Autologous Mixed Lymphocyte Reaction Research Articles

Abstract 2729: Discovery and characterization of LILRB2XPD-L1 bispecific antibody SPX-303

Abstract The advent of immunotherapy has led to deep and sometimes durable remissions in certain cancers. However, the variable response rates and development of resistance mechanisms pose significant challenges to the current therapies. The immunosuppressive nature in the tumor microenvironment (TME) can drive many of these observations. Reversing this immunosuppression is a promising avenue for enhancing and broadening the applicability of IO therapies. Macrophages are critically modulated by LILRB2, a pivotal receptor in their maturation and polarization to the M2 immunosupressive phenotype. We report the discovery and development of a novel anti-LILRB2/PD-L1 bispecific antibody, SPX-303, designed using an anti-LILRB2 IgG4 derived from a mouse hybridoma screen and linked with an anti-PD-L1 epitope. SPX-303 demonstrates sub-nanomolar binding affinity to both LILRB2 and PD-L1 targets, effectively blocking the interaction between LILRB2 and its ligand, as well as inhibiting PD-1 binding to PD-L1. In in vitro functional assays, SPX-303 exhibits effects comparable to the parental monomeric anti-LILRB2 (SPX-104) in redirecting macrophage polarization from M2 to M1 stage, as observed in both cultured PBMC and human monocyte-derived macrophage (HMDM) models. Furthermore, SPX-303 demonstrates a synergistic effect in inducing T cell activation in the autologous mixed lymphocyte reaction assay (auto-MLR), surpassing the effects of the anti-LILRB2 epitope (SPX-104) and the anti-PDL1 fragment (single-domain anti-PDL1) alone or in combination. In vivo experiments reveal that SPX-303 significantly inhibits tumor growth. Collectively, these results support SPX-303 as a novel therapeutic antibody that effectively enhances anti-cancer immunity in the treatment of advanced solid tumors. The pre-clinical data as well as the clinical plan of SPX-303 will be presented. Citation Format: Anthony Haight, Qian Chen, Brandon William, Dinh-Duc Nguyen, Robert Cai, Jichun Ma, Michael White, Jindong Jin, Jadon Shen, Martin Siekierzycki, Victoria Hall, Hua Jin, Roland Meier, Justin Moser. Discovery and characterization of LILRB2XPD-L1 bispecific antibody SPX-303 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2729.

Exploring the immunomodulatory properties of glucan particles in human primary cells

The immunomodulatory properties of β-glucans have sparked interest among various medical fields. As vaccine adjuvants, glucan particles offer additional advantages as antigen delivery systems. This study reported the immunomodulatory properties of glucan particles with different size and chemical composition. The effect of glucan microparticles (GPs) and glucan nanoparticles (Glu 130 and 355 NPs) was evaluated on human immune cells. While GPs and Glu 355 NPs demonstrated substantial interaction with Dectin-1 receptor on monocytes, Glu 130 NPs exhibited reduced activation of this receptor. This observation was substantiated by blocking Dectin-1, resulting in inhibition of reactive oxygen species production induced by GPs and Glu 355 NPs. Notably, monocyte-derived dendritic cells (moDCs) stimulated by Glu 355 NPs exhibited phenotypic and functional maturation, essential for antigen cross-presentation. The immunomodulatory efficacy was investigated using an autologous mixed lymphocyte reaction (AMLR), resulting in considerable rates of lymphocyte proliferation and an intriguing profile of cytokine and chemokine release. Our findings highlight the importance of meticulously characterizing the size and chemical composition of β-glucan particles to draw accurate conclusions regarding their immunomodulatory activity. This in vitro model mimics the human cellular immune response, and the results obtained endorse the use of β-glucan-based delivery systems as future vaccine adjuvants.

Abstract 5663: Discovery and characterization of anti-LILRB2 antibody SPX-104

Abstract T-cell based immunotherapies have produced durable remission in certain cancer patients, but owing at least partially to immunosuppression in the tumor microenvironment, response rates with current therapies remains challenging. Reversing the immunosuppression is a recognized pathway for enhancing the activity of existing IO therapies. LILRB2 (ILT4) is a key receptor for macrophage maturation and polarization and an emerging therapeutic target in immuno-oncology. This research aims to produce anti-LILRB2 antibodies to redirect the polarization of myeloid cells, specifically macrophages, in the tumor microenvironment. The LILRB2 antibodies were generated through the mouse hybridoma method. After humanization and maturation, potent and selective IgG4 anti-LILRB2 antibodies were obtained. Binding affinities were evaluated through ELISA and 293T-LILRB2 cell flow cytometry binding assays which demonstrated sub-nanomolar binding affinity as well as effective blocking of the interaction of LILRB2 with its ligands HLA-G and HLA-A2 for multiple antibodies. SPX-104 was shown to redirect macrophage polarization in primary cultured human PBMC and human monocyte derived macrophage (HMDM) models. SPX-104 treatment induced hTNFa expression and suppressed hIL10 expression in the LPS stimulated PBMC model. In the HMDM model, SPX-104 effectively induced pro-inflammatory cytokine hTNFα expression in M-CSF. Furthermore, an autologous mixed lymphocyte reaction (auto-MLR) assay to evaluate T cell response showed dose-dependent increases in hIFNγ and hGM-CSF expression following SPX-104 treatment. In a CD34-humanized hLILRB2 transgenic mouse efficacy model, SPX-104 in combination with an immune checkpoint inhibitor, effectively inhibited tumor growth. These results confirm enhanced T cell activity following macrophage redirected polarization. The in vitro and in vivo evaluation, of SPX-104 relative to other anti-LILRB2 antibodies will be presented. Citation Format: Anthony Haight, Qian Chen, Ernesto Rodriguez, Martin Siekierzycki, Victoria Hall, Brandon Williams, Jun Shi, Dinh-Duc Nguyen, Robert Cai, Jichun Ma, Michael White, Jingdong Qin, Jadon Shen, Chuanke Zhao, Zhuona Rong, Lin Meng, Chengchao Shou. Discovery and characterization of anti-LILRB2 antibody SPX-104. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5663.

Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity.

There remains an unmet need for reliable fully synthetic adjuvants that increase lasting protective immune responses from vaccines. We previously reported a high-throughput screening for small molecules that extended nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation after a Toll-like receptor 4 (TLR4) ligand, lipopolysaccharide (LPS), stimulation using a human myeloid reporter cell line. We identified compounds with a conserved aminothiazole scaffold including 2D216 [N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide], which increased murine antigen-specific antibody responses when used as a co-adjuvant with LPS. Here, we examined the mechanism of action in human cells. Although 2D216 activated the major mitogen-activated protein kinases, it did not interact with common kinases and phosphatases and did not stimulate many of the pattern recognition receptors (PRRs). Instead, the mechanism of action was linked to intracellular Ca2+ elevation via Ca2+ channel(s) at the plasma membrane and nuclear translocation of the nuclear factor of activated T-cells (NFAT) as supported by RNA-seq data, analysis by reporter cells, Ca2+ flux assays, and immunoblots. Interestingly, 2D216 had minimal, if any, activity on Jurkat T cells but induced cytokine production and surface expression of costimulatory molecules on cells with antigen-presenting functions. A small series of analogs of 2D216 were tested for the ability to enhance a TLR4 ligand-stimulated autologous mixed lymphocyte reaction (MLR). In the MLR, 2E151, N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide, was more potent than 2D216. These results indicate that a small molecule that is not a direct PRR agonist can act as a co-adjuvant to an approved adjuvant to enhance human immune responses via a complementary mechanism of action.

Autoreactivity of Peripheral Helper T Cells in the Joints of Rheumatoid Arthritis.

Autoreactive CD4 T cells are thought to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). Recently, a subset of CD4 T cells that express high levels of programmed death-1 (PD-1) but are distinct from follicular helper T cells have been identified in the joints of RA patients and named peripheral helper T (Tph) cells. Because PD-1 is expressed on T cells chronically stimulated with the Ags, we tested a hypothesis that Tph cells are the pathogenic autoreactive CD4 T cells in RA. We found that human Tph cells in RA joints produce proinflammatory effector cytokines, including IFN-γ, TNF-α, and GM-CSF, in addition to B cell-helping cytokines, such as IL-21 and CXCL13. Flow cytometric analysis showed different bias of TCR Vβ usage between PD-1high Tph cells and PD-1low/neg CD4 T cells, including Th1 cells, in the joint or memory CD4 T cells in the peripheral blood, whereas there was little difference between the latter two subsets. In line with this, deep sequencing of TCR demonstrated an overlap of expanded clones between peripheral blood memory CD4 T cells and PD-1low/neg CD4 T cells but not Tph cells in the joint. Interestingly, Tph cells preferentially exhibited autologous MLR in vitro, which required recognition of self-MHC class II and was pronounced by blocking PD-1 signaling. Taken together, these results suggest that Tph cells are the pathogenic autoreactive CD4 T cells in RA, which expand locally in the joints and are regulated by PD-1 signaling.

Indoleamine 2,3-Dioxygenase (IDO) Enzyme Links Innate Immunity and Altered T-Cell Differentiation in Non-ST Segment Elevation Acute Coronary Syndrome.

Atherosclerosis is a chronic inflammatory disease characterized by a complex interplay between innate and adaptive immunity. Dendritic cells (DCs) play a key role in T-cell activation and regulation by promoting a tolerogenic environment through the expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme involved in tryptophan catabolism. IDO expression and activity was analyzed in monocytes derived DCs (MDDCs) from non-ST segment elevation myocardial infarction (NSTEMI) patients, stable angina (SA) patients and healthy controls (HC) by real-time quantitative polymerase chain reaction (RT-qPCR) before and after in vitro maturation with lipopolysaccharide (LPS). The amount of tryptophan catabolite; kynurenine; was evaluated in the culture supernatants of mature-MDDCs by ELISA assay. Autologous mixed lymphocyte reaction (MLR) between mature-MDDCs and naïve T-cells was carried out to study the differentiation towards T-helper 1 (Th1) and induced regulatory T-cells (iTreg). Analysis of IDO mRNA transcripts in mature-MDDCs revealed a significant reduction in cells isolated from NSTEMI (625.0 ± 128.2; mean ± SEM) as compared with those from SA (958.5 ± 218.3; p = 0.041) and from HC (1183.6 ± 231.6; p = 0.034). Furthermore; the concentration of kynurenine was lower in NSTEMI patients (2.78 ± 0.2) and SA (2.98 ± 0.25) as compared with HC (5.1 ± 0.69 ng/mL; p = 0.002 and p = 0.016; respectively). When IDO-competent mature-MDDCs were co-cultured with allogeneic naïve T-cells, the ratio between the percentage of generated Th1 and iTreg was higher in NSTEMI (4.4 ± 2.9) than in SA (1.8 ± 0.6; p = 0.056) and HC (0.9 ± 0.3; p = 0.008). In NSTEMI, the tolerogenic mechanism of the immune response related to IDO production by activated MDDCs is altered, supporting their role in T-cell dysregulation.

Antigen-presenting human B cells are expanded in inflammatory conditions.

Traditionally, B cells have been best known for their role as producers of antibodies. However, in recent years, a growing body of evidence has accumulated showing that B cells fulfill a range of other immunologic functions. One of the functions that has attracted increasing attention is the capacity of B cells to induce antigen-specific activation of T cells through presentation of antigens. However, the analysis of this B cell function has been hampered by the lack of a phenotypically well-defined antigen-presenting B cell subset. Here, we report the identification of a human antigen-presenting B cell subset with strong immunostimulatory properties. This B cell subset is characterized by low expression of CD21 and high expression of the activation marker CD86 and exhibits strong T cell-stimulatory activity, as demonstrated by means of an autologous mixed-lymphocyte reaction. Phenotypically, CD21lowCD86pos immunostimulatory B cells (BAPC) represented CD27+ class-switched IgMnegIgDneg B lymphocytes and displayed a higher expression of cell surface receptors, which mediate the migration from peripheral blood to sites of inflammation. Flow cytometric analysis of peripheral blood obtained from individuals with inflammatory conditions revealed that the BAPC subset was expanded following vaccination and in patients with rheumatoid arthritis. Taken together, our work shows that BAPC represents a strongly immunostimulatory B cell subset, which could be a promising target for immunotherapeutic intervention in inflammatory diseases.

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Basophils inhibit proliferation of CD4+T cells in autologous and allogeneic mixed lymphocyte reactions and limit disease activity in a murine model of graft versus host disease

Basophils are known to modulate the phenotype of CD4(+) T cells and to enhance T helper type 2 responses in vitro and in vivo. In this study, we demonstrate that murine basophils inhibit proliferation of CD4(+) T cells in autologous and allogeneic mixed lymphocyte reactions. The inhibition is independent of Fas and MHC class II, but dependent on activation of basophils with subsequent release of interleukin-4 (IL-4) and IL-6. The inhibitory effect of basophils on T-cell proliferation can be blocked with antibodies against IL-4 and IL-6 and is absent in IL-4/IL-6 double-deficient mice. In addition, we show that basophils and IL-4 have beneficial effects on disease activity in a murine model of acute graft-versus-host disease (GvHD). When basophils were depleted with the antibody MAR-1 before induction of GvHD, weight loss, GvHD score, mortality and plasma tumour necrosis factor levels were increased while injection of IL-4 improved GvHD. Basophil-depleted mice with GvHD also have increased numbers of CD4(+) T cells in the mesenteric lymph nodes. Our data show for the first time that basophils suppress autologous and allogeneic CD4(+) T-cell proliferation in an IL-4-dependent manner.

Treg cell function in rheumatoid arthritis is compromised by ctla-4 promoter methylation resulting in a failure to activate the indoleamine 2,3-dioxygenase pathway.

Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function. CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction. Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway. We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.

Management of Clozapine-Induced Fever in a Child

“Marie,” an 11-year-old African American girl of Caribbean descent with no previous psychiatric history, premorbid developmental impairments, or significant family psychiatric history, was admitted at the National Institute of Mental Health (NIMH) for participation in a longitudinal study of individuals with childhood-onset schizophrenia. She was reported to be a normally developing straight-A student who, not long before the onset of symptoms, had won a spelling bee. At age 10 years 3 months, after a brief trip to her father’s country of origin to attend a family funeral, she began to decompensate both emotionally and behaviorally. She had traveled to the region multiple times before, had not received any prophylactic medications or vaccines before the trip, and had no known sick contacts or exposures. Over the next 13 months, Marie progressively deteriorated psychiatrically. Through a detailed review of the records, discussions with her outpatient providers, and multiple interviews with both parents, it was found that the patient’s initial presentation was thought to be attributable to a major depressive episode with psychotic features, particularly given the context of the death of a close relative. Marie exhibited diminished emotional expression, avolition, alogia, and asociality. She subsequently received multiple antidepressant treatment trials, which were ineffective as she continued to deteriorate. Soon after her initial symptoms arose, she began to discuss paranoia and delusions and respond to internal stimuli, and she became increasingly disorganized. Antipsychotics were added to her regimen, without appreciable benefit. Although Marie’s symptoms did not fit clearly into a diagnosis of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) (1), her providers, struggling with the severity and character of her symptoms, administered several trials of antibiotics, again with no effect. Over the following several months, she had numerous inpatient psychiatric admissions for physical aggression and psychosis, the longest of which lasted 3 months. During her admissions and the periods that followed, combinations of antidepressants, maximally dosed, and antipsychotics, minimally dosed, were administered with no effect. MRI and lumbar puncture were performed. The MRI was unremarkable. Results of the lumbar puncture suggested the possibility of an elevated intracranial pressure; the records were unclear, and on direct communication the providers could recall no further specifics. A repeat lumbar puncture was performed, and the results were normal. Nevertheless, apparently because of the patient’s lack of response to other treatments, she had received treatment with acetazolamide, which made no difference to her symptoms. Lumbar puncture was repeated at NIMH several months after the discontinuation of the acetazolamide and while the patient was symptomatic, and the results were again within normal limits. Subsequently, Marie’s providers discontinued antipsychotic and antidepressant prescriptions, instead providing as-need doses of clonazepam and directing her to emergency departments for agitation whenever her parents could not manage, and they referred her to NIMH for participation in the childhood-onset schizophrenia study. Since 1991, patients meeting DSM-IV criteria for schizophrenia with an onset of psychosis before age 13 have been recruited nationally for participation in a longitudinal study of individuals with childhood-onset schizophrenia conducted at NIMH. The protocol includes screening, evaluation, diagnosis, treatment optimization, and followup phases. Patients and first-degree relatives are interviewed over a period of 3 days for current and lifetime psychiatric disorders. Extended inpatient evaluation lasts up to 4 months and includes initial observation, medication taper, medication-free observation, and stabilization/ treatmentoptimizationphases. Beforeadefinitivediagnosis is made, medical causes for a patient’s presentation are aggressively explored, often with the aid of consulting specialties, and without limitation. Once diagnosed, the patient enters the treatment optimization phase and is given appropriate clinical treatment until stabilized. Longitudinal outpatient follow-ups are offered every 2 years for subsequent evaluation, and more frequently if indicated (2). Marie was screened by the childhood-onset schizophrenia team at NIMH. The patient and her parents were interviewed individually and as a group over a 3-day period using both clinical and structured interviews. The

The Immunosupressive Effect Of Wharton Jerry -Derived Mesenchymal stem Cells In vitro

BackgroundHuman mesenchymal stem cells (MSCs) can be isolated from several tissues, including bone marrow, umbilical cord, placenta, and adipose tissue. In recent reports, MSCs have the ability to migrate to inflammatory tissues and suppress adverse immune reactions. In fact, BM-derived MSCs are already applied for the patients with acute graft versus host disease (aGVHD) with promising efficacy. To avoid the potential risk of BM harvest, we intended to use umbilical cord Wharton's jelly (WJ) as an alternative source for MSCs. However, the mechanism by which WJ-MSCs exert their immunosuppressive effects is not completely understood. MethodsUmbilical cord was collected after informed consent from the mother. WJ-MSCs were isolated by explants method and in part, continued to culture for further expansion by passage 3 (P3). Otherwise, the remaining WJ-MSCs were cryopreserved in P1. Mixed lymphocyte reaction (MLR) was performed on WJ-MSCs or control. CFSE-labeled human peripheral blood (PB) or cord blood(CB) derived mononuclear cells (MNCs) as the responder cells were mixed with irradiated (50Gy) human CB-derived dendrite cells as stimulator cells at a 10:1 ratio, in the presence of low-dose anti-CD3 antibody. CFSE-labeled responder cells were analyzed by FACS. ResultsWJ-MSCs corresponded to the minimal criteria of MSCs defined by the International Society for Cell Therapy. Thus WJ-MSCs showed spindle-shaped plastic adherent with positive CD105, CD73, CD90, and negative for CD45, CD34, CD14, CD19, and HLA -DR. They also have the ability of differentiation into osteoblasts, adipocytes, chondroblasts in vitro. In MLR, WJ-MSCs efficiently inhibited the responder T cells derived from the same donor of WJ-MSCs, triggered by autologous or allogeneic dendritic cells (DC)(Autologous and allogeneic MLR). Moreover, 3rd party-derived WJ-MSCs also strongly suppressed allogeneic responder T cells proliferation triggered by allogeneic DC(3rd party MLR) (n=3). And WJ-MSCs could inhibit the proliferation of T cells upon PHA-L stimulation. In order to study whether the inhibitory effects of WJ-MSCs on MLR requires the cell to cell contact or not, we next performed MLR on separated WJ-MSCs using by transwell. We observed the inhibitory effect of WJ-MSCs on MLR, although the immunosuppressive effect of the separated WJ-MSCs was much less than that of cell to cell contact. The supernatants of WJ-MSCs, which were derived from 80% confluent WJ-MSCs indicated the mild inhibitory effects on MLR. These inhibitory effects of the WJ-MSCs on MLR were reversed by the addition of indoleamine 2,3-dioxygenase (IDO) inhibitor, 1-methyltryptophan (1-MT) , in a dose dependent manner as shown in Figure 1 (n=3). We also studied the influence of the passage of WJ-MSCs on MLR. The inhibitory effect was comparable among the early passage (∼P3) and long-term passage (P10), although the long-term passage did not show the osteogenic differentiation. DiscussionWe demonstrated WJ-MSCs have the immunosuppressive effect on the stimulated T cells in autologous, allogeneic and 3rd party MLR setting, indicating that immunosuppressive effect of WJ-MSCs is not restricted by MHC. Soluble IDO may play an important role in immunosuppressive effect of MLR, although we need to detect the additional factors especially in cell to cell contact between WJ-MSCs and the responder T cells. Conclusively, WJ-MSCs may be a feasible alternative non-invasive source of BM-MSCs for GVHD treatment and WJ-MSCs banking can be considered for the prompt clinical applications.▪ Disclosures:No relevant conflicts of interest to declare

Conditional Deletion of PTEN in Peripheral T Cells Augments TCR-Mediated Activation but Does Not Abrogate CD28 Dependency or Prevent Anergy Induction

PTEN is thought to play a critical role in T cell activation by negatively regulating the PI3K signaling pathway important for cellular activation, growth, and proliferation. To directly eliminate PTEN in postthymic T cells for studies of functional effects, we used CAR transgenic × PTEN(flox/flox) mice, which enabled gene deletion using a Cre adenovirus in vitro. These mice were also immunized to generate stable Th1 clones that could have PTEN deleted when desired. PTEN-deleted T cells exhibited enhanced IL-2 production, proliferation, and Akt phosphorylation upon TCR/CD28 engagement, whereas T cell survival was not potentiated. Gene expression profiling revealed a small subset of induced genes that were augmented upon PTEN deletion. However, PTEN-deficient T cells still required CD28 costimulation for IL-2 production and remained susceptible to anti-CD3-induced anergy. The absence of PTEN within the CD8 T cell compartment led to markedly increased cytolytic activity following an allogeneic MLR in vitro, without increasing autologous MLR activity. Our results indicate that deletion of PTEN can augment the activation of postthymic T cells but does not mediate CD28 independence or anergy resistance. Nonetheless, PTEN inhibition may be a viable target for immune potentiation owing to increased cytokine production by activated CD4(+) cells and increased cytotoxicity by CD8(+) T cells.

Psoriasis patients exhibit impairment of the high potency CCR5+ T regulatory cell subset

CCR5 expression on CD4(+)CD25(high)Foxp3(+) regulatory T cells (Tregs) has been reported to be crucial for limiting Th1 inflammation associated with autoimmunity and bacterial infections. We inquired whether abnormalities in chemokine receptors expressed on Tregs might be involved in the psoriatic pathogenesis. Indeed, the proportion of CCR5(+) Treg was 58.8% in healthy individuals (n=9), whereas only half as many CCR5(+) Treg cells were found in psoriatic individuals (29.1%, n=8, p<0.01). The flow-enriched control CCR5(+) Tregs consistently exceeded the suppressive capacity of unsorted Tregs in autologous MLR assays (n=5, p<0.05) showing that CCR5(+) Treg subset is a high potency regulatory T cell population. Interestingly, psoriatic CCR5(+) Treg cells exhibited significantly less migratory capacity toward CCR5 ligands MIP-1β and RANTES in vitro compared to CCR5(+) Treg controls (n=3, p<0.05). Our data demonstrate that psoriatic CCR5(+) Tregs cells are numerically-, functionally- and chemotactically-deficient compared to controls and may pose a triple impairment on the ability of psoriatic Tregs to restrain inflammation.

Chemotherapy ± Cetuximab Modulates Peripheral Immune Responses in Metastatic Colorectal Cancer

Objective: To identify changes in peripheral immune responses in patients with metastatic colorectal cancer (mCRC) treated with irinotecan/5-fluorouracil/leucovorin (IFL) alone or in combination with cetuximab (C-IFL). Methods: Peripheral blood mononuclear cells (PBMCs) collected from healthy donors (n = 20) and patients with mCRC receiving treatment with either IFL (n = 30) or C-IFL (n = 30) were tested for cytokine production upon polyclonal stimulation with anti-CD3 monoclonal antibody, T cell proliferation in the autologous mixed lymphocyte reaction (auto-MLR) and T regulatory cell (Treg) frequency. The respective results were evaluated over two treatment cycles and further assessed in relation to response to treatment. Results: PBMCs prior to treatment exhibited significantly lower production of IL-2, IFN-γ, IL-12 and IL-18 cytokines and lower auto-MLR responses, whereas Treg frequency, IL-4, IL-10 cytokines were increased compared to healthy donors. During treatment, IL-2, IFN-γ, IL-12, IL-18 and auto-MLR responses increased, while Treg frequency and IL-10 secretion decreased significantly compared to the baseline. Responders to treatment exhibited a significantly higher increase in IL-2, IFN-γ, IL-12 and IL-18 production and auto-MLR responses, and higher decrease in IL-4, IL-10 secretion and Treg frequency. Among all patient subgroups analysed, responders to C-IFL demonstrated significantly higher increase in auto-MLR responses, IL-12 and IL-18 secretion and higher decrease in Treg frequency. Conclusion: The disturbed immune parameters observed in patients with mCRC at presentation can be significantly improved during treatment with IFL and this effect can be potentiated by the addition of cetuximab. Monitoring of the peripheral immune system function could be used as surrogate marker in predicting treatment-related outcome.

Increased Myeloid-Derived Suppressor Cells in Gastric Cancer Correlate with Cancer Stage and Plasma S100A8/A9 Proinflammatory Proteins

Immune dysfunction may contribute to tumor progression in gastric cancer (GC) patients. One mechanism of immune dysfunction is the suppression of T cell activation and impairment of the efficacy of cancer immunotherapy by myeloid-derived suppressor cells (MDSCs). We assessed the phenotype and immunosuppressive function of MDSCs in GC patients. We further investigated the role of S100A8/A9 in GC and the relationship between S100A8/A9 and MDSC function. Lastly, the effect of MDSCs on survival rates and its potential as a prognostic factor in GC patients were investigated. MDSCs from PBMCs of GC patients were identified by comparing the expression of specific surface markers with PBMCs from healthy individuals. The ability of MDSCs to suppress T lymphocyte response and the effect of S100A8/A9 and RAGE blocking were tested in vitro by (autologous) MLR. GC patients had significantly more MDSCs than healthy individuals. These MDSCs suppressed both T lymphocyte proliferation and IFN-γ production and had high arginase-I expression. Levels of S100A8/A9 in plasma were higher in GC patients compared with healthy individuals, and they correlated with MDSC levels in the blood. Blocking of S100A8/A9 itself and the S100A8/A9 receptor RAGE on MDSCs from GC patients abrogated T cell effector function. We found that high levels of MDSCs correlated with more advanced cancer stage and with reduced survival (p = 0.006). S100A8/A9 has been identified as a potential target to modulate antitumor immunity by reversing MDSC-mediated immunosuppression.

In vitro suppression of immune responses using monocyte-derived tolerogenic dendritic cells from patients with primary Sjögren's syndrome.

IntroductionTherapeutic vaccination with antigen-specific tolerogenic dendritic cells (tolDC) might become a future option of individualized therapy for patients with autoimmune diseases. In this study, we tested the possibility of generating monocyte-derived tolDC from patients with primary Sjögren's syndrome (pSS). We analyzed phenotype, cytokine production and ability to suppress Ro/La-specific immune responses.MethodsMonocyte-derived tolDC from patients with pSS were generated in the presence of dexamethasone, vitamin D3 and lipopolysaccharide (DexVD3 DC). The phenotype was analyzed by flow cytometry and the cytokine profile was investigated using a 25-plex Luminex assay and ELISA. The capacity to both stimulate Ro/La-specific T cells and suppress this response was evaluated by autologous mixed lymphocyte reaction (MLR).ResultsDC generated from patients with pSS had a similar phenotype and cytokine profile to those from healthy controls. DexVD3 DC from pSS patients induced little antigen-specific T cell proliferation, but DexVD3 DC-primed lymphocytes successfully suppressed Ro/La-specific T cell responses.ConclusionsDexVD3 DC presenting Ro/La antigens might be a promising new therapeutic option for patients with pSS.

Combined treatment with Bevacizumab and standard chemotherapy restores abnormal immune parameters in advanced colorectal cancer patients

Bevacizumab, a monoclonal antibody (mAb) targeting vascular endothelial growth factor (VEGF), has produced promising results when combined with chemotherapy in the treatment of advanced colorectal cancer (CRC). The aim of the present study was to define the immunological profile of metastatic CRC patients at baseline and following chemotherapy with either irinotecan/5-fluorouracil/leucovorin (IFL) alone or IFL in combination with.bevacizumab (B-IFL). Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (HD) (n = 20) and patients (n = 40) were tested for T-cell proliferation in the autologous mixed lymphocyte reaction (auto-MLR), and cytokine production following stimulation with anti-CD3 mAb. PBMCs obtained from CRC patients prior to treatment exhibited lower auto-MLR responses and low production of IL-2, IFN-γ, IL-12 and IL-18 cytokines, whereas IL-4 and IL-10 cytokines were increased as compared to HD (p < 0.001, for all parameters) following in vitro stimulation with anti-CD3 mAb. During treatment, and in particular in week 12 of evaluation, IL-2 (p < 0.001 for both IFL and B-IFL groups), IFN-γ (p < 0.001 for IFL and p = 0.001 for B-IFL), IL-12 (p < 0.001 for both IFL and B-IFL) and IL-18 (p < 0.001 for both IFL and B-IFL) production, as well as auto-MLR responses increased (p < 0.001 for both IFL and B-IFL), whereas IL-4 (p < 0.001 for IFL and p = 0.001 for B-IFL) and IL-10 [p < 0.001 for IFL and p = 0.067 (non-significant) for B-IFL] production decreased over baseline in the two treatment groups, yet their respective values never reached those of HD. Moreover, IL-2, IFN-γ production, and auto-MLR were higher in the B-IFL over the IFL treatment group (p < 0.001, p < 0.04, p < 0.001, respectively). Our study demonstrates that the abnormal immune parameters observed in metastatic CRC patients at presentation can substantially improve during treatment with either IFL or B-IFL. The immune parameters examined can provide a sensitive and valuable tool for monitoring immune function in CRC patients, and could be applied as surrogate markers predicting treatment-related outcome.

Development of TRX518, an aglycosyl humanized monoclonal antibody (Mab) agonist of huGITR.

e13028 Background: Glucocorticoid-induced tumor necrosis factor receptor (GITR) is expressed on several cell types, including regulatory and effector T cells, B cells, NK cells, and antigen-presenting cells. In murine models, our agonistic anti-mouse (m) GITR antibody was a robust vaccine adjuvant and, in combination with chemotherapy, significantly reduced tumor burden and prolonged survival compared with antibody or chemotherapy alone. The anti-mGITR Mab enhanced effector T cell responses and ablation of T regulatory cell-mediated suppression. A similar effect was observed when a chimeric anti-human (hu) GITR Mab (Ch-6C8-Agly) was used in nonhuman primates (NHP). NHP treated with anti-huGITR had augmented humoral and cellular immune responses compared with controls. Mab treatment was well tolerated with no observed adverse events. Methods: TRX518, a fully humanized IgG1 anti-huGITR Mab, was constructed. In addition to humanization, heavy chain asparagine 297 was substituted with alanine to eliminate N-linked glycosylation. This mutation abrogates Fc region functionality, including antibody-dependent cellular cytotoxicity and complement-mediated lysis. Further in vitro and in vivo characterization of TRX518 was conducted. Results: TRX518 bound to GITR on CD4+ and CD8+ naïve and memory T cells, B cells, NK cells, NKT cells, monocytes, and macrophages, yet was nondepleting. Though TRX518 costimulated suboptimally activated peripheral blood lymphocytes it was not a superagonist, as it did not induce proliferation by itself. Plate-bound TRX518 did not induce appreciable cytokine release (IL-2, IL-6, IL-10, TNFa, IFNg). TRX518 blocked the interaction of GITR with its ligand and enhanced the cytotoxicity of NK cells. However, it did not block an allogeneic mixed lymphocyte reaction (MLR) or enhance an autologous MLR. Cynomolgous macaque and human tissue cross studies and an IND-enabling GMP toxicology study are complete. Conclusions: TRX518 is a promising candidate for evaluation in human cancer indications. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Tolerx Tolerx

Chemokine Receptor CCR5 Mediates AlloImmune Responses in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematologic malignancies. However graft-versus-host disease (GVHD) is a major limiting factor for a successful patient outcome. GVHD is a result of alloimmune responses of donor T lymphocytes attacking the recipient's cells and tissues. Chemokine receptor CCR5 plays a role in solid organ allograft rejection and mediates murine GVHD pathogenesis. Herein, we report that infiltrating lymphocytes in the skin of human acute GVHD (aGVHD) samples are predominantly CCR5(+) T cells. In addition, we characterized the features of the CCR5 expression on alloreactive T lymphocytes. We found that the CCR5(+) population exhibits the characteristics of the activated effector T cell phenotype. CCR5 expression is upregulated upon allogenic stimulation, and CCR5(+) cells are proliferating with coexpression of T cell activation markers. Furthermore, the activated T cells producing inflammatory cytokine tumor necrosis factor (TNF)alpha, interleukin (IL)-2, or interferon (IFN)-gamma, are positive for CCR5. Thus, CCR5 is a marker for GVHD effector cells and CCR5(+) T cells are active participants in the pathogenesis of human aGVHD.