CD3 Monoclonal Antibody Research Articles

Anti-human CD14 monoclonal antibody improves survival following sepsis induced by endotoxin, but not following polymicrobial infection

Cluster of differentiation 14 (CD14), a pattern recognition receptor expressed on myeloid cells and a critical component of the innate immune system, mediates local and systemic host responses to gram-negative bacterial products, including lipopolysaccharide (LPS). Therefore, CD14 is an attractive target for development of sepsis therapies, and several monoclonal anti-CD14 antibodies have been reported. In this study, we prepared an anti-human CD14 monoclonal antibody, F1024-1-3, which suppressed LPS-induced upregulation of pro-inflammatory cytokines and an adhesion molecule in human peripheral mononuclear cells and human vascular endothelial cells. Half-maximal inhibitory concentrations in these assays ranged from 0.1 to 1μg/ml. In rabbits, intravenous administration (3mg/kg) as well as in vitro exposure of F1024-1-3 suppressed LPS-induced cytokine production in whole blood. In endotoxemia models generated by three sequential injections of LPS, intravenous administration of F1024-1-3 at 0.3–3mg/kg sharply reduced pro-inflammatory responses in a dose-dependent manner and moderately attenuated pro-coagulant responses; at 1mg/kg, the protein protected rabbits from lethality even when administered 2h after the initial LPS injection. However, F1024-1-3 (10mg/kg) given 2h post-surgery did not prevent death of rabbits in a cecal ligation and puncture model. Thus, suppression of CD14-mediated activation of leukocytes and endothelial cells alone may not be clinically efficacious for the treatment of severe sepsis and septic shock.

Nonnucleoside Reverse Transcriptase Inhibitors Reduce HIV-1 Production from Latently Infected Resting CD4+ T Cells following Latency Reversal.

Therapeutic strategies that target the latent HIV-1 reservoir in resting CD4+ T cells of infected individuals are always administered in the presence of combination antiretroviral therapy. Using a primary cell of HIV-1 latency, we evaluated whether different antiviral drug classes affected latency reversal (as assessed by extracellular virus production) by anti-CD3/CD28 monoclonal antibodies or bryostatin 1. We found that the nonnucleoside reverse transcriptase inhibitors efavirenz and rilpivirine significantly decreased HIV-1 production, by ≥1 log.

Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes

The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry. FeCD4 bound to its cognate antigen post-fixation with paraformaldehyde (PFA) which is routinely used to inactivate highly pathogenic viruses. We have also used FeCD4 in conjunction with other immune cell markers to characterise ferret T cells in both primary and secondary lymphoid organs. In summary, we have developed an important reagent for the study of cellular immunological responses in the ferret model of infectious disease.

Abstract 158: Pre-existing Cancer Exacerbates Cerebral Ischemic Stroke in Mice via Regulatory T Cell Redistribution

Background and purpose: Accumulating epidemic evidence suggest that a considerable number of ischemic stroke patients had 1 or more cancer diagnosis before stroke attack. How the pre-existing cancer impact the cerebral ischemic injury is remarkably unknown. We tested the hypothesis that pre-existing cancer exacerbates cerebral ischemic stroke via regulatory T cell (Treg) redistribution. Methods: MC38 colon cancer cells or B16 melanoma cells were injected subcutaneously at the dose of 5*10 5 cells in 200μl PBS 3 weeks before distal middle cerebral artery occlusion (d-MCAO) surgery. Infarct volume was assessed at 3 days after surgery by staining the mice brain with 2,3,5-triphenyltetrazolium chloride. Sensorimotor assessments, such as body proprioception, climbing, forelimb walking, lateral turning, foot fault and adhesive removal were examined at 3, 5, 7, 14, 21 and 28 days after stroke. Neuro-inflammation was examined by measuring inflammatory cytokines with RT-PCR and immune cell infiltration using immunofluorescence and flow cytometry. Results: Pre-existing colon cancer and melanoma both exacerbated infarct volume growth in d-MCAO mice at 3 days after surgery. Mice with colon cancer exhibited prominently deterioration in their performance in sensorimotor functions after stroke compared with mice without cancer. Pre-existing colon cancer augmented peripheral immune cell infiltration into the ischemic brain but hindered Tregs’ recruitment into the brain. Cerebral ischemic stroke induced reduction in the number of Tregs in the peripheral blood were significantly aggravated in mice with pre-existing colon cancer. Depletion of Tregs with CD25 monoclonal antibody increased infarct volume in stroke mice but did not further exacerbate infarct growth in colon cancer stroke mice. Conclusion: Pre-existing cancer exacerbates ischemic brain injury and neuro-inflammation after stroke. Tregs redistribution plays an indispensable role in the cancer related deterioration of ischemic brain injury and may represent a promising target for treating stroke patients with pre-existing cancer.

Depletion of CD52-positive cells inhibits the development of central nervous system autoimmune disease, but deletes an immune-tolerance promoting CD8 T-cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis.

SummaryThe objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction by way of intravenous antigen administration such that it could be used to either improve efficiency of multiple sclerosis (MS) inhibition or inhibit secondary autoimmunities that may occur following alemtuzumab use in MS. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses were assessed clinically. A dose–response of CD4 monoclonal antibody depletion indicated that the 60–70% functional CD4 T‐cell depletion achieved in perceived failed trials in MS was perhaps too low to even stop disease in animals. However, more marked (~75–90%) physical depletion of CD4 T cells by CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly, in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness through a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T‐cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rationale for the rapid B‐cell hyper‐repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T‐cell regulatory mechanisms that promote immune tolerance may account for the secondary B‐cell autoimmunities, which occur following alemtuzumab treatment of MS.

Synergistic effect of cytokine-induced killer cell with valproate inhibits growth of hepatocellular carcinoma cell in a mouse model

ABSTRACTObjective: Long-term prognosis of hepatocellular carcinoma (HCC) remains poor owing to the lack of treatment options for advanced HCC. Cytokine-induced killer (CIK) cells are ex vivo expanded T lymphocytes expressing both NK– and T-cell markers. CIK cell therapy alone is insufficient for treating advanced HCC. Thus, this study aimed to determine whether treatment with CIK cells combined with valproic acid (VPA) could provide a synergistic effect to inhibit tumor growth in a mouse model of HCC. Methods: Upregulation of natural killer group 2D (NKG2D) ligands (retinoic acid early inducible 1 [RAE-1], mouse; major histocompatibility complex class I polypeptide-related sequence A [MIC-A], human) were evaluated by FACS. VPA concentrations that did not reduce tumor volume were calculated to avoid VPA cytotoxicity in a C3H mouse model of HCC. CIK cells were generated from mouse splenocytes using interferon gamma, a CD3 monoclonal antibody, and interleukin 2. The potential synergistic effect of CIK cells combined with VPA was evaluated in the mouse model and tissue pathology was investigated. Results: After 40 h of incubation with VPA, RAE-1 and MIC-A expression were increased in 4 HCC cell lines compared with that in control (2.3-fold in MH-134, 2.4-fold in Huh-7, 3.7-fold in SNU-761, and 6.5-fold in SNU-475). The maximal in vivo VPA dosage that showed no significant cytotoxicity compared with control was 10 mg/kg/day. CIK cells were well generated from C3H mouse splenocytes. After 7 d of treatment with CIK cells plus VPA, a synergistic effect was observed on relative tumor volume in the mouse model of HCC. While the relative tumor volume in untreated control mice increased to 11.25, that in the combination treatment group increased to only 5.20 (P = 0.047). Conclusions: The VPA-induced increase in NKG2D ligands expression significantly enhanced the effects of CIK cell therapy in a mouse model of HCC.

Identification and characterization of two CD4 alleles in Microminipigs.

BackgroundWe previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies.ResultsWe identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation.ConclusionsWe identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0856-8) contains supplementary material, which is available to authorized users.

Digital Microscopy Versus Conventional Microscopy In Assessment Of Jejune Intraepithelial Lymphocytes

Introduction/ Background Leukocytes infiltrates assessment in the lining epithelium of the digestive tract is a common request in current pathology practice both in terms of diagnosis and quantification of the therapeutic response. Frequently analyzed are lymphocytes positive for CD4 and CD8 in the context of gluten sensitive enteropathy. Microscopic quantification remains a time consuming and a laborious work. The possibility of human error puts at question the accuracy of data provided. Implementation of digital image acquisition techniques brings a relative comfort in delimitation the areas of interest, quantifying those areas and introduces digital algorithms that can determine the number of positive signals on the area of interest. Aims Establishing quantitative differences between the data acquired using optical microscopy versus digital evaluation of intraepithelial leukocytes (CD3+, CD4+, CD8+). Methods We analyzed paraffin embedded jejune segments fixed in buffered formalin (10%), taken from healthy patients (n= 15), monitoring the positive lymphocyte count and CD4 + CD8 + ratio. From each block, 4 sections of 3 µm thickness were taken. One section was stained with hematoxylin and eosin, and the other 3 sections were stained immunohistochemically using three steps-indirect streptavidin method for anti CD3 monoclonal antibodies, anti CD4 and CD8. The sections were referred to five independent pathologists in order to quantify positive leukocyte number in 100 enterocytes using optical microscopy and subsequent transmission microscopy images projected on high-definition monitors. Readings were performed on optical microscopes Leica DM750 objective 10X 40X eyepieces FN20. Image acquisition was made with 5-megapixel Leica microscope camera, ICC50 model, in direct transmission (stream) USB 2.0, monitors Dell 27 “HDMI IPS. For normalization and comparison of data, reporting was done in an area of 1 mm2. The variation between observers, for the two methods, were calculated (k). Results Interobserver variability for CD3 in optical was estimated as slight agreement (k0.36) in traditional microscopy compared to substantial agreement for digital assessment (0.68). For CD4 and CD8 markers the differences were less significant, substantial agreement in both categories (CD4 0.72 for optical evaluation vs 0.78 in digital evaluation data and for CD8 0.65 vs 0.72 respectively). In our opinion the use of digital image acquisition techniques in quantification of intraepithelial leukocytes increases concordance degree among observers through diminishing the subjective factors and by choosing identical ROIs.

Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken

Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100μg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4μg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4μg of antigen.

OR2 Variable expression of HLA-C impacts T versus B cell crossmatch outcomes

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibodies (DSA) and determine eligibility for transplantation and desensitization regimens. We measured HLA class I and HLA-C surface expression on T and B cells from various sources and examined the impact of HLA-C expression on FCXM reactivity in the presence of HLA-C DSA. HLA class I and HLA-C expression was examined on cells isolated from 18 donors using monoclonal antibodies (clone G46.2 and mAb DT9). T and B cells were differentiated using CD3 and CD19 specific monoclonal antibodies. Cells were acquired on a BD FACSCanto II; HLA expression was determined using median channel fluorescence (MCF) values and FCXM reactivity was assessed as a ratio of test serum to negative control serum. The mean HLA-C expression was significantly higher on T (11693 MCF) than B cells (5480 MCF, p < 0.05, Figure 1) while class I expression was lower on T (10167 MCF) than B cells (23602 MCF, p < 0.001) when comparing cells isolated from the same donors. This difference in HLA-C expression was mirrored in FCXM tests using sera containing HLA-C specific DSA. Mean reactivity with T cells as targets yielded consistently higher ratios than tests using B cells as targets (p < 0.001). Seven of 10 FCXM tests yielded positive T and negative B results. HLA-C expression is known to vary among HLA-C alleles (Apps et al., Science 2013); however, this study shows variability also exists between lymphocyte lineages. HLA-C is expressed higher on T than B cells and this difference impacts crossmatch reactivity. These findings should be considered when interpreting FCXM results.Download : Download high-res image (108KB)Download : Download full-size image

Abstract 1485: Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment

Abstract CD40-activating immunotherapy has potent anti-tumor effects due to its ability to activate dendritic cells and induce cytotoxic T-cell responses. However, its efficacy is limited by accumulation of immunosuppressive cells in the tumor and by endothelial anergy induced by pro-angiogenic growth factors. Here, we show that combining agonistic CD40 monoclonal antibody (mAb) therapy with vascular targeting using the tyrosine kinase inhibitor sunitinib significantly decrease tumor growth and improve survival in B16.F10 melanoma and T241 fibrosarcoma. Treatment of tumor-bearing mice with anti-CD40 mAb lead to increased activation of CD11c+ dendritic cells in the tumor draining lymph node, as evidenced by enhanced CD86 expression, while sunitinib treatment reduced vessel density. CD11b+Gr1+ myeloid derived suppressor cells accumulated in the tumor draining lymph nodes after anti-CD40 mAb treatment. This effect was reversed by co-treatment with sunitinib. Tumor endothelial expression of ICAM1 and VCAM1 adhesion molecules were increased only when anti-CD40 mAb treatment was combined with sunitinib. This was associated with enhanced tumoral infiltration of CD8+ T-lymphocytes. Our results show that combining CD40-stimulating immunotherapy with sunitinib treatment exert potent complementary antitumor effects mediated by dendritic cell activation, reduction in myeloid derived suppressor cells and increased endothelial activation, resulting in enhanced recruitment of cytotoxic T cells. Citation Format: Luuk van Hooren, Maria Georganaki, Hua Huang, Sara Mangsbo, Anna Dimberg. Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1485.

Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment.

CD40-activating immunotherapy has potent antitumor effects due to its ability to activate dendritic cells and induce cytotoxic T-cell responses. However, its efficacy is limited by immunosuppressive cells in the tumor and by endothelial anergy inhibiting recruitment of T-cells. Here, we show that combining agonistic CD40 monoclonal antibody (mAb) therapy with vascular targeting using the tyrosine kinase inhibitor sunitinib decreased tumor growth and improved survival in B16.F10 melanoma and T241 fibrosarcoma. Treatment of tumor-bearing mice with anti-CD40 mAb led to increased activation of CD11c+ dendritic cells in the tumor draining lymph node, while sunitinib treatment reduced vessel density and decreased accumulation of CD11b+Gr1+ myeloid derived suppressor cells. The expression of ICAM-1 and VCAM-1 adhesion molecules was up-regulated on tumor endothelial cells only when anti-CD40 mAb treatment was combined with sunitinib. This was associated with enhanced intratumoral infiltration of CD8+ cytotoxic T-cells. Our results show that combining CD40-stimulating immunotherapy with sunitinib treatment exerts potent complementary antitumor effects mediated by dendritic cell activation, a reduction in myeloid derived suppressor cells and increased endothelial activation, resulting in enhanced recruitment of cytotoxic T-cells.

Anti–CD3 Antibody Treatment: a Promising Immunotherapeutic for Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by spontaneous pancreatic beta cells destruction. The central cause of T1D is mainly the weakening of central and peripheral tolerance. So, the induction of tolerance is a major goal for most of the immunotherapies. Investigations over the last couple of decades have shown that anti–CD3 monoclonal antibodies (mAbs) and their therapeutic variants effectively treat autoimmune disease in animal models, and have also shown promise in clinical trials. Tolerance induction by anti–CD3 mAbs is mainly related to the induction of Th2 type immune response and regulatory T cells (Tregs) development that control pathogenic autoimmune responses. Here, we review main findings of last couple of decades research, and current scenario of anti–CD3 mediated immune tolerance, and preclinical and clinical studies in which anti–CD3 mAbs have been used.

Platelet microparticles inhibit IL-17 production by regulatory T cells through P-selectin

AbstractSelf-tolerance and immune homeostasis are orchestrated by FOXP3+ regulatory T cells (Tregs). Recent data have revealed that upon stimulation, Tregs may exhibit plasticity toward a proinflammatory phenotype, producing interleukin 17 (IL-17) and/or interferon γ (IFN-γ). Such deregulation of Tregs may contribute to the perpetuation of inflammatory processes, including graft-versus-host disease. Thus, it is important to identify immunomodulatory factors influencing Treg stability. Platelet-derived microparticles (PMPs) are involved in hemostasis and vascular health and have recently been shown to be intimately involved in (pathogenic) immune responses. Therefore, we investigated whether PMPs have the ability to affect Treg plasticity. PMPs were cocultured with healthy donor peripheral blood–derived Tregs that were stimulated with anti-CD3/CD28 monoclonal antibodies in the presence of IL-2, IL-15, and IL-1β. PMPs prevented the differentiation of peripheral blood–derived Tregs into IL-17– and IFN-γ–producing cells, even in the presence of the IL-17–driving proinflammatory cytokine IL-1β. The mechanism of action by which PMPs prevent Treg plasticity consisted of rapid and selective P-selectin–dependent binding of PMPs to a CCR6+HLA-DR+ memory-like Treg subset and their ability to inhibit Treg proliferation, in part through CXCR3 engagement. The findings that ∼8% of Tregs in the circulation of healthy individuals are CD41+P-selectin+ and that distinct binding of patient plasma PMPs to Tregs was observed support in vivo relevance. These findings open the exciting possibility that PMPs actively regulate the immune response at sites of (vascular) inflammation, where they are known to accumulate and interact with leukocytes, consolidating the (vascular) healing process.

Characterisation of CD4 T cells in healthy and diseased koalas (Phascolarctos cinereus) using cell-type-specific monoclonal antibodies

The koala (Phascolarctos cinereus) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4+ lymphocytes collected from koala spleens (41.1%, range 20–45.1%) lymph nodes (36.3%, range 19–55.9%) and peripheral blood (23.8%, range 17.3–35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4+ cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4+ T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4+ T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system.

Changes in the expression of three markers in T lymphocytes of peripheral blood and immunoregulatory mechanisms of burned mice with sepsis at early stage

Changes in the expression of three markers in T lymphocytes of peripheral blood and immunoregulatory mechanisms of burned mice with sepsis at early stage

Molecular mechanism of AFP-specific liver tumor vaccines killing HepG2 hepatocellular carcinoma(HCC) cells in vitro

Objective To prepare AFP-specific CD8+ T lymphocyte liver tumor vaccine, and investigate its anti-tumor activity in vitro and the related molecular mechanism. Methods We used AFP-specific dendritic cells (DC) which were activated by either AFP peptide (AFP542-550)-pulsed or lenti-AFP-engineered to activate AFP-specific CD8+ T lymphocyte in vitro, and investigated the anti-tumor activity of AFP-specific CD8+ T lymphocytes (AFP-CD8+ -CTL). The anti-human CD134 or anti-human CD28 monoclonal antibody was used to block or activate AFP-CD8+ -CTL and FasL signaling, and cytokines released such as IL-2, IFN-γ, perforin, granzyme B and FasL were detected to determine the direct role of perforin/granzyme B pathway in this anti-tumor effect. Results The results demonstrated that anti-human CD134 monoclonal antibody could block the activity of AFP-CD8+ -CTL, which could not efficiently kill HepG2 cells by decreasing the level of IL-2, IFN-γ, perforin and granzyme B significantly (P<0.05). When AFP-CD8+ -CTL was cocultured with anti-human CD28 monoclonal antibody, the AFP-specific T cells could efficiently kill HepG2 cells by significantly increasing the level of IL-2, IFN-γ, perforin and granzyme B (P<0.05). No obvious change was observed on FasL level. Conclusions The anti-human CD134 and anti-human CD28 monoclonal antibody could inhibit and activate AFP-CD8+ -CTL. The perforin/granzyme B pathway might play an important role in the anti-tumor activity of liver tumor vaccines. Key words: Hepatocellular carcinoma; Alpha-fetoprotein; Tumor vaccine; Molecular Mechanism

A study of Foxp3+ regulatory T-cell in lesional skin of mycosis fungoides in comparison with some histological mimickers

Introduction Regulatory T-cells (Tregs) are essential to balance between proinflammatory and anti-inflammatory responses by protecting against autoimmune reactions. They have different roles in skin tumors, including cutaneous T-cell lymphomas. Foxp3 (forkhead box P3) is thought to be a reliable marker for Tregs. Contradictory results were reported in the literature when the density of Tregs in the skin of mycosis fungoides (MF, a type of cutaneous T-cell lymphoma) was compared with some pathologically mimicking inflammatory dermatoses. Patients and methods This is a comparative cross-sectional study of lesional skin biopsies from 36 cases of MF versus those from 36 cases of histopathological mimickers, including 10 cases of psoriasis, 10 cases of spongiotic dermatitis, and 16 cases of lichenoid dermatoses (10 with pityriasis lichenoides chronica and six with discoid lupus erythematosus). We compared the densities of Foxp3+ Tregs in relation to CD4+ cells in the epidermis and dermis of these groups using Foxp3 and CD4 monoclonal antibodies. Results The MF group showed a lower statistically significant difference as regards epidermal and dermal Foxp3/CD4+ ratios compared with the histologic mimicker group. Conclusion MF lesions showed a significantly lower density of CD4+ Foxp3+ Tregs compared with its histological mimickers, which might be attributed to their limited proliferation at the expense of malignant T-lymphocytes.

Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits?

Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits?

Presence of PD-1 Expressing T Cells Predicts for Inferior Overall Survival in Newly Diagnosed Multiple Myeloma

Background: Programmed cell death 1 (PD-1) protein downregulates T cell activation and is related to immune tolerance. PDL1 up regulation, T cell infiltration, and T cell exhaustion are features, which suggest susceptibility to PD-1 blockade antibodies. Blockade of PD-1 or its ligand PD-L1 has shown promising responses in several malignancies. Although little clinical activity has been seen in patients with relapsed multiple myeloma (MM), the role of the PD-1 pathway and T cell exhaustion in newly diagnosed MM has not been explored.Objective: To determine whether T-cell infiltrate or expression of PD-1 correlates with clinical features and prognosis among patients with newly diagnosed multiple myeloma.Methods: We screened a clinically annotated database of 341 patients seen at MSKCC between 1998 and 2012 that had Multiple Myeloma, received a bone marrow transplant and were consented to a biospecimen research protocol for availability of pre-treatment bone marrow specimens. A total of 64 bone marrow biopsy specimens were identified. Immunohistochemistry (IHC) was performed in formalin-fixed paraffin-embedded specimens using an anti human CD3 monoclonal antibody (mAb) (Dako, Clone F7.2.38) and an anti human PD-1 mAb (Cell Marque, catalog #315M-95). CD3 and PD1 IHC staining were graded as negative (<5% for CD3, < 1% for PD1), or positive (≥5% for CD3, ≥1% for PD1). Correlative analyses were performed between CD3/PD1 expression and clinical outcome using the following parameters: International Staging System (ISS), cytogenetic risk, progression free survival (PFS), overall survival (OS), and response to treatment. Groups were compared by Fisher's exact test. OS and PFS were assessed by Cox regression and estimated by Kaplan-Meier methods.Results: 23 specimens (36%) were CD3 positive and 10 specimens (16%) were PD-1 positive. All PD-1 positive specimens were CD3 positive. 41 specimens (64%) were CD3 negative (<5%) and PD1 negative (<1%). Based on these results, specimens were divided into three groups: Exhausted T-cell infiltrate (CD3+/PD1+), non exhausted T-cell infiltrate (CD3+/PD1-) and no T-Cell infiltrate (CD3-/PD1-). In the exhausted T-cell infiltrate group 30% of patients had ISS stage 3 and 40% had high risk cytogenetics. In the non-exhausted T-cell group 15% of patients had ISS stage 3 and 15% high cytogenetic risk. In the no T-cell infiltrate group 10% had ISS stage 3 disease and 22% high cytogenetic risk. These proportions were not significantly different across the 3 groups. Median OS from 1st auto infusion was 7 years while median PFS was 2.3 years. On univariate analysis, there was no significant difference in PFS between the 3 groups. The presence of CD3 and PD1 T-cells were significantly associated with OS (p-value = 0.04). Median OS from 1st auto infusion was 43 months for the exhausted T-Cell infiltrate group followed by 83 months for the no T-Cell infiltrate group. The non exhausted T-cell group had the highest OS, median not reached; OS by 7-years was 75%. Cytogenetic risk at diagnosis was significantly associated with OS (p-value = 0.03). In a multivariable model, CD3/PD1 staining continued to trend toward an association with OS (p-value = 0.08) and cytogenetic risk remained significant (p-value = 0.05).Conclusions: The presence of T-cells with PD-1 expression was not associated with higher risk disease at MM diagnosis based on cytogenetics and ISS stage. The presence of PD-1 expressing CD3+ T cells trends toward an association with poorer overall survival in newly diagnosed MM, especially compared to non exhausted T-cell infiltrate, suggesting the possibility that T cell exhaustion represents a novel high risk disease characteristic. Further investigation is necessary to assess if the presence of CD3+PD-1+ T cells is an independent prognostic feature in newly diagnosed MM. [Display omitted] DisclosuresGiralt:JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding. Landgren:International Myeloma Foundation: Research Funding; Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medscape: Honoraria; Bristol-Myers Squibb: Honoraria; Medscape: Consultancy; BMJ Publishing: Consultancy; Celgene: Honoraria; Onyx: Honoraria; BMJ Publishing: Honoraria; Onyx: Research Funding. Hassoun:Novartis: Consultancy; Takeda: Research Funding; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Lesokhin:Genentech: Research Funding; Aduro: Consultancy; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Efranat: Consultancy.