Ipilimumab Combination Dosing: Less is More.

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.

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.

Dynamical system modeling to simulate donor T cell response to whole exome sequencing-derived recipient peptides: Understanding randomness in alloreactivity incidence following stem cell transplantation.

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<0.01); resulting peptide antigens that may be presented on HLA class I molecules in each DRP were derived in silico (NetMHCpan ver2.0) and the tissue expression of proteins these were derived from determined (GTex). MRD DRP had an average 3,670 HLA-binding-alloreactive peptides, putative mHA (pmHA) with an IC50 of <500 nM, and URD, had 5,386 (p<0.01). To simulate an alloreactive donor cytotoxic T cell response, the array of pmHA in each patient was considered as an operator matrix modifying a hypothetical cytotoxic T cell clonal vector matrix; each responding T cell clone's proliferation was determined by the logistic equation of growth, accounting for HLA binding affinity and tissue expression of each alloreactive peptide. The resulting simulated organ-specific alloreactive T cell clonal growth revealed marked variability, with the T cell count differences spanning orders of magnitude between different DRP. Despite an estimated, uniform set of constants used in the model for all DRP, and a heterogeneously treated group of patients, higher total and organ-specific T cell counts were associated with cumulative incidence of moderate to severe GVHD in recipients. In conclusion, exome wide sequence differences and the variable alloreactive peptide binding to HLA in each DRP yields a large range of possible alloreactive donor T cell responses. Our findings also help understand the apparent randomness observed in the development of alloimmune responses.

Dynamical System Modeling to Simulate Donor T Cell Response to Whole Exome Sequencing-Derived Recipient Peptides Demonstrates Different Alloreactivity Potential in HLA-Matched and -Mismatched Donor-Recipient Pairs.

Immune reconstitution kinetics and subsequent clinical outcomes in HLA-matched recipients of allogeneic stem cell transplantation (SCT) are variable and difficult to predict. Considering SCT as a dynamical system may allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response, which may allow better clinical outcome prediction. To accomplish this, whole exome sequencing was performed on 34 HLA-matched SCT donor-recipient pairs (DRPs) and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each DRP was determined. The resulting array of peptide-HLA binding affinity values in each patient was considered as an operator modifying a hypothetical T cell repertoire vector, in which each T cell clone proliferates in accordance with the logistic equation of growth. Using an iterating system of matrices, each simulated T cell clone's growth was calculated with the steady-state population being proportional to the magnitude of the binding affinity of the driving HLA-peptide complex. Incorporating competition between T cell clones responding to different HLA-peptide complexes reproduces a number of features of clinically observed T cell clonal repertoire in the simulated repertoire, including sigmoidal growth kinetics of individual T cell clones and overall repertoire, Power Law clonal frequency distribution, increase in repertoire complexity over time with increasing clonal diversity, and alteration of clonal dominance when a different antigen array is encountered, such as in SCT. The simulated, alloreactive T cell repertoire was markedly different in HLA-matched DRPs. The patterns were differentiated by rate of growth and steady-state magnitude of the simulated T cell repertoire and demonstrate a possible correlation with survival. In conclusion, exome wide sequence differences in DRPs may allow simulation of donor alloreactive T cell response to recipient antigens and may provide a quantitative basis for refining donor selection and titration of immunosuppression after SCT.

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.

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

Donor T-cell mediated graft versus host (GVH) effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the human leukocyte antigen (HLA) molecules in each donor-recipient pair undergoing stem-cell transplantation (SCT). Whole exome sequencing has previously demonstrated a large number of non-synonymous single nucleotide polymorphisms (SNP) present in HLA-matched recipients of SCT donors (GVH direction). The nucleotide sequence flanking each of these SNPs was obtained and the amino acid sequence determined. All the possible nonameric peptides incorporating the variant amino acid resulting from these SNPs were interrogated in silico for their likelihood to be presented by the HLA class I molecules using the Immune Epitope Database stabilized matrix method (SMM) and NetMHCpan algorithms. The SMM algorithm predicted that a median of 18,396 peptides weakly bound HLA class I molecules in individual SCT recipients, and 2,254 peptides displayed strong binding. A similar library of presented peptides was identified when the data were interrogated using the NetMHCpan algorithm. The bioinformatic algorithm presented here demonstrates that there may be a high level of mHA variation in HLA-matched individuals, constituting a HLA-specific alloreactivity potential.

Whole exome sequencing to estimate alloreactivity potential between donors and recipients in stem cell transplantation.

Whole exome sequencing (WES) was performed on stem cell transplant donor-recipient (D-R) pairs to determine the extent of potential antigenic variation at a molecular level. In a small cohort of D-R pairs, a high frequency of sequence variation was observed between the donor and recipient exomes independent of human leucocyte antigen (HLA) matching. Nonsynonymous, nonconservative single nucleotide polymorphisms were approximately twice as frequent in HLA-matched unrelated, compared with related D-R pairs. When mapped to individual chromosomes, these polymorphic nucleotides were uniformly distributed across the entire exome. In conclusion, WES reveals extensive nucleotide sequence variation in the exomes of HLA-matched donors and recipients.

Disulfide bond oxidoreductase DsbA2 of Legionella pneumophila exhibits protein disulfide isomerase activity.

The extracytoplasmic assembly of the Dot/Icm type IVb secretion system (T4SS) of Legionella pneumophila is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential disulfide bond oxidoreductase DsbA2 and not by DsbA1, a second nonessential DSB oxidoreductase. DsbA2, which is widely distributed in the microbial world, is phylogenetically distinct from the canonical DsbA oxidase and the DsbC protein disulfide isomerase (PDI)/reductase of Escherichia coli. Here we show that the extended N-terminal amino acid sequence of DsbA2 (relative to DsbA proteins) contains a highly conserved 27-amino-acid dimerization domain enabling the protein to form a homodimer. Complementation tests with E. coli mutants established that L. pneumophila dsbA1, but not the dsbA2 strain, restored motility to a dsbA mutant. In a protein-folding PDI detector assay, the dsbA2 strain, but not the dsbA1 strain, complemented a dsbC mutant of E. coli. Deletion of the dimerization domain sequences from DsbA2 produced the monomer (DsbA2N), which no longer exhibited PDI activity but complemented the E. coli dsbA mutant. PDI activity was demonstrated in vitro for DsbA2 but not DsbA1 in a nitrocefin-based mutant TEM ß-lactamase folding assay. In an insulin reduction assay, DsbA2N activity was intermediate between those of DsbA2 and DsbA1. In L. pneumophila, DsbA2 was maintained as a mixture of thiol and disulfide forms, while in E. coli, DsbA2 was present as the reduced thiol. Our studies suggest that DsbA2 is a naturally occurring bifunctional disulfide bond oxidoreductase that may be uniquely suited to the majority of intracellular bacterial pathogens expressing T4SSs as well as in many slow-growing soil and aquatic bacteria.

DsbA2 (27 kDa Com1-like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide-bond formation in proteins including the Dot/Icm type IV secretion system.

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.

Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice.

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 ß 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 PSMß 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.

RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus.

Cell-density-dependent gene regulation by quorum-sensing systems has a crucial function in bacterial physiology and pathogenesis. We demonstrate here that the Staphylococcus aureus agr quorum-sensing regulon is divided into (1) control of metabolism and PSM cytolysin genes, which occurs independently of the small regulatory RNA RNAIII, and (2) RNAIII-dependent control of additional virulence genes. Remarkably, PSM expression was regulated by direct binding of the AgrA response regulator. Our findings suggest that quorum-sensing regulation of PSMs was established before wide-ranging control of virulence was added to the agr regulon, which likely occurred by development of the RNAIII-encoding region around the gene encoding the PSM delta-toxin. Moreover, the agr regulon in the community-associated methicillin-resistant S. aureus MW2 considerably differed from that previously determined using laboratory strains. By establishing a two-level model of quorum-sensing target gene regulation in S. aureus, our study gives important insight into the evolution of virulence control in this leading human pathogen.

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, CO2, 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 sigma70-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.