MVA vector to stimulate highly functional HIV-1-specific T-cell responses. However, the clinical benefit of these functional T cells remains to be determined.

Candida albicansHyr1p Confers Resistance to Neutrophil Killing and Is a Potential Vaccine Target

Candida albicans is the most common cause of invasive fungal infections in humans. It is unclear how C. albicans escapes from phagocytic attack and survives in the hostile blood environment during life-threatening systemic infections. Using a conditional overexpression or suppression genetic strategy, we discovered that HYR1 gene reduced phagocytic killing activity of C. albicans in vitro and increased tissue fungal burden in vivo. Concordant with its positive regulation by the transcription factor Bcr1p, autonomous expression of HYR1 complemented the hypersusceptibility to phagocyte-mediated killing of a bcr1 null mutant of C. albicans in vitro. As for C. albicans, heterologous expression of HYR1 in Candida glabrata rendered the organism more resistant to neutrophil killing activity. Vaccination with a recombinant Hyr1p significantly protected mice against hematogenously disseminated candidiasis (P = .001). Finally, anti-rHyr1p polyclonal antibodies enhanced mouse neutrophil killing activity by directly neutralizing rHyr1p effects in vitro. Thus, Hyr1 is an important virulence factor for C. albicans, mediating resistance to phagocyte killing. Hyr1p is a promising target for vaccine or other immunological or small molecule intervention to improve the outcomes of disseminated candidiasis.

Acetaminophen-induced hepatic neutrophil accumulation and inflammatory liver injury in CD18-deficient mice

Acetaminophen (APAP) hepatotoxicity is currently the most frequent cause of acute liver failure in the US and many European countries. Although intracellular signalling mechanisms are critical for hepatocellular injury, a contribution of inflammatory cells, especially neutrophils, has been suggested. However, conflicting results were obtained when using immunological intervention strategies. The role of neutrophils was investigated using a CD18-deficient mouse model. Treatment of C57Bl/6 wild type mice with 300 mg/kg APAP resulted in severe liver cell necrosis at 12 and 24 h. This injury was accompanied by formation of cytokines and chemokines and accumulation of neutrophils in the liver. However, there was no difference in the inflammatory response or liver injury in CD18-deficient mice compared with wild-type animals. In contrast to treatment with endotoxin, no upregulation of CD11b or priming for reactive oxygen was observed on neutrophils isolated from the peripheral blood or the liver after APAP administration. Furthermore, animals treated with endotoxin 3 h after APAP experienced an exaggerated inflammatory response as indicated by substantially higher cytokine and chemokine formation and twice the number of neutrophils in the liver. However, liver injury in the two-hit model was the same as with APAP alone. Our data do not support the hypothesis that neutrophils contribute to APAP hepatotoxicity or that a neutrophil-mediated injury phase could be provoked by a second, pro-inflammatory hit. Thus, APAP-induced liver injury in mice is dominated by intracellular mechanisms of cell death rather than by neutrophilic inflammation.

A randomized phase II study of a novel antigen-presenting cell-targeted hCG-β vaccine (the CDX-1307 regimen) in muscle-invasive bladder cancer.

TPS228 Background: hCG-β is frequently expressed and correlates with poor prognosis in a number of common epithelial cancers (Iles 2007). Patients (pts) with invasive bladder cancer and elevated hCG-β levels are much more likely to develop metastasis and die earlier (Iles 1996). We have developed a dendritic cell-targeted vaccine, CDX-1307, by genetically coupling hCG-β to a human monoclonal antibody targeting mannose receptors. CDX-1307 efficiently stimulates both MHC-I and II hCG-β-specific T-cell responses in vitro (Ramakrishna 2004/2007). CDX-1307 has completed a phase I study to define an immunogenic regimen and safety profile for phase II studies. The study design included administration in combination with GM-CSF (GM), the TLR3 agonist Poly-ICLC (PIC), and/or the TLR7/8 agonist resiquimod (R), in pts with advanced cancers known to frequently express hCG-β (Morse, iSBTc 2009). Treatment was well tolerated with no DLT. Significant hCG-β–specific cellular and humoral immune responses were detected in pts receiving the combination with all 3 adjuvants. Indications of clinical benefit (disease stabilization and/or reduction in tumor markers) were seen in a subset of patients. These data support further evaluation in newly diagnosed, resectable, hCG-β+ bladder cancer pts, who may have greatest potential for response to immunologic intervention. Methods: A phase II, multicenter trial randomizes (1:1) 60 chemo-naive pts with newly diagnosed, nonmetastatic, resectable, muscle invasive, hCG-β+ bladder cancer to (a) neoadjuvant gemcitabine/cisplatin chemotherapy (GC) or (b) CDX-1307 (2.5 mg) with GM, PIC and R (the CDX-1307 regimen), both with neoadjuvant GC and in the adjuvant setting to prolong immunity. The primary efficacy objective is to estimate the 2-year PFS (95% confidence interval extending ≤18.5% about the point estimate). Besides safety and immune response (during neoadjuvant GC and adjuvantly), additional antitumoral effect will be assessed including radiographic and pathologic tumor response to neoadjuvant vaccination, immune response at the tumor site and persistence of hCG-b expression. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Celldex Therapeutics Celldex Therapeutics Celldex Therapeutics

Abstract 5618: Arming the melanoma sentinel lymph node against tumor spread: The effects of local administration of combined low-dose CpG-B PF-3512676 and GM-CSF

Abstract The sentinel lymph node (SLN) procedure has proven a useful prognostic tool for the assessment of melanoma relapse and mortality risk, but is also of great value for the assessment of immunological interventions. Since early melanoma development is accompanied by a suppressed immune state of the SLN, there is a strong rationale for therapeutic immune modulation of the SLN in order to strengthen local as well as systemic cell-mediated anti-tumor immunity. Previous Phase II studies showed promising immunostimulatory effects on the melanoma SLN of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) and the CpG-B oligodeoxynucleotide PF-3512676. The present 3-arm Phase II study was designed to determine the effects of combined low-dose GM-CSF+CpG-B administration. Stage I-III melanoma patients (n=28) were randomized to receive i.d. injections around the primary tumor excision site of saline, CpG-B alone (1 mg), or CpG-B combined with GM-CSF (100 μg), 7 and 2 days prior to surgical excision of the SLN. Flowcytometric DC analyses revealed significantly increased maturation of all identifiable cDC and pDC SLN subsets upon administration of CpG-B with or without GM-CSF, but remarkably, the combination resulted in significantly higher levels of co-stimulatory molecules on both cDC and pDC. In addition, a significant CpG-related increase in frequencies of CD1a−CD11chiCD14+/− cDC subsets was observed in the SLN, possibly resulting from DC and/or monocyte mobilisation from blood. Indeed, increased activation of pro-inflammatory 6-sulfo LacNac+slanDC, monocytes, and pDC was observed in peripheral blood upon administration of CpG-B alone, and additionally of CD1c+ cDC when combined with GM-CSF. In the case of slanDC this activation was accompanied by significantly reduced frequencies. Peripheral blood rates of CD14+DRlo myeloid suppressors remained unchanged after saline or combined CpG and GM-CSF administration, but significantly decreased after administration of CpG-B alone. T cell analyses revealed a CpG-related increase in CTLA-4 and FoxP3 levels on Tregs in SLN, without changes in their actual rates. Nevertheless, higher intracellular Th1 cytokine levels were detected in ex vivo expanded T cells of CpG-administered patients than of patients receiving saline or CpG-B+GM-CSF. In keeping with this, also higher frequencies were found in SLN of MAA-specific CD8+ T cells in low dose CpG-B administered patients (n=4) than in saline controls (n=5), as determined by tetramer binding and cytokine release. We conclude that the imunomodulatory effects observed in melanoma SLN upon local administration of combined low-dose GM-CSF and CpG-B are mostly attributable to CpG-B and that they are consistent with the induction of protective cell-mediated anti-tumor immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5618.

M1 macrophages as a major host defense effector cell in bacterial translocation induced by whole body 137Cs γ-irradiation (89.34)

Abstract In thermally injured hosts, mesenteric lymph node macrophages (MLNMϕ) are identified as major effector cells against bacterial translocation. In this study, the antibacterial function of MLNMϕ from mice exposed to whole body γ-irradiation was investigated. BALB/c mice, 14 days after 0.5 (0.5 GY-mice) or 5 Gy (5 GY-mice) whole body γ-irradiation, were tested for their resistance against oral infection with 106 CFU/mouse of Enterococcus faecalis. Also, the resistance of decontaminated NOD-SCID IL-2rγ-/- mice (NOD-SCID mice), inoculated with MLNMϕ from 0.5 GY-mice or 5 GY-mice, to E. faecalis oral infection was examined. Furthermore, MLNMϕ isolated from 0.5 GY-mice and 5 GY-mice were biophysically characterized. In the results, 100% of 5 GY-mice died after E. faecalis oral infection, while 0.5 GY-mice similarly infected with the pathogen were shown to be resistant. All NOD-SCID mice inoculated with 5 GY-mouse MLNMϕ died after the infection, while 100% of these mice inoculated with 0.5 GY-mouse MLNMϕ survived. MLNMϕ from 0.5 GY-mice produced IL-12 (but not IL-10) and expressed iNOS mRNA. MLNMϕ from 5 GY-mice produced IL-10 (but not IL-12) and did not express iNOS mRNA. These results indicate that 0.5 GY-mouse MLNMϕ are M1Mϕ, and 5 GY-mouse MLNMϕ are M2Mϕ. Bacterial translocation in whole body γ-irradiated mice may be controlled by immunological intervention of M1Mϕ distributed in mesenteric lymph nodes.

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

CD34+ cell dose and establishment of full donor chimerism at day +100 are important factors for survival with reduced-intensity conditioning with fludarabine and melphalan before allogeneic hematopoietic SCT for hematologic malignancies

The combination of fludarabine and melphalan as a reduced-intensity conditioning (RIC) regimen extends allogeneic hematopoietic SCT (HSCT) as a therapeutic option for elderly or frail patients with relapsed, refractory or other high-risk hematologic malignancies. Whether any modifiable factors exist that could improve survival before or immediately after HSCT is unknown. We reviewed the medical records of the first 50 patients at our institution to undergo fludarabine/melphalan RIC from September 2000 to September 2007 to determine factors associated with survival. A total of 25 (50%) patients had undergone prior HSCT and as such was a high-risk group of patients. On multivariate analysis, CD34+ cell dose greater than 5.5 × 106 per kg (risk ratio (RR) 0.44, 95% CI 0.19–0.98, P=0.02) and full donor chimerism at day +100 (RR 0.17, 95% CI 0.06–0.64, P=0.002) remained independent prognostic factors. In our series, achievement of full donor chimerism at day +100 was associated with an approximately 70% 2-year survival, a favorable outcome in this high-risk group of patients. Although the infused CD34+ cell dose is a modifiable variable, whether donor lymphocyte infusions or other immunologic interventions should be performed to promote the establishment of full chimerism early post transplant remains unknown.

Assessment Of The Utility Of Subset Donor Chimerism For Predicting Event-Free Survival After Non-Myeloablative Allogeneic Hematopoietic Cell Transplantation Using TLI And ATG

Allogeneic hematopoietic cell transplantation (HCT) following conditioning with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) is an effective treatment modality for leukemia and lymphoma. We recently reported 3-year event-free survival rates for AML and NHL of 40% and 42%, respectively (Kohrt et al., Blood, 114:1099-1109). Disease relapse remains the primary cause of treatment failure, thus the ability to predict when patients are at risk for relapse would allow for timely immunologic interventions, such as alterations of immunosuppressive therapy or donor lymphocyte infusions.