In Silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem Cell Transplant Donors and Recipients: Understanding the Quantitative Immuno-biology of Allogeneic Transplantation

Donor T cell mediated graft vs. host effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the HLA in each donor-recipient pair (DRP) undergoing stem cell transplantation (SCT). Whole exome sequencing has demonstrated extensive nucleotide sequence variation in HLA-matched DRP. Non-synonymous single nucleotide polymorphisms (nsSNPs) in the GVH direction (polymorphisms present in recipient and absent in donor) were identified in 4 HLA-matched related and 5 unrelated DRP. The nucleotide sequence flanking each SNP was obtained utilizing the ANNOVAR software package. All possible nonameric-peptides encoded by the non-synonymous SNP were then interrogated in-silico for their likelihood to be presented by the HLA class I molecules in individual DRP, using the Immune-Epitope Database (IEDB) SMM algorithm. The IEDB-SMM algorithm predicted a median 18,396 peptides/DRP which bound HLA with an IC50 of <500nM, and 2254 peptides/DRP with an IC50 of <50nM. Unrelated donors generally had higher numbers of peptides presented by the HLA. A similarly large library of presented peptides was identified when the data was interrogated using the Net MHCPan algorithm. These peptides were uniformly distributed in the various organ systems. The bioinformatic algorithm presented here demonstrates that there may be a high level of minor histocompatibility antigen variation in HLA-matched individuals, constituting an HLA-specific alloreactivity potential. These data provide a possible explanation for how relatively minor adjustments in GVHD prophylaxis yield relatively similar outcomes in HLA matched and mismatched SCT recipients.Comment: Abstract: 235, Words: 6422, Figures: 7, Tables: 3, Supplementary figures: 2, Supplementary tables:

Determining the Quantitative Principles of T Cell Response to Antigenic Disparity in Stem Cell Transplantation

Alloreactivity compromising clinical outcomes in stem cell transplantation is observed despite HLA matching of donors and recipients. This has its origin in the variation between the exomes of the two, which provides the basis for minor histocompatibility antigens (mHA). The mHA presented on the HLA class I and II molecules and the ensuing T cell response to these antigens results in graft vs. host disease. In this paper, results of a whole exome sequencing study are presented, with resulting alloreactive polymorphic peptides and their HLA class I and HLA class II (DRB1) binding affinity quantified. Large libraries of potentially alloreactive recipient peptides binding both sets of molecules were identified, with HLA-DRB1 generally presenting a greater number of peptides. These results are used to develop a quantitative framework to understand the immunobiology of transplantation. A tensor-based approach is used to derive the equations needed to determine the alloreactive donor T cell response from the mHA-HLA binding affinity and protein expression data. This approach may be used in future studies to simulate the magnitude of expected donor T cell response and determine the risk for alloreactive complications in HLA matched or mismatched hematopoietic cell and solid organ transplantation

Stem Cell Transplantation As A Dynamical System: Are Clinical Outcomes Deterministic?

Outcomes in stem cell transplantation (SCT) are modeled using probability theory. However the clinical course following SCT appears to demonstrate many characteristics of dynamical systems, especially when outcomes are considered in the context of immune reconstitution. Dynamical systems tend to evolve over time according to mathematically determined rules. Characteristically, the future states of the system are predicated on the states preceding them, and there is sensitivity to initial conditions. In SCT, the interaction between donor T cells and the recipient may be considered as such a system in which, graft source, conditioning and early immunosuppression profoundly influence immune reconstitution over time. This eventually determines clinical outcomes, either the emergence of tolerance or the development of graft versus host disease. In this paper parallels between SCT and dynamical systems are explored and a conceptual framework for developing mathematical models to understand disparate transplant outcomes is proposed.Comment: 23 pages, 4 figures. Updated version with additional data, 2 new figures and editorial revisions. New authors adde

Cytomegalovirus Antigenic Mimicry of Human Alloreactive Peptides: A Potential Trigger for Graft versus Host Disease

The association between human cytomegalovirus (hCMV) reactivation and the development of graft-versus-host-disease (GVHD) has been observed in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n=50; matched related donor (MRD), n=27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) Database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (6 or greater) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500 nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, 29,658.8 +/- 9038.5 were found to match MRD DRP alloreactive peptides and 52,910.2 +/- 16121.8 matched MUD DRP peptides (Student's T-test, p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner (proteins expressed at 10 RPKM or greater). hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset with a subset analysis of 7 instances of hCMV viremia prior to acute GVHD onset (n=3), chronic GVHD (n=2) and acute + chronic GVHD (n=2) indicating cross reactive peptide expression within affected organs. We propose that based on our analysis and preliminary clinical correlations that hCMV immune cross-reactivity may cause antigenic mimicry of human alloreactive peptides triggering GVHD.Comment: Pre-submission manuscript, 4 tables, 5 figures, 2 supplements & 2 Appendices-available upon request from first autho

Determining the Quantitative Principles of T Cell Response to Antigenic Disparity in Stem Cell Transplantation

Alloreactivity compromising clinical outcomes in stem cell transplantation is observed despite HLA matching of donors and recipients. This has its origin in the variation between the exomes of the two, which provides the basis for minor histocompatibility antigens (mHA). The mHA presented on the HLA class I and II molecules and the ensuing T cell response to these antigens results in graft vs. host disease. In this paper, results of a whole exome sequencing study are presented, with resulting alloreactive polymorphic peptides and their HLA class I and HLA class II (DRB1) binding affinity quantified. Large libraries of potentially alloreactive recipient peptides binding both sets of molecules were identified, with HLA-DRB1 generally presenting a greater number of peptides. These results are used to develop a quantitative framework to understand the immunobiology of transplantation. A tensor-based approach is used to derive the equations needed to determine the alloreactive donor T cell response from the mHA-HLA binding affinity and protein expression data. This approach may be used in future studies to simulate the magnitude of expected donor T cell response and determine the risk for alloreactive complications in HLA matched or mismatched hematopoietic cell and solid organ transplantation

The US Program in Ground-Based Gravitational Wave Science: Contribution from the LIGO Laboratory

Recent gravitational-wave observations from the LIGO and Virgo observatories have brought a sense of great excitement to scientists and citizens the world over. Since September 2015,10 binary black hole coalescences and one binary neutron star coalescence have been observed. They have provided remarkable, revolutionary insight into the "gravitational Universe" and have greatly extended the field of multi-messenger astronomy. At present, Advanced LIGO can see binary black hole coalescences out to redshift 0.6 and binary neutron star coalescences to redshift 0.05. This probes only a very small fraction of the volume of the observable Universe. However, current technologies can be extended to construct "3rd Generation" (3G) gravitational-wave observatories that would extend our reach to the very edge of the observable Universe. The event rates over such a large volume would be in the hundreds of thousands per year (i.e. tens per hour). Such 3G detectors would have a 10-fold improvement in strain sensitivity over the current generation of instruments, yielding signal-to-noise ratios of 1000 for events like those already seen. Several concepts are being studied for which engineering studies and reliable cost estimates will be developed in the next 5 years

The Influence of Manga on the Graphic Novel

This material has been published in The Cambridge History of the Graphic Novel edited by Jan Baetens, Hugo Frey, Stephen E. Tabachnick. This version is free to view and download for personal use only. Not for re-distribution, re-sale or use in derivative works. © Cambridge University PressProviding a range of cogent examples, this chapter describes the influences of the Manga genre of comics strip on the Graphic Novel genre, over the last 35 years, considering the functions of domestication, foreignisation and transmedia on readers, markets and forms

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Regulation and Physiologic Significance of the Agmatine Deiminase System of Streptococcus mutans UA159

We previously demonstrated that Streptococcus mutans expresses a functional agmatine deiminase system (AgDS) encoded by the agmatine-inducible aguBDAC operon (A. R. Griswold, Y. Y. Chen, and R. A. Burne, J. Bacteriol. 186:1902-1904, 2004). The AgDS yields ammonia, CO(2), and ATP while converting agmatine to putrescine and is proposed to augment the acid resistance properties and pathogenic potential of S. mutans. To initiate a study of agu gene regulation, the aguB transcription initiation site was identified by primer extension and a putative σ(70)-like promoter was mapped 5′ to aguB. Analysis of the genome database revealed an open reading frame (SMU.261c) encoding a putative transcriptional regulator located 239 bases upstream of aguB. Inactivation of SMU.261c decreased AgD activity by sevenfold and eliminated agmatine induction. AgD was also found to be induced by certain environmental stresses, including low pH and heat, implying that the AgDS may also be a part of a general stress response pathway of this organism. Interestingly, an AgDS-deficient strain was unable to grow in the presence of 20 mM agmatine, suggesting that the AgDS converts a growth-inhibitory substance into products that can enhance acid tolerance and contribute to the competitive fitness of the organism at low pH. The capacity to detoxify and catabolize agmatine is likely to have major ramifications on oral biofilm ecology