MHC Class I Molecules Research Articles

Abstract B59: Co-evolution between tumor cells and immune system in the setting of T-cell immunotherapy

Abstract It is now well established that the host’s adaptive immune system plays an important role in identifying and eliminating cancer cells in much the same way that intracellular pathogens are cleared during an adaptive immune response to infection. From a therapeutic standpoint, the adaptive immune system is unique in that it can co-evolve alongside a developing tumor. However, tumor acquisition of immune evasive phenotypes, such as class-I MHC downregulation, remains a major limitation of successful T-cell immunotherapy. We propose a stochastic model that couples tumor and CD8+ T-cell adaptive immune compartments. This framework accurately models relevant empirical findings, including the growth-threshold conjecture of immune activation, and predicts experimental observations including “sneak-through,” wherein intermediate-growth threats are penalized relative to their slower- and faster-growing counterparts. We demonstrate that predicted optimal growth strategies depend on whether or not the threat may acquire an immune-evasive phenotype as well as the mode of immune detection. The model is able to accurately characterize age-dependent cancer incidence data as a function of decreasing T-cell turnover and repertoire diversity. Lastly, we quantify therapeutic efficacy of immunotherapy in the setting of an immune-evasive threat. Our model serves as a first attempt at modeling stochastic cancer evolution sculpted by an adaptive immune compartment. Citation Format: Jason T. George, Haven R. Garber, Jeffrey J. Molldrem, Herbert Levine. Co-evolution between tumor cells and immune system in the setting of T-cell immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B59.

Phosphoinositide 3-Kinase Signaling Can Modulate MHC Class I and II Expression

Molecular events activating the PI3K pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to antitumor immunity. Given the ability of IFNγ to regulate antigen processing and presentation and the pivotal role of MHC class I (MHCI) and II (MHCII) expression in T-cell-mediated antitumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFNγ. We found that the induction of cell surface MHCI and MHCII molecules by IFNγ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFNγ treatment and increases accessibility at genomic STAT1-binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFNγ, and likewise, the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFNγ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intratumoral regions with high phospho-AKT IHC staining had reduced MHCI IHC staining. IMPLICATIONS: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.

Epitope\u2010based peptide vaccine design and target site depiction against Middle East Respiratory Syndrome Coronavirus: an immune-informatics study

BackgroundMiddle East Respiratory Syndrome Coronavirus (MERS-COV) is the main cause of lung and kidney infections in developing countries such as Saudi Arabia and South Korea. This infectious single-stranded, positive (+) sense RNA virus enters the host by binding to dipeptidyl-peptide receptors. Since no vaccine is yet available for MERS-COV, rapid case identification, isolation, and infection prevention strategies must be used to combat the spreading of MERS-COV infection. Additionally, there is a desperate need for vaccines and antiviral strategies.MethodsThe present study used immuno-informatics and computational approaches to identify conserved B- and T cell epitopes for the MERS-COV spike (S) protein that may perform a significant role in eliciting the resistance response to MERS-COV infection.ResultsMany conserved cytotoxic T-lymphocyte epitopes and discontinuous and linear B-cell epitopes were predicted for the MERS-COV S protein, and their antigenicity and interactions with the human leukocyte antigen (HLA) B7 allele were estimated. Among B-cell epitopes, QLQMGFGITVQYGT displayed the highest antigenicity-score, and was immensely immunogenic. Among T-cell epitopes, MHC class-I peptide YKLQPLTFL and MHC class-II peptide YCILEPRSG were identified as highly antigenic. Furthermore, docking analyses revealed that the predicted peptides engaged in strong bonding with the HLA-B7 allele.ConclusionThe present study identified several MERS-COV S protein epitopes that are conserved among various isolates from different countries. The putative antigenic epitopes may prove effective as novel vaccines for eradication and combating of MERS-COV infection.

Complementary approaches to study NKT cells in cancer.

NKT cells are a small but influential member of the T cell family, recognizing lipids presented by the non-classical MHC-like molecule CD1d rather than peptides presented by classical MHC molecules. They bridge between the innate and adaptive immune systems, serving as rapid responders but also allowing the T cell immune system to recognize lipid antigens, for example derived from tumors or bacteria. They also serve as potent regulatory cells, controlling other immune responses. Type I NKT cells use a semi-invariant T cell receptor (TCR) whereas type II use diverse TCRs. Most often, type I NKT cells promote tumor immunity whereas type II tend to suppress it, and the two subtypes crossregulate each other, forming an immunoregulatory axis. Lack of tools to study these important cells has limited the understanding of these, but newer tools have allowed great advances, especially in mouse models. These range from transgenic and knock-out mice to CD1d tetramers carrying ligands for type I or II NKT cells, to antibodies and NKT cell hybridomas. Here we describe these complementary tools and approaches and their use to study NKT cells and their role in the immunology and immunotherapy of cancer.

Identification of Novel Multi Epitopes Vaccine against the Capsid Protein ( ORF2 ) of Hepatitis E Virus

Hepatitis E virus (HEV) is non-enveloped, small virus with a positive RNA sense in the family Hepeviredae genus Orthohepevirus. More than 20 million individuals annually infected by HEV with increased mortality rate ranged from 8% to 20% in pregnant women. The aim of the present study was to design multi peptides vaccine against HEV using immunoinformatic tools that elicited humoral and cellular immunity. The capsid protein sequences of HEV were retrieved from NCBI and subjected to various immunoinformatics tools from IEDB to assess their conservancy, surface accessibility and antigenicity as promising epitopes against B cells. Moreover the binding affinity of the conserved predicted epitopes was analyzed against MHC-I and MHC-II alleles of the T cells. The predicted epitopes were further assessed for their population coverage. For B-cell 32, 23 and 12 epitopes were predicted as linear conserved epitopes, surface accessibility and antigenic respectively. However the best B cell epitopes that overlapped the prediction tools were 165PLQD168, 219PTSVD223, 452PTPSPAPS459, 556GYPYNY561 and 615DYPA619. For T cell, the MHC-I alleles interacted with 37 conserve epitopes. Four epitopes (367GIALTLFNL375, 379LLGGLPTEL387, 389SSAGGQLFY397 and 394QLFYSRPVV402) interacted with MHC class-I with high affinity and specificity and hence were proposed as vaccine candidates. Moreover seven epitopes out of 125 predicted epitopes (were 205YAISISFWP213, 299LLDFALELE307, 341LTTTAATRF349, 367GIALTLFNL375, 368IALTLFNLA376, 379LLGGLPTEL387 and 394QLFYSRPVV402) were proposed as vaccine since they demonstrated high affinity to MHC-II alleles. The epitopes 367GIALTLFNL375, 379LLGGLPTEL387 and 394QLFYSRPVV402 were recognized interacting with both MHC-I and MHC-II alleles. The population coverage epitopes set for MHC-I and MHC-II alleles was 78.97% and 99.99%, respectively. While the epitopes set for all T cell proposed epitopes was 100%. Thirteen epitopes were predicted eliciting B and T cells and proposed as vaccine candidates against HEV. However these proposed epitopes require clinical trials studies to ensure their efficacy as vaccine candidates.

HLA-B locus products resist degradation by the human cytomegalovirus immunoevasin US11.

To escape CD8+ T-cell immunity, human cytomegalovirus (HCMV) US11 redirects MHC-I for rapid ER-associated proteolytic degradation (ERAD). In humans, classical MHC-I molecules are encoded by the highly polymorphic HLA-A, -B and -C gene loci. While HLA-C resists US11 degradation, the specificity for HLA-A and HLA-B products has not been systematically studied. In this study we analyzed the MHC-I peptide ligands in HCMV-infected cells. A US11-dependent loss of HLA-A ligands was observed, but not of HLA-B. We revealed a general ability of HLA-B to assemble with β2m and exit from the ER in the presence of US11. Surprisingly, a low-complexity region between the signal peptide sequence and the Ig-like domain of US11, was necessary to form a stable interaction with assembled MHC-I and, moreover, this region was also responsible for changing the pool of HLA-B ligands. Our data suggest a two-pronged strategy by US11 to escape CD8+ T-cell immunity, firstly, by degrading HLA-A molecules, and secondly, by manipulating the HLA-B ligandome.

Contriving a chimeric polyvalent vaccine to prevent infections caused by herpes simplex virus (type-1 and type-2): an exploratory immunoinformatic approach

Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) cause a variety of infections including oral-facial infections, genital herpes, herpes keratitis, cutaneous infection and so on. To date, FDA-approved licensed HSV vaccine is not available yet. Hence, the study was conducted to identify and characterize an effective epitope based polyvalent vaccine against both types of Herpes Simplex Virus. The selected proteins were retrieved from ViralZone and assessed to design highly antigenic epitopes by binding analyses of the peptides with MHC class-I and class-II molecules, antigenicity screening, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach. The final vaccine was constructed by the combination of top CTL, HTL and BCL epitopes from each protein along with suitable adjuvant and linkers. Physicochemical and secondary structure analysis, disulfide engineering, molecular dynamic simulation and codon adaptation were further employed to develop a unique multi-epitope peptide vaccine. Docking analysis of the refined vaccine structure with different MHC molecules and human immune TLR-2 receptor demonstrated higher interaction. Complexed structure of the modeled vaccine and TLR-2 showed minimal deformability at molecular level. Moreover, translational potency and microbial expression of the modeled vaccine was analyzed with pET28a(+) vector for E. coli strain K12 and the vaccine constructs had no similarity with entire human proteome. The study enabled design of a novel chimeric polyvalent vaccine to confer broad range immunity against both HSV serotypes. However, further wet lab based research using model animals are highly recommended to experimentally validate our findings. Communicated by Ramaswamy H. Sarma

Abstract 3252: TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming

Abstract SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of the small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 is a novel, selective small molecule inhibitor of the SUMOylation enzymatic cascade, which has demonstrated induction of potent anti-tumor immunity in preclinical models. In this study, we evaluated changes in immune cell composition and states after TAK-981 treatment in preclinical models. In both mouse bone-marrow and human peripheral blood mononuclear cell derived dendritic cells (DCs), TAK-981 treatment ex vivo induced markers of activation and maturation including CD40, CD80 and CD86, as well as increased secretion of inflammatory cytokines like IP-10, MCP1, MIP-1α, MIP1β, IFNα and IFNβ. These effects were reversed by an interferon alpha receptor (IFNAR) blocking antibody, demonstrating dependence of TAK-981 induced pharmacodynamic effects on Type I interferon signaling in DCs. In vivo, a single sub-cutaneous injection of TAK-981 in naïve Balb/c mice at the brachial lymph nodes induced activation of DCs, measured as significant increases in CD40 and CD86 expression. Concomitantly, an increase in expression of the early lymphocyte activation marker CD69 was observed on T cells and NK cells, demonstrating pleiotropic effects of TAK-981 on immune cells. Increased expression of CD69 was also observed in purified human T and NK cells treated with TAK-981 ex vivo. To investigate if enhanced activation of DCs by TAK-981 leads to improved cross-presentation of exogenous antigens, we challenged naïve C57BL/6 mice with chicken-ovalbumin (OVA) protein alone or in combination with TAK-981. Interestingly, an increase in the frequency of Kb-SIINFEKL tetramer positive CD8 T cells was observed in the draining lymph nodes overnight, and in the spleen on day 14, after treatment with TAK-981+OVA, suggesting that TAK-981 promotes cross-presentation of SIINFEKL, the class-I MHC epitope of OVA, to cognate antigen-specific CD8 T cells. We also observed an increase in the frequency of CD8α+ DCs loaded with the peptide ‘SIINFEKL’ on H-2Kb (class-I MHC of C57BL/6 mice), providing direct evidence for enhanced antigen-cross presentation. Similar findings were observed with TAK-981 administered either sub-cutaneously or via the therapeutic route of intra-venous injection in the above OVA immunization model. These results suggest a mechanism by which TAK-981 may promote anti-tumor immune responses in mice via enhanced cross-presentation of exogenous antigens released by dying tumor cells, leading to priming and activation of antigen reactive cytotoxic T cells. Currently, TAK-981 is being evaluated in Phase 1 clinical trials in adult patients with metastatic solid tumors or lymphomas (ClinicalTrials.gov Identifier: NCT03648372). Citation Format: Mithun Khattar, Keli Song, Stephen Grossman, Kristina Xega, Xingyue He, Neeraja Idamakanti, Dennis Huszar. TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming [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 3252.

Getting in the groove: Editing of MHC-I antigen repertoires by molecular chaperones is governed by a network of protein dynamics

Abstract Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with class-I major histocompatibility complex (MHC-I) molecules to promote optimization (editing) of peptide cargo. Using solution NMR, we investigate the molecular mechanism of peptide exchange performed by the 90 kDa chaperone protein complex. We identify TAPBPR-induced conformational changes on conserved MHC-I surfaces, consistent with our independently determined X-ray structure of the empty complex. Conformational dynamics present in the empty MHC-I are stabilized by TAPBPR in a peptide-deficient complex, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized by the chaperoned groove according to the global stability of the final pMHC-I product, and anneal in a native-like conformation. Our results demonstrate an inverse relationship between MHC-I occupancy by peptide and the affinity of TAPBPR for such pMHC-I molecules, where the lifetime of transiently bound peptides controls the dynamic regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. We further discuss the role of protein dynamics in shaping chaperone specificity towards different human and murine class-I MHC allotypes, and present the high-resolution cryoEM structure of a human A*02/TAPBPR complex with novel insights into the antigen editing mechanism. These results suggest a similar mechanism for the editing function of tapasin in the peptide-loading complex, and provide a molecular blueprint for the design of novel chaperones with tailored antigen editing functions.

Function of the cargo sorting dileucine motif in a cytomegalovirus immune evasion protein.

As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the β2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.

Transcripts expressed in cytomegalovirus latency coding for an antigenic IE/E phase peptide that drives "memory inflation".

Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.

Evidence of functional Cd94 polymorphism in a free-living house mouse population.

The CD94 receptor, expressed on natural killer (NK) and CD8+ T cells, is known as a relatively non-polymorphic receptor with orthologues in humans, other primates, cattle, and rodents. In the house mouse (Mus musculus), a single allele is highly conserved among laboratory strains, and reports of allelic variation in lab- or wild-living mice are lacking, except for deficiency in one lab strain (DBA/2J). The non-classical MHC-I molecule Qa-1b is the ligand for mouse CD94/NKG2A, presenting alternative non-americ fragment of leader peptides (Qa-1 determinant modifier (Qdm)) from classical MHC-I molecules. Here, we report a novel allele identified in free-living house mice captured in Norway, living among individuals carrying the canonical Cd94 allele. The novel Cd94LocA allele encodes 12 amino acid substitutions in the extracellular lectin-like domain. Flow cytometric analysis of primary NK cells and transfected cells indicates that the substitutions prevent binding of CD94 mAb and Qa-1b/Qdm tetramers. Our data further indicate correlation of Cd94 polymorphism with the two major subspecies of house mice in Europe. Together, these findings suggest that the Cd94LocA/NKG2A heterodimeric receptor is widely expressed among M. musculus subspecies musculus, with ligand-binding properties different from mice of subspecies domesticus, such as the C57BL/6 strain.

Mutational Landscape of the Transcriptome Offers a Rich Neoantigen Resource for Immunotherapy of Myeloproliferative Neoplasms

BackgroundMyeloproliferative Neoplasms (MPN) encompass several disease subgroups (ET, PV, and PMF) with distinct phenotypic and clinical features. Although the landscape of somatic mutations in MPN has been mapped in detail, the current therapies show limited efficacy in eliminating MPN cells. Immunotherapy for MPN have been suggested as an alternative to standard therapies, however, a broader view on the neoantigen landscape and MHC class I (MHCI) restriction of putative MPN neoantigens is missing.AimsWe aimed to map the mutation and neoantigen landscape of MPN by analyzing whole transcriptome data and examine the applicability of targeted immunotherapy by analyzing co-occurrence of specific neoantigens with high affinity MHCI molecules in individual patients.MethodsWe have performed whole transcriptome sequencing of granulocytes for 113 MPN patients (32 ET, 17 PV, 55 PMF, and 9 post-MPN AML) and 15 controls. We implemented customized and established algorithms for genome wide fusion gene discovery, variant calling on 87 myeloid relevant genes, detection of splicing abnormalities and inferred these mutation classes directly from RNA-seq data for each patient. We extracted HLA genotypes from the same RNA-seq dataset. We examined the impact on protein sequence for each mutation class and predicted peptide affinities to MHCI. Top predicted peptide:MHCI interactions were validated in vitro using the UV-induced peptide exchange method.ResultsFollowing data processing and filtering, we identified a total of 13 gene fusions, 231 non-synonymous SNVs, and 21 Indels in 106/113 patients. Fusions were validated with Sanger sequencing, for SNVs and Indels, we re-sequenced DNA of 77 patients using a TruSight targeted sequencing panel. An unexpected high frequency of SF3B1 (0% ET, 14.6% PMF, 0% PV) hotspot mutations (K666N/R/T, K700E) was identified. We performed differential splice junction analysis comparing SF3B1 mutated and non-mutated patients and found 895 significantly differentially spliced junctions (750/895 were mediated through alternative 3'splicing). For each junction we calculated the percent splice in value (PSI) expressing the relative abundance of the alternative junction. We identified 20 junctions with retained intronic sequences with PSI>20%. For each of the 20 splicing defects we evaluated the impact of intron retention on the protein sequence. These splicing defects caused insertion of novel amino acids either in frame or through frameshifts. From all the mutations detected, we generated a virtual peptide library. In 113 patients, we detected 541 patient specific peptides predicted to bind patients' own MHCI proteins of which 102 had a predicted strong and 439 weak binding affinity. The recurrent CALR frameshifts and MPL-W515K/L/A mutations were also a rich resource for potential neoantigens due to their unique property to bind many shared common MHCI molecules (42, and 17 predicted for CALR and MPL, respectively). SF3B1 mutated patients had the highest number of predicted recurrent neoantigens with an average of 38 weak and 16 strong peptide/MHCI pairs. In total, we could identify 149 unique neoantigens covering 62% of MPN patients as a potential target in personalized cancer immunotherapy approaches. Of the 149 predicted unique neoantigens we tested 35 top ranking peptides for binding to recombinant MHCI monomers. We validated 70% of these peptides to be strong MHCI binders.ConclusionsOur results provide a framework for systematic mining of neoantigens from different mutation classes using RNA analysis. The data presented in this study may serve as a resource for development of personalized vaccines or adoptive cell-base therapies, in particular for PMF patients positive for CALR, MPL and SF3B1 mutations. DisclosuresRosebrock:MyeloPro Diagnostics and Research GmbH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Gisslinger:Takeda: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Shire: Honoraria. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership.

Stochastic modeling of tumor progression and immune evasion

It is now well-established that the host’s adaptive immune system plays an important role in identifying and eliminating cancer cells in much the same way that intracellular pathogens are cleared during an adaptive immune response to infection. From a therapeutic standpoint, the adaptive immune system is unique in that it can co-evolve alongside a developing tumor. Tumor acquisition of immune evasive phenotypes, such as class-I MHC down-regulation, remains a major limitation of successful T-cell immunotherapy. Here, we consider a population dynamical model coupling tumor and adaptive immune compartments in order to study the dynamics and survival of an evolving threat when faced with adaptive immune pressure. We demonstrate that predicted optimal growth strategies depend on whether or not the threat may acquire an immune-evasive phenotype as well as the mode of immune detection. We parameterize adaptive immune functioning by T-cell turnover and repertoire diversity and predict that decreases in the latter quantity which occur in advanced age may substantially affect the ability to recognize, and therefore control, an immune evasive threat like cancer. This framework recapitulates general features of age-dependent AML incidence, thereby providing a probable association between cancer frequency and adaptive immune functioning. Lastly, we quantify therapeutic efficacy of adjuvant immunotherapeutic strategies, and predict their benefits and limitations with regard to handling immune evasion. Our model generates survival behavior consistent with known growth-dependent characteristics, and serves as a first attempt at modeling stochastic cancer evolution alongside an adaptive immune compartment.

TAP-Dependent and -Independent Peptide Import into Dendritic Cell Phagosomes.

Cross-presentation of phagocytosed Ags by MHC class I (MHC-I) molecules is thought to involve transport of cytosolic peptides into dendritic cell phagosomes, mediated by TAP transporters recruited from the endoplasmic reticulum. However, because pure and tightly sealed phagosomes are difficult to obtain, direct evidence for peptide transport into phagosomes has remained limited. Moreover, the parameters determining peptide uptake by, and survival in, phagosomes remain little characterized. In this study, we monitored peptide import into phagosomes by flow cytometry using two types of fluorescent reporter peptides, one of which directly bound to intraphagosomal beads. We observed that a peptide with high TAP affinity is imported into phagosomes in a TAP- and ATP-dependent manner, as expected. However, surprisingly, import of the OVA peptide SIINFEKL, a CD8+ T cell epitope frequently used to study cross-presentation, is ATP-dependent but substantially TAP-independent. The half-life of both reporter peptides is shortened by enhanced phagosome maturation triggered by TLR signaling. Conversely, formation of complexes with MHC-I molecules enhances peptide accumulation in phagosomes. Collectively, these results confirm that TAP can import peptides into phagosomes, but they suggest that some peptides, including the popular SIINFEKL, can enter phagosomes also via a second unknown energy-dependent mechanism. Therefore, the frequently reported TAP dependence of cross-presentation of phagocytosed OVA may principally reflect a requirement for recycling MHC-I molecules rather than SIINFEKL import into phagosomes via TAP.

Cell-Extrinsic MHC Class I Molecule Engagement Augments Human NK Cell Education Programmed by Cell-Intrinsic MHC Class I

The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by "self" MHC classImolecules in a process called "education." In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA)mediate NK cell education. In HLA-B(∗)27:05(+) transgenicmice and in patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34(+) stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1(+) NK cells gained reactivity after adoptive transfer to HLA-B(∗)27:05(+) mice or bone marrow chimeric mice where HLA-B(∗)27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell-intrinsic HLA in maintaining NK cell education.

The comings and goings of MHC class I molecules herald a new dawn in cross-presentation.

MHC class I (MHC-I) molecules are the centerpieces of cross-presentation. They are loaded with peptides derived from exogenous sources and displayed on the plasma membrane to communicate with CD8 T cells, relaying a message of tolerance or attack. The study of cross-presentation has been focused on the relative contributions of the vacuolar versus cytosolic pathways of antigen processing and the location where MHC-I molecules are loaded. While vacuolar processing generates peptides loaded onto vacuolar MHC-I molecules, how and where exogenous peptides generated by the proteasome and transported by TAP meet MHC-I molecules for loading has been a matter of debate. The source and trafficking of MHC-I molecules in dendritic cells have largely been ignored under the expectation that these molecules came from the Endoplasmic reticulum (ER) or the plasma membrane. New studies reveal a concentrated pool of MHC-I molecules in the endocytic recycling compartment (ERC). These pools are rapidly mobilized to phagosomes carrying microbial antigens, and in a signal-dependent manner under the control of Toll-like receptors. The phagosome becomes a dynamic hub receiving traffic from multiple sources, the ER-Golgi intermediate compartment for delivering the peptide-loading machinery and the ERC for deploying MHC-I molecules that alert CD8 T cells of infection.

Characterizing the anti-tumor response of T cell receptor gene-modified invariant natural killer T cells

Abstract Human invariant NKT cells represent a distinct lymphocyte population that co-express an invariant antigen receptor and NK surface markers. Classic NKT cells express a restricted T cell receptor that recognizes lipids, such as α-Galactosylceramide (α-GalCer), in the context of CD1d. Activated iNKT cells produce a high level of cytokines, such as IFN- γ, thereby bridging innate and acquired immunity through the activation of other effector cells including dendritic cells (DCs), CD8 T cells, and NK cells. One major hurdle to studying the anti-tumor activity of iNKT cells is that they do not recognize antigens presented by classical MHC molecules and they have unknown tumor antigen specificity. Therefore, we expressed the MHC class I-restricted TIL 1383I TCR in human NKT cells to characterize their biology and tumor antigen-specific response. The 1383I TCR is a CD8 independent, high affinity TCR that recognizes the melanoma antigen, tyrosinase, presented by HLA-A2. Here, we report the expression and function of TIL 1383I TCR transduced human iNKT cells. iNKT cells stimulated with α-GalCer- pulsed DCs are transduced with the retroviral vector encoding the TIL1383I TCR. After confirming TIL 1383I TCR expression in iNKT cells by flow cytometry, we assess their anti-tumor activity and polyfunctionality by intracellular cytokine staining. TIL 1383I TCR transduced iNKT cells produced IFN-γ in response to tyrosinase loaded T2 cells and HLA-A2+/tyrosinase+ melanoma. These findings indicate that genetically engineered iNKT cells are useful to study their biology in the context of antigen specificity, as well as to generate new effector populations for the treatment of cancer patients.

NLRC5 elicits antitumor immunity by enhancing processing and presentation of tumor antigens to CD8+ T lymphocytes

ABSTRACTCancers can escape immunesurveillance by diminishing the expression of MHC class-I molecules (MHC-I) and components of the antigen-processing machinery (APM). Developing new approaches to reverse these defects could boost the efforts to restore antitumor immunity. Recent studies have shown that the expression of MHC-I and antigen-processing molecules is transcriptionally regulated by NOD-like receptor CARD domain containing 5 (NLRC5). To investigate whether NLRC5 could be used to improve tumor immunogenicity, we established stable lines of B16-F10 melanoma cells expressing NLRC5 (B16-5), the T cell co-stimulatory molecule CD80 (B16-CD80) or both (B16-5/80). Cells harboring NLRC5 constitutively expressed MHC-I and LMP2, LMP7 and TAP1 genes of the APM. The B16-5 cells efficiently presented the melanoma antigenic peptide gp10025–33 to Pmel-1 TCR transgenic CD8+ T cells and induced their proliferation. In the presence of CD80, B16-5 cells stimulated Pmel-1 cells even without the addition of gp100 peptide, indicating that NLRC5 facilitated the processing and presentation of endogenous tumor antigen. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that the NLRC5-expressing tumor cells elicited antitumor immunity. Following intravenous injection, B16-5 and B16-5/80 cells formed fewer lung tumor foci compared to control cells. In mice depleted of CD8+ T cells, B16-5 cells formed large subcutaneous and lung tumors. Finally, immunization with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Collectively, our findings indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity.

CD8(+) Tcells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) Tcells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) Tcells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) Tcells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) Tcells. Such mechanism may reduce the chances for pathogen immune evasion.