Screening patient peripheral blood using our proposed non-invasive method can boost the accuracy in selecting proper candidates for immune checkpoint therapy in the clinical setting, thereby leading to higher success in treatment. The immune system is regulated by a sophisticated network of modulatory molecules. In chronic infections and cancers, T cell exhaustion can arise when clearance of antigens is incomplete due to sustained expression of co-inhibitory molecules. T cell exhaustion has been exploited by some cancers, and also described in chronic infections with latency. Such exhausted T cells may be reversed with the right immune checkpoint blockade, thereby restoring effector T cell function. We have optimized a T cell Exhaustion Recall Antigen Assay, demonstrating T cell exhaustion may be reversed by immune checkpoint blockades in certain hosts. We hypothesize that understanding of the capacity and expressions of co-inhibitory molecules assist in predicting responders for specific immune checkpoint therapies. We have used a flow cytometry-based approach to examine changes in an array of immune checkpoint molecules on activated T cells and antigen presenting cells. We first tested the effect of anti-PD1 on tumor antigen-specific activated T cells using splenocytes from PMEL T cell receptor (TCR) transgenic mouse, which expresses mouse homologue of human premelanosome protein, or gp100. Activated CD3+CD8+ gp100-specific T cell population and IFNγ production were measured by flow cytometry. Secondly, we evaluated the expression levels of various immune checkpoint molecules post anti-CD3 stimulation of human peripheral blood mononuclear cells (PBMC) from characterized healthy hosts. Surprisingly, we found high expressions of co-inhibitory and co-stimulatory molecules including CTLA-4, PD1, PDL-1/PDL2, TIGIT, LAG3, TIM3, GITR, are associated with poor response to immune checkpoint blockades. Levels of activated T cells and cytokine production in donors with high checkpoint receptors showed lower T cell activations and cytokine production. Screening patient peripheral blood using our proposed non-invasive method can boost the accuracy in selecting proper candidates for immune checkpoint therapy in the clinical setting, thereby leading to higher success in treatment.Citation Format: Pirouz M. Daftarian, Marybeth George, Eden Kleiman, Wushouer Ouerkaxi, Amy Yamamura, Zhongliang Li, Mingfa Zang, Derron Yu, Eunmi Hwang, Annie X. An, Ann E. Lin, Henry Li. Expressions of co-inhibitory / co-stimulatory molecules may impact immune checkpoint therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4124.
Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.
Bacteria in humans play an important role in health and disease. Considerable emphasis has been placed in understanding the role of bacteria in host-microbiome interkingdom communication. Here we show that serotonin, responsible for mood in the brain and motility in the gut, can also act as a bacterial signaling molecule for pathogenic bacteria. Specifically, we found that serotonin acts as an interkingdom signaling molecule via quorum sensing and that it stimulates the production of bacterial virulence factors and increases biofilm formation in vitro and in vivo in a novel mouse infection model. This discovery points out at roles of serotonin both in bacteria and humans, and at phenotypic implications not only manifested in mood behavior but also in infection processes in the host. Thus, regulating serotonin concentrations in the gut may provide with paradigm shifting therapeutic approaches.
Introduction: Stem cell therapy has emerged as a promising approach for treatment of a number of diseases, including delayed and non-healing wounds. However, targeted systemic delivery of therapeutic cells to the dysfunctional tissues remains one formidable challenge. Methods: We have developed a targeted nanocarrier-mediated cell delivery method by coating the surface of the cell to be delivered with dendrimer nanocarriers modified with adhesion molecules. Infused nanocarrier-coated cells reach to destination via recognition and association with the counterpart adhesion molecules highly or selectively expressed on the activated endothelium in diseased tissues. Once anchored on the activated endothelium, nanocarriers-coated transporting cells undergo transendothelial migration, extravasation and homing to the targeted tissues to execute their therapeutic role. Wound healing was measured by digital photograph and ImageJ calculation. Targeted tissue homing of LacZ + bone marrow cells (BMC) was detected and quantified by X-gal staining, and BMC-enhanced neovascularization was examined by Dil perfusion and scanning confocal microscopy. Results: Soluble E-selectin (sE-sel) was successful installed on BMC surface by dendrimer nanocarriers. sE-sel-nanocarriers can associate with E-selectin ligands highly expressed on the wound endothelium, by which coated BMC are targeted delivered to skin wound tissues and grafted corneas to promote wound healing and neovascularization. Conclusions: We demonstrate feasibility, efficacy and safety of our targeted nanocarrier for delivery of BMC to cutaneous wound tissues and grafted corneas and its advantages over conventional BMC transplantation in mouse models for wound healing and neovascularization. This versatile platform is suited for targeted systemic delivery of virtually any type of therapeutic cell.
Abstract The surge of therapeutic regimens involving immune checkpoint antagonists and co-stimulatory pathways (ACP) antagonists used as single agents or in combination necessitates better functional screening assays. In vitro functional screening assays need to be non-invasive and high throughput. The recall antigen assay is arguably the one that, in an antigen specific controlled system, directly assesses the activity of potential immune checkpoint inhibitors (ICI) candidates, in a relatively physiologically relevant manner. The recall antigen assay has been often used in the past to prove the potency of ICI or ACP as indicated in publications and submissions to regulatory bodies. We have conducted a thorough optimization combined with modifications to the recall antigen assay to enable the need for a more effective and efficient assay.Our recall antigen assay has been optimized across multiple parameters including donor selection based on expression of co-inhibitory/co-stimulatory molecules. This facilitates the selection of the right donor for the right candidate. We have optimized both stimulation and readout components to enhance signal to noise ratio. For example, we reduced the duration of in vitro antigen specific CD8+ T-cell clonotype expansion from 2 weeks to 5 to 7 days. To maximize the direct readout, we have used an MHC Tetramer guided analysis, using a cocktail of MHC tetramers made with stimulating peptides. To further empower the tetramer assay with functionality, we also have co-stained tetramer positive cells with activation markers. Finally, we verified the assay using various activity-confirmed ICIs and controls. The assay uses characterized PBMCs stimulated with a pool of peptides with positive and negative treatments (e.g., PD-1 blockades and IgG4) and “test samples,” which are the candidates to be tested. Depending on the donor’s recall responses, a typical assay results in two to 6-fold increase of the antigen specific MHC tetramer positive population. Furthermore, after developing this recall antigen assay, we validated it by performing the assay in different laboratories, using different reagents lots, different instrumentations, and different operators. Recall antigen assay data indicated that not all donors respond to approved immune checkpoint blockades. Healthy donors stimulated with anti CD3 or cytokine cocktails varied showed a wide a wide variability of immune checkpoint molecules (ICM) cell surface levels. Further studies using characterized PBMCs in our recall antigen potency assay revealed that the expression of immune checkpoint molecules may contribute in unresponsiveness to a given ICI. In conclusion, we have developed an improved in vitro recall antigen potency assay for immunomodulatory biologics functional screening including ICIs and APCs. Furthermore, we observed that in our studies this potency assay divides hosts into responders and nonresponders. Interestingly, we also showed significant variation of persons’ PBMC responses to ICIs, which may be related to their inherent expression levels of immune checkpoint molecules. We intend to confirm this finding with further studies using well-annotated clinical samples. Citation Format: Marc Delcommenne, Eden Kleiman, Wushouer Ouerkaxi, Pirouz Daftarian. A recall antigen-based potency assay for immunomodulatory biologics that could discriminate responders from nonresponders [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B074.
