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Ipilimumab with and without anti–Programmed Death 1 (PD-1) improved overall survival (OS) in melanoma. Despite this, the optimal dose and therapeutic mechanism of ipilimumab in patients remains unclear. KEYNOTE-029 and other studies suggest that low-dose ipilimumab with anti-PD1 maintains efficacy while decreasing toxicity, emphasizing T-cell trafficking and reinvigoration as likely mechanisms. See related article by Long et al., p. 5280

The development of anti-programmed death 1 (PD1) antibodies has rapidly changed standard oncology clinical practice however only a fraction of patients benefit from these treatments and some tumor types remain unresponsive.

TPS9596 Background: Targeted therapy (BRAF + MEK inhibitors) and immunotherapy (anti-PD1 + anti-CTLA4) have improved overall survival for metastatic or unresectable BRAFV600E/Kmutant melanoma. Whereas targeted therapy has a high response rate, immunotherapy may deliver longer term disease control for a larger number of patients. Despite these treatments, patients with high risk metastatic melanoma such as those with brain or liver metastases, elevated lactate dehydrogenase (LDH) and bulky disease have inferior treatment outcomes with current therapies. A BRAF+MEK+PDL1 regimen has recently emerged however the role for this treatment remains unclear. Several recent trials combining MEK inhibition and immunotherapy have failed possibly because MEK inhibition can compromise T cell activation. Meanwhile the addition of CTLA4 blockade to PD1 inhibition appears to disproportionately benefit patients with non-T cell-inflamed tumors and potentially high-risk disease. For patients with high risk BRAF-mutant metastatic melanoma, further investigation of BRAF/MEK targeted and PD-1/CTLA-4 directed immunotherapy combination strategies remains a priority. Methods: This is an open label, multi-site, Phase 1/2 study of encorafenib (Enco) +/- binimetinib (Bini) + nivolumab (Nivo) + ipilimumab (Ipi) for the treatment of patients with unresectable or metastatic BRAF-mutated melanoma in high-risk cohorts (NCT04655157). An initial regimen confirming Phase I approach will be pursued on two schedules concurrently, with patients accruing equally to each group. Group 1 will receive 3mg/kg Nivo, and 1 mg/kg Ipi and 300mg Enco (12 participants, triple therapy) and Group 2 will receive 3mg/kg Nivo and 1mg/kg Ipi and 450mg Enco and 45mg Bini, (12 participants, quadruple therapy). Dose limiting toxicity (DLT) will be evaluated weeks 1-6. A recommended Phase II regimen (RP2R) [either triple or quadruple therapy] will be carried forward into two high risk metastatic disease cohort expansions of 30 participants each. Cohort 1 will include patients with symptomatic brain metastases, while cohort 2 will include patients with elevated LDH as well as either liver metastases OR bulky visceral disease (sum of longest diameters > 44mm). Patients meeting criteria for cohorts 1 and 2 will be placed in cohort 1. Patients with symptomatic brain metastases will be included with an ECOG up to 2 and on ≤ 4mg of dexamethasone or equivalent. Continuous Bayesian toxicity monitoring will be used throughout to monitor DLT. Pre and on-treatment tumor biopsies will assess changes in the tumor microenvironment while peripheral blood ctDNA and T cell Ki67% changes will assess early response and immune activation during triplet and quadruplet therapy. Clinical trial information: NCT04655157.

Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA) and graft versus host disease (GVHD) pathophysiology in stem cell transplant (SCT) donor-recipient pairs (DRP) is not established. In order to elucidate this relationship, whole exome sequencing (WES) was performed on 27 HLA matched related (MRD), & 50 unrelated donors (URD), to identify nonsynonymous single nucleotide polymorphisms (SNPs). An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p

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, Pre-submission manuscript, 4 tables, 5 figures, 2 supplements & 2 Appendices-available upon request from first author

In Gram-negative bacteria, thiol oxidoreductases catalyse the formation of disulphide bonds (DSB) in extracytoplasmic proteins. In this study, we sought to identify DSB-forming proteins required for assembly of macromolecular structures in Legionella pneumophila. Here we describe two DSB-forming proteins, one annotated as dsbA1 and the other annotated as a 27 kDa outer membrane protein similar to Com1 of Coxiella burnetii, which we designate as dsbA2. Both proteins are predicted to be periplasmic, and while dsbA1 mutants were readily isolated and without phenotype, dsbA2 mutants were not obtained. To advance studies of DsbA2, a cis-proline residue at position 198 was replaced with threonine that enables formation of stable disulphide-bond complexes with substrate proteins. Expression of DsbA2 P198T mutant protein from an inducible promoter produced dominant-negative effects on DsbA2 function that resulted in loss of infectivity for amoeba and HeLa cells and loss of Dot/Icm T4SS-mediated contact haemolysis of erythrocytes. Analysis of captured DsbA2 P198T-substrate complexes from L. pneumophila by mass spectrometry identified periplasmic and outer membrane proteins that included components of the Dot/Icm T4SS. More broadly, our studies establish a DSB oxidoreductase function for the Com1 lineage of DsbA2-like proteins which appear to be conserved among those bacteria also expressing T4SS.

Graft versus host disease (GVHD) continues to be a life-threatening complication of stem cell transplantation for hematological malignancies. GVHD occurs in perfectly HLA-matched, related (MRD) transplants, and conversely can be absent in matched un-related (MUD) or haploidentical stem cell transplants (SCT). Minor histocompatibility antigens (mHA) have been investigated for their contribution to GVHD in SCT, which is mediated by differential presentation of these antigens in the recipient tissues to the donor derived T cells. Our group and others have shown that predicted mHA in bone marrow transplant donor-recipient pairs (DRP) range in the 1,000s, with MUD on the average having 2x as many mHA as MRD. However, repeated studies have shown that absolute number of mHA does not correlate with GVHD - that is more minor histocompatibility antigens do not lead to higher rates of GVHD. It is unknown if the tissue distribution of mHA in SCT differs significantly between patients, and what role this may have in the biology of GVHD. Therefore, our group investigated if the distribution of in silico derived mHA might correlate with GVHD occurrence. Whole exome sequencing (WES) of 77 HLA matched SCT DRP was performed and revealed thousands of nucleotide polymorphisms unique to the recipient when compared with the donor. When translated into peptide fragments and queried for binding to HLA-A, B, and C, in each DRP, these polymorphic peptides generated thousands of putative mHA/DRP in silico. Lists with the genes of origin for the mHA were then compiled for each DRP and sorted by the binding affinity of polymorphic recipient peptides to HLA; presented binders (IC50 Normal gene expression data for 13 distinct tissues was obtained from the GTEx database v6 consisting of 54,354 transcripts averaged over hundreds of samples. The R package "pSI" was utilized to generate lists of genes that were disproportionately expressed in various tissues. A "Specificity index" (SI) was calculated for each gene in specific tissues by comparing its expression level in that tissue, against expression level in all other tissues, with cutoffs given by P value (Wells et al., 2015 Nucleic Acids Research). Using these SI gene lists as reference, SB mHA in the patient data set showed significant differences in predicted tissue expression of the polymorphic genes between the 77 patients. The probabilities of mHA being presented by specific tissues was seen to follow a normal distribution across patient samples, allowing for further statistical analyses that assume normal distributions. The intersection of the mHA lists (PB or SB) and the SI lists (normal tissue specific expression) were calculated (Figure 1). 16 models were then analyzed for ability to predict GVHD, and best fit was discovered using C-statistics. The SI list at a specificity of P=0.001 together with the proportion analysis had the best C-statistic (0.831) and was used for further investigation. Univariate and multivariate analysis revealed that higher proportions, but not absolute number, of antigens expressed in colonic tissue was statistically correlated with GVHD in this pilot study (Table 1). In conclusion, this pilot study reveals that the tissue distribution of WES derived mHA are not uniform across DRP. It also suggests that differences in mHA tissue distribution may contribute to GVHD. Specifically, a heavier burden of polymorphisms in the genes differentially expressed in the colon compared to other tissues may make recipients more likely to experience GVHD. Disclosures No relevant conflicts of interest to declare.

Stem cell transplants may be considered as dynamical systems to allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response. Whole exome sequencing was performed on HLA matched stem cell transplant donor-recipient pairs, and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each pair was determined. The resulting array of peptide-HLA binding affinity values in each patient was used to simulate an alloreactive donor derived T cell repertoire. This simulated T cell repertoire reproduces a number of features of clinically observed T cell repertoire. The simulated, alloreactive T cell repertoire was markedly different in HLA matched stem cell transplant donors and recipients and demonstrates a possible correlation with survival., 50 pages (including references and supplementary materials), 12 figures, 3 supplementary figures

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, 235, Words: 6422, Figures: 7, Tables: 3, Supplementary figures: 2, Supplementary tables: 2

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 added

In Gram-negative bacteria, thiol oxidoreductases catalyse the formation of disulphide bonds (DSB) in extracytoplasmic proteins. In this study, we sought to identify DSB-forming proteins required for assembly of macromolecular structures in Legionella pneumophila. Here we describe two DSB-forming proteins, one annotated as dsbA1 and the other annotated as a 27 kDa outer membrane protein similar to Com1 of Coxiella burnetii, which we designate as dsbA2. Both proteins are predicted to be periplasmic, and while dsbA1 mutants were readily isolated and without phenotype, dsbA2 mutants were not obtained. To advance studies of DsbA2, a cis-proline residue at position 198 was replaced with threonine that enables formation of stable disulphide-bond complexes with substrate proteins. Expression of DsbA2 P198T mutant protein from an inducible promoter produced dominant-negative effects on DsbA2 function that resulted in loss of infectivity for amoeba and HeLa cells and loss of Dot/Icm T4SS-mediated contact haemolysis of erythrocytes. Analysis of captured DsbA2 P198T-substrate complexes from L. pneumophila by mass spectrometry identified periplasmic and outer membrane proteins that included components of the Dot/Icm T4SS. More broadly, our studies establish a DSB oxidoreductase function for the Com1 lineage of DsbA2-like proteins which appear to be conserved among those bacteria also expressing T4SS.

Biofilms are surface-attached agglomerations of microorganisms embedded in an extracellular matrix. Biofilm-associated infections are difficult to eradicate and represent a significant reservoir for disseminating and recurring serious infections. Infections involving biofilms frequently develop on indwelling medical devices in hospitalized patients, and Staphylococcus epidermidis is the leading cause of infection in this setting. However, the molecular determinants of biofilm dissemination are unknown. Here we have demonstrated that specific secreted, surfactant-like S. epidermidis peptides — the β subclass of phenol-soluble modulins (PSMs) — promote S. epidermidis biofilm structuring and detachment in vitro and dissemination from colonized catheters in a mouse model of device-related infection. Our study establishes in vivo significance of biofilm detachment mechanisms for the systemic spread of biofilm-associated infection and identifies the effectors of biofilm maturation and detachment in a premier biofilm-forming pathogen. Furthermore, by demonstrating that antibodies against PSMβ peptides inhibited bacterial spread from indwelling medical devices, we have provided proof of principle that interfering with biofilm detachment mechanisms may prevent dissemination of biofilm-associated infection.

Stem cell transplant (SCT) recipients who develop human cytomegalovirus (hCMV) reactivation are at risk of developing graft versus host disease (GVHD). This may stem from immune cross reactivity (Figure 1) towards both pathogen-derived peptides and nearly identical recipient-derived alloreactive peptide minor histocompatibility antigens from SCT donor-recipient pairs (DRP). Whole exome sequencing was performed on 9 SCT DRP, and the resulting nucleotide sequences aligned and compared, identifying all the single nucleotide polymorphisms (SNPs) present in the recipient and absent in the donor. Alloreactive peptide libraries were compiled for each DRP (9-mer peptides) and HLA binding affinity calculated for relevant HLA (Front Immunol 2014). A library representing the hCMV proteome was generated from the NCBI Protein database and directly compared to libraries of recipient specific peptide-HLA complexes from the SCT D-R pairs. BLAST Protein sequence alignment was performed to interrogate each pair's peptide-HLA complex library for matches with hCMV peptides, identifying CMV peptides with a 6/6-9/9 amino acid sequence matching to recipient peptides bound to HLA (IC50 Disclosures Buck: CHRB: Research Funding. Neale:CHRB: Research Funding.

Allogeneic hematopoietic stem cell transplants may result in graft versus host disease, mediated by donor T cells alloreactivity towards minor histocompatibility antigens (miHA) in the recipient. Up to 60% of allogeneic stem cell transplants experience acute graft vs host disease (GVHD). Even in perfectly HLA-matched related transplants, GVHD is still unpredictable; conversely, haploidentical mismatched transplants with reduced intensity conditioning can have a GVHD-free course. As of 2015, only 54 miHA have been described, such as HY, shown to affect GVHD and outcomes in specific diseases and donor-recipient pairs. To quantify potential miHA, whole exome sequencing was performed on 34 allogeneic transplants. For patient characteristics, 27 patients were molecularly matched at 8/8 loci and 7 patients were 7/8 HLA matched; 5 female to male donor transplants were included; as well as 10 African American and 24 Caucasian matches. Ten patients did not develop GVHD, while 24 patients experienced GVHD, either acutely or chronically. A bioinformatics pipeline was developed to discover all non-synonymous single nucleotide polymorphisms (nsSNPs) within unique donor-recipient pairs (DRP) in the graft vs host (GVH) direction. These nsSNPs were translated into 9-mer peptides, with the protein-coding change modeled at each site (position 1-9); this generated nine 9-mers per nsSNP. The peptides were then queried for binding affinity (represented as an IC50) to each patient's HLA with the NetMHC algorithm, with an IC50 < 500 called-presented and IC50 Using logistic regression analysis, significant differences were found between matched-related and matched-unrelated donors' presented peptides: 1646 vs. 2249, respectively (p-value .036). There were no significant differences in the average number of predicted peptides presented by HLA-A, B, or C. Importantly, the ratio of strong binders/presented peptides was a significant predictor of GVHD (p-value 0.046) with a model controlling for donor type, HLA-mismatch, patient race, and donor gender. There were 17,457 presented peptides shared between GVHD and GVHD-free patients. In 10 patients that did not develop GVHD 8,331 peptides were unique. In the 24 patients that did develop GVHD, 20,300 peptides were unique to that group (Figure 1). Potential miHA had varying tissue distribution based on average tissue values from the GTEx RNA expression database. The data from this 34 patient cohort quantify the large magnitude of potential miHA present in every BMT and their HLA binding affinity distribution, which can be thought of as an alloreactivity potential. Additional DRP whose analysis is not yet complete may aid in refining the analysis presented here. Current HLA matching strategies ignore genomic variation and strategies to account for this variation may aid both in better donor selection for optimal GVH/GVL effects and also in tailoring immunosuppression in an individualized, patient-specific manner. Figure 1. Figure 1. Disclosures Buck: Commonwealth Health Research Board: Research Funding. Neale:CHRB: Research Funding.

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.

Whole exome sequencing was performed on HLA-matched stem cell donors and transplant recipients to measure sequence variation contributing to minor histocompatibility antigen differences between the two. A large number of nonsynonymous single nucleotide polymorphisms were identified in each of the nine unique donor-recipient pairs tested. This variation was greater in magnitude in unrelated donors as compared with matched related donors. Knowledge of the magnitude of exome variation between stem cell transplant recipients and donors may allow more accurate titration of immunosuppressive therapy following stem cell transplantation., 12 pages- main article, 29 pages total, 5 figures, 1 supplementary figure