No abstract

Graft vs. host diseases (GvHD) accounts for 15-30% of deaths following allogenic hematopoietic stem cell transplantation (allo-HSCT) for treatment of malignant diseases. Acute GvHD (aGvHD) typically involves skin, gastrointestinal and hepatic inflammation, and occurs within 100 days of transplantation; Chronic GvHD (cGvHD) involves multiple organs and occurs beyond 100 days. aGvHD is largely due to the rapid activation of donor T cells (Th1, CD8+ biased), causing tissue damage and often leading to mortality; in contrast, cGvHD is Th2 biased, typically display autoimmune-like syndrome, involving both T- and B-cell. Currently, GvHD models are mostly allo-transplantations between mice, which are criticized for poor physiological relevance. Here, we present Xenotransplantation of human donor into NSG mice, which will address some of the limitation of current models. Human peripheral blood mononuclear cells (PBMC) derived from normal donors were transplanted into NSG or NSG-like strains for modeling aGvHD. Cord blood derived HSCs (hCD34+) NSG (Jackson) were transplanted for modeling cGvHD. Hosts were monitored twice weekly, including clinical observations (e.g. animal postures, activity, fur texture, and skin integrity), body weight changes, gross pathology and histopathology upon termination, along with human-immunological phenotyping of peripheral blood, spleen, lung, liver and skin by flow cytometry, histopathology and/or immunohistochemistry. In addition, GvHD novel biomarkers Elafin, IL-18, REG3α and ST2, measured by ELISA (MBL International) at baseline and post cells engraftment. NSG mice engrafted with human PBMC, or purified T-cells, from normal donors rapidly developed typical symptoms of aGvHD, as early as 4 weeks post transplantation, including severe body loss, reduced activity, hunched posture, loss of fur, severe ruffling and overall poor grooming. Significant engraftment of human CD45+ cells were detected up to 6 weeks post-engraftment dominated with CD3+ human T-cells of single positive CD4+ or CD8+ T-cells. The corresponding kinetics of clinical symptoms in parallel with the degree of engraftment of T cells suggests xenografting were responsible for aGvHD. As for the cGvHD, NSG-mice engrafted with CD34+ cells derived from cord blood with cGvHD high- risk HLA haplotypes C*0602 and C*0401. Manifestation of cGvHD was observed 18 weeks to 39 weeks post-engraftment; symptoms were similar to aGvHD, but also include facial/full body alopecia, and scaly skin. Interestingly, in correlation of this, human engraftment of CD45+, particularly T-cells including CD4+ and their CD30+ subsets, are kinetically increased in blood and spleen with similar timeline, suggesting their roles in the observed cGvHD. The xenograft murine model using adult human PBMC and cord blood derived CD34+ HSCs could be alternative experimental systems to model human aGvHD and cGvHD for investigating disease mechanisms and evaluating treatment strategy. Citation Format: Ann E. Lin, Annie X. An, Mingfa Zang, Derron Yu, Eunmi Hwang, Israel Romero, Linda Quirino, Marybeth George, Pirouz Daftarian, Wenqing Yang, Henry Li. Experimental modeling of acute- and chronic-GvHD by xenotransplanting human donor PBMCs or cord blood CD34+ cells (HSC) into NSG mice [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 2342.

Extracellular adenosine suppresses T cell immunity in the tumor microenvironment and in vitro treatment of memory T cells with adenosine can suppress antigen-mediated memory T cell expansion. We describe utilizing the recall antigen assay platform to screen small molecule drug off-target effects on memory T cell expansion/function using a dosing regimen based on adenosine treatment. As a proof of principle, we show low dose GS-5734, a monophosphoramidate prodrug of an adenosine analog, does not alter memory T cell recall at lower doses whereas toxicity observed at high dose favors antigen-specific memory T cell survival/proliferation over non-specific CD8+ T cells. Conversely, parent nucleoside GS-441524 at high dosage does not result in cellular toxicity and reduces antigen-specific T cell recall in most donors. Despite similar chemical structure, these drugs displayed opposing effects on memory T cell expansion and viability highlighting the sensitivity of this assay setup in screening compounds for off-target effects.

Background Peptide-MHC (pMHC) tetramers have been widely used in infectious disease and immune-oncology area to detect antigen-specific T cells. However, little is reported about their application in the context of pMHC presentation to stimulate T cells. Here we compared the expansion of antigen-specific T cells stimulated by an unlabeled pMHC tetramer and the peptide. We also analyzed the relationship among the programmed death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), PD-L1, CD80 and CD38 expression on antigen-specific and non-specific T cells. Results Compared to the peptide stimulation, the pMHC tetramer induced lower expansion of the peptide-specific T cells. PD-1, PD-L1, CD80, and LAG3 expression was significantly higher on peptide-specific T cells than on non-specific T cells. Interestingly, peptide-specific T cells expanded by pMHC tetramer induced lower numbers of dysfunctional PD-1+CD38high CD8+ T cells. Furthermore, blockade of the PD-1 pathway by using an FDA approved antibody reversed such a restriction of expansion. Conclusion The pMHC tetramer stimulation resulted in the reduced expansion of peptide-specific CD8 T cells when compared to the peptide stimulation. Since the impaired expansion was reversed by the anti-PD1 therapeutic antibody, this artificial stimulation method could be used as a functional in vitro immune-oncology drug screening tool.

The success of SARS-CoV-2 vaccines is measured by their ability to mount long-lasting memory responses. To this end, it is important to identify viral protein fragments, or peptides, that generate the best immune response and lead to the development of memory T cells. For this purpose, researchers need a system that would allow them to rapidly test the ability of an antigenic peptide to be presented by the immune system and to identify T cells that can be stimulated by such a peptide in the context of the right MHC molecule. The QuickSwitch™ platform contains two modules for performing both functions. First a large number of SARS-CoV-2 peptides, including mutant peptides were incubated with QuickSwitch™ monomers or tetramers and exchanged against their exiting peptides in the first module. Peptide binding affinities to various human MHC Class I and Class II alleles, which are correlated with their exchange rates, were determined by the capture binding assay, the second QuickSwitch™ module. MHC tetramers built with SARS-CoV-2 high affinity MHC binding peptides were then used for T cell staining. Cell staining indicated that tetramers generated with the QuickSwitch™ platform were able to stain and identify T cells specific for the tested peptides. Further, peptides that bound to multiple MHC alleles, including potentially dominant peptides, were also detected. In conclusion, since QuickSwitch™ allows the screening of MHC binding SARS-CoV-2 peptides and, at the same time, the generation of corresponding tetramers, this platform is a useful tool for developing SARS-CoV-2 vaccines, monitoring T cell populations from vaccinated subjects and assessing the durability of mounted responses.