Murine Major Histocompatibility Complex Research Articles

Mice humanized for MHC and hACE2 with high permissiveness to SARS-CoV-2 omicron replication

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike. The murine ACE2 does not interact efficiently with SARS-CoV-2 Spike and therefore the laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in “HHD-DR1” background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. These new hACE2 transgenic strains display high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants, while the previously available B6.K18-ACE22Prlmn/JAX mice have been reported to be poorly susceptible to infection with Omicron. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral-based COVID-19 vaccine.

Development of mouse models with restricted HLA-B∗57:01 presentation for the study of flucloxacillin-driven T-cell activation and tolerance in liver injury

Flucloxacillin (FLX)-induced liver injury is immune-mediated and highly associated to HLA-B∗57:01 expression. Host factors leading to drug-induced liver injury are not yet well understood. Characterize invivo immune mechanisms determining the development of CD8+ T cells reactive to FLX in animals expressing the risk human leukocyte antigen (HLA) allotype. HLA-B∗57:01 transgenic mice (Tg) or Tg strains with H2-KbDb knockout (Tg/KO) or H2-KbDb/PD-1 double knockout (Tg/DKO) were treated with drug and/or anti-CD4 antibody. Drug-induced liver injury was evaluated on the basis of liver enzyme and histologic changes at day 10 of treatment. FLX-reactive CD8+ T cells were characterized invitro by release of effector molecules on drug restimulation, gene expression, and flow cytometry analysis, and functionality tested for hepatic cytotoxicity. CD8+ T-cell responses to FLX in Tg were dependent on both HLA and mouse major histocompatibility complex I presentation and invivo priming. Eliminating H2-KbDb in Tg/KO to allow exclusive presentation of FLX by HLA resulted in a less robust drug-specific CD8+T-cell response unless CD4+ cells, including regulatory T cells, were depleted. Treatment of Tg/KO with anti-CD4 antibody and FLX led to subclinical liver inflammation associated with an increase in PD1+CD8+ T cells in the lymphoid organs and liver. Impaired PD-1 expression in Tg/DKO led to liver histopathologic and transcriptional alterations but without hepatic enzyme elevations. Moreover, effector lymphocytes accumulated in the liver and showed FLX-dependent hepatic cytotoxicity invitro when tolerogenic liver cells were depleted. In our invivo models, FLX primes CD8+ T cells to recognize drug presented by HLA-B∗57:01 and murine major histocompatibility complex I. HLA-B∗57:01-dependent CD8+ T-cell reaction to FLX is limited by the presence of CD4+ cells, presumably regulatory T cells, and PD-1 expression. Tolerogenic hepatic cells limit clinical disease through PD-L1 or additional unexplored mechanisms.

Prevention of acute GVHD using an orthogonal IL-2/IL-2Rβ system to selectively expand regulatory T cells in vivo

AbstractAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow (BM) failure syndromes. Graft-versus-host disease (GVHD) remains a leading cause of morbidity posttransplant. Regulatory T cell (Treg) therapies are efficacious in ameliorating GVHD but limited by variable suppressive capacities and the need for a high therapeutic dose. Here, we sought to expand Treg in vivo by expressing an orthogonal interleukin 2 receptor β (oIL-2Rβ) that would selectively interact with oIL-2 cytokine and not wild-type (WT) IL-2. To test whether the orthogonal system would preferentially drive donor Treg expansion, we used a murine major histocompatibility complex–disparate GVHD model of lethally irradiated BALB/c mice given T cell–depleted BM from C57BL/6 (B6) mice alone or together with B6Foxp3+GFP+ Treg or oIL-2Rβ–transduced Treg at low cell numbers that typically do not control GVHD with WT Treg. On day 2, B6 activated T cells (Tcons) were injected to induce GVHD. Recipients were treated with phosphate-buffered saline (PBS) or oIL-2 daily for 14 days, then 3 times weekly for an additional 14 days. Mice treated with oIL-2Rβ Treg and oIL-2 compared with those treated with PBS had enhanced GVHD survival, in vivo selective expansion of Tregs, and greater suppression of Tcon expansion in secondary lymphoid organs and intestines. Importantly, oIL-2Rβ Treg maintained graft-versus-tumor (GVT) responses in 2 distinct tumor models (A20 and MLL-AF9). These data demonstrate a novel approach to enhance the efficacy of Treg therapy in allo-HSCT using an oIL-2/oIL-2Rβ system that allows for selective in vivo expansion of Treg leading to GVHD protection and GVT maintenance.

S163: EFFICACY OF JAK 1/2 INHIBITION IN MURINE IMMUNE BONE MARROW FAILURE

Background: Immune aplastic anemia (AA) is a severe blood disorder characterized by cytotoxic T-lymphocyte mediated stem cell destruction. Therapies including hematopoietic stem cell transplant and immunosuppression are effective but entail costs and risks, and are not effective or possible in all patients. The Janus Kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T cell activation and inhibits production of interferon gamma and tumor necrosis factor alpha in models of graft-versus-host disease. Aims: Assess RUX in murine immune AA for potential therapeutic benefit. Methods: In a murine major histocompatibility complex mismatched C67BL/6(B6) to CByB6F1 lymph node (LN) cell infusion AA model, and a C.B10 minor histocompatibility antigen mismatched B6 to C.B10 LN cell infusion AA model, RUX was administered as a food additive (Rux-chow), which achieves therapeutic levels in 48-72 hours after administration. Control BMF mice received chow without RUX. Animals were fed with Rux-chow two days before LN cell infusion as a prophylaxis (BMF+RUXD-2), or two days after LN cell infusion as therapy (BMF+RUXD+2). Recipient mice were either bled and euthanized at day 14 following LN infusion to collect tissues or were kept for 56 days to record animal survival. Blood counts were measured biweekly. Samples for flow cytometry, histology, and gene expression assays were collected. Results: In both AA murine models, RUX attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, and prevented mortality when used either prophylactically or therapeutically. Treated mice had significantly higher blood counts (neutrophils, red blood cells, hemoglobin, platelets) as well as bone marrow (BM) and RBM (residual BM, excluding T-cells), and cellularity relative to control BMF mice. RUX suppressed infiltration, proliferation and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. All mice who received RUX were alive at 56 days while control BMF mice all died (Figure 1). With the exception of two mice with low RBC at day 56, discontinuing Rux-chow at day 28 or day 42 did not affect animal blood counts measurements, nor survival. Gene expression in mice who received RUX revealed downregulated T cell function and JAK/STAT pathway-related genes (Stat1, Stat3, Stat4, Fas, Ly6a, Infg, Gzmb, Gzma, Gzmk, Infgr1, Il2rb, Il2rg, and Lag3). On network analysis of differentially expressed genes in downregulated pathways, Stat1 and Ifn-g genes were at the center of the network, connecting immune responses and cell cycle pathways. When toxicity was assessed, RUX exerted modest suppression of lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice, but the drug showed impressive clinical efficacy despite this. Image:Summary/Conclusion: RUX showed striking therapeutic efficacy, improving blood counts and prolonging survival in two different BMF murine models. In patients, clonal T cell expansion and activation, IFN-g and TNF-α upregulation, and Fas mediated cell death, lead to severe BM destruction and the development of AA. Our study demonstrated that JAK 1/2 inhibition with RUX produced its suppressive effects by inhibiting T cell activation, reducing inflammatory cytokine secretion, and limiting FasL/Fas-mediated BM destruction. Given these results, JAK 1/2 inhibition with RUX is an exciting and novel potential therapy for BMF patients based on a well characterized mechanism of action.

Combinatorial in silico and in vivo evaluation of immune response elicitation by the affibody ZHER2

Due to the high affinity for binding to target molecules and also other unique attributes, affibodies have a great potential to be used in immunotherapeutic and diagnostic approaches. However, the possibility of undesirable immune response is still a great concern. In the current study, we investigated the possible antigenicity, allergenicity and cytotoxicity of the HER2-targeting affibody ZHER2. The binding affinity of potential epitopes of the affibody to murine major histocompatibility complex (MHC) molecules was investigated by immunoinformatics tools and docking approaches. The possible interaction of ZHER2 with human leukocyte antigens HLA-DP, HLA-DM, HLA-DQ and HLA-DR was also studied by protein-protein docking. Additionally, the synthesized affibody gene was expressed and the protein was purified for boosted immunization of Balb/c mice. Induced secretion of IFN-γ, IL-2, IL-4 and IL-10, and total serum IgG were assessed in the immunized mice. Furthermore, MTT cell viability test was performed to evaluate the cytotoxicity of ZHER2 in splenocytes of the treated mice. In silico analyses showed the possible induction of the immune response by ZHER2. While the affibody could elicit the secretion of cellular immune cytokines, it could not induce a significant humoral response in the treated mice and did not show any cytotoxic effects on the exposed splenocytes. These findings explain the practicability of ZHER2 for therapeutic and in vivo diagnostic usages, though its ubiquitous application may need more studies.

A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes.

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.

Combining Well-Tempered Metadynamics Simulation and SPR Assays to Characterize the Binding Mechanism of the Universal T-Lymphocyte Tetanus Toxin Epitope TT830-843.

Peptide TT830-843 from the tetanus toxin is a universal T-cell epitope. It helps in vaccination and induces T-cell activation. However, the fine molecular interaction between this antigen and the major histocompatibility complex (MHC) remains unknown. Molecular analysis of its interaction with murine MHC (H-2) was proposed to explore its immune response efficiency. Molecular dynamics simulations are important mechanisms for understanding the basis of protein-ligand interactions, and metadynamics is a useful technique for enhancing sampling in molecular dynamics. SPR (surface plasmon resonance) assays were used to validate whether the metadynamics results are in accordance with the experimental results. The peptide TT830-843 unbinding process was simulated, and the free energy surface reconstruction revealed a detailed conformational landscape. The simulation described the exiting path as a stepwise mechanism between progressive detachment states. We pointed out how the terminus regions act as anchors for binding and how the detachment mechanism includes the opening of α-helices to permit the peptide's central region dissociation. The results indicated the peptide/H-2 receptor encounter occurs within a distance lesser than 27.5 Å, and the encounter can evolve to form a stable complex. SPR assays confirmed the complex peptide/H-2 as a thermodynamically stable system, exhibiting enough free energy to interact with TCR on the antigen-presenting cell surface. Therefore, combining in silico and in vitro assays provided significant evidence to support the peptide/H-2 complex formation.

Natural Loss-of-Function Mutations in Qa2 and NCF1 Cause the Spread of Mannan-Induced Psoriasis

A basis for the genetic predisposition to psoriasis is a single locus, PSORS1, within the major histocompatibility complex I region. This murine major histocompatibility complex locus encodes nonclassical molecules such as Qa2. We hypothesized that a natural loss-of-function variant of Qa2 gene clusters promotes psoriasis. In this study, we have developed a mannan-induced psoriasis model with the double deficiency of Qa2 and ROS owing to natural mutations of Qa2 gene clusters and the Ncf1 gene in the C57BL/6 background, respectively. We report three key findings in mice with mannan-induced psoriasis. A complete deficiency of Qa2 resulted in the expansion of IL-17‒producing γδ T cells and group 3 innate lymphoid cells in draining lymph nodes, leading to ear psoriasis. A single copy of Qa2-encoding genes was enough to protect against mannan-induced psoriasis, and such a protection was erased by a mutated Ncf1. Double defects with Qa2 and Ncf1 elicited a spread of exaggerated ear psoriasis to the nails, and the deficiency of γδ T cells reduced the severity of nail psoriasis. Collectively, these findings in mice provide evidence for the importance of Ncf1 mutations and Qa2 gene clusters, possibly corresponding to the PSORS1 locus in the spread of psoriasis.

Abstract 432: Development of a novel engineered B2M mouse strain on B-NDG background that specifically eliminates MHC I expression while retaining normal FcRn-mediated antibody retention

Abstract Severely immunodeficient mouse strains, such as NOD.Cg-Prkdcscid Il2rg-/-, have been widely used to evaluate human immune responses, including applications in immuno-oncology in recent years. However, when human peripheral blood mononuclear cells (PBMCs) are engrafted in these mice, severe acute xenogeneic graft versus host disease (GvHD) arises due to engagement of human T cell receptors (TCRs) and murine major histocompatibility complex (MHC) I and II. GvHD shortens animal life span and results in only a brief window of evaluation of human immune cell functions in such mouse models. Efforts have been undertaken to reduce human PBMC-induced GvHD and to subsequently extend the window of human immunity assessment by eliminating mouse MHC I and MHC II. While these MHC deficient mice show few symptoms of GvHD, the use of beta-2-microglobulin (B2M) null allele in order to eliminate expression of all MHC I molecules resulted in an unintended consequence in that these mice had rapid antibody clearance. This is because B2M is also associated with neonatal Fc receptor (FcRn) and is important to FcRn-mediated in vivo antibody recycling and retention. Low half-lives of antibodies in B2M null mice render them unsuitable for antibody efficacy assessment. To overcome this undesired pharmacokinetic (PK) limitation of B2M null allele, while retaining its desired effect on eliminating MHC I expression and alleviating GvHD, we engineered a B2M knock-out strain in the background of B-NDG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/Bcgen) that was deficient in MHC I expression but not antibody retention. In present study, we describe genetic, phenotypic, pharmacokinetic, and functional characterizations of the resulting mouse strain, which we believe will be a valuable addition to the collection of severely immunodeficient mouse models that permit extended and appropriate interrogation of antibody therapeutics on human immune cells. Citation Format: W. Frank An, Lan Gao, Xiaoen Wang, Shruti Sharma, Kaiqi Li, Xiqiang Gao, Chengzhang Shang, Yuting Hu. Development of a novel engineered B2M mouse strain on B-NDG background that specifically eliminates MHC I expression while retaining normal FcRn-mediated antibody retention [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 432.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits Tcell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in Tcells remains enigmatic. Here weinvestigated how PD-1 affects transcriptome changes induced by Tcell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of Tcell populations.

Natural Spleen Cell Ligandome in Transporter Antigen Processing-Deficient Mice.

Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using high-throughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P1 position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.

Distinct Immune Regulatory Potential of Invariant Natural Killer T (iNKT) Cell Subsets: iNKT2 and iNKT17, but Not iNKT1, Protect from Graft-Versus-Host-Disease

Invariant natural killer T (iNKT) cells can potently inhibit graft-versus-host-disease (GVHD) in animal models through the production of Interleukin-4. Once considered a homogeneous population, it is now well established that murine iNKT cells differentiate during thymic development into three distinct sublineages, distinguished based on the expression of transcription factors and effector molecules: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th17-like iNKT (iNKT17) cells. In this study, we investigated the immune regulatory potential of iNKT1, iNKT2 and iNKT17 cell subsets. In order to identify surface molecules whose expression would allow us to isolate viable iNKT1, iNKT2 and iNKT17 cells, we performed single cell RNA sequencing analysis on murine thymic iNKT cells isolated from FVB/N mice by flow cytometry based on PBS-57-CD1d-Tetramer staining. The results of this analysis together with previous knowledge allowed us to develop a sorting strategy to isolate iNKT1 (ICOS- PD1- CD27+ CD24-), iNKT2 (ICOS+ PD1+ CD27+ CD4+ CD24-), iNKT17 (ICOS+ PD1+ CD4- CD27- CD24-) cells, whose purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORgT. To assess the immune regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)–mismatched bone marrow transplantation model. Lethally irradiated BALB/c (H-2Kd) mice were injected intravenously with T cell depleted-bone marrow (TCD-BM) cells and conventional CD4 and CD8 T cells (Tcon, 10e6 cells/mouse) from FVB/N (H-2kq) mice. Additionally, 5 × 10e4 sorted iNKT1, iNKT2 or iNKT17 cells from FVB/N donors were injected the same day. A significant survival benefit was observed when iNKT2 (p=0.0166) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 × 10e4 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. In summary, we demonstrate here that only iNKT2 and iNKT17 cells mitigated GVHD, whereas iNKT1 had no detectable immune regulatory potential. To our knowledge, this is the first study to show functional differences between iNKT sublineages, indicating that iNKT1, iNKT2 and iNKT17 cells have diverse functions and providing new biological insights that will be useful for developing iNKT cell-based cell therapies.

Sleep restriction alters the temporal expression of major histocompatibility complex class II molecules in murine lymphoid tissues

Inadequate sleep is a major health concern of modern societies in view of the increased morbidity and mortality rates from physiological disturbances, including compromised adaptive immune responses. Many studies investigated the effect sleep restriction (SR) on the normal immune response in terms of leukocyte number and circulating cytokine and T helper cell (Th) profiles, but none considered the major histocompatibility complex (MHC), namely class II molecules. As no information exists about the normal temporal expression of MHC class II, the present study aimed at understanding how SR affects the adaptive immune response via altering the 1) normal daily expression profile and 2) overall constitutive levels of murine MHC class II by leukocytes’ isolates from spleen and axillary lymph nodes. Male C57BL/6 mice were acclimatized to 12:12 light/dark cycle (lights on at 0700, corresponding to Zeitgeber time (ZT) 0) for a week before splitting into 2 groups: control (C) and SR (exposed to a 12 and 18 h of activity, respectively). SR was carried for one week before lymphoid tissues from both C and SR mice were sampled at the following time points: ZT0, ZT5, ZT10, ZT13, and ZT18. Spleen and lymph node cells were assessed for leukocyte number and MHC class II expression at the preselected time points using flow cytometry. SR resulted in a 21% decrease in granulocyte and 24% increase in agranulocyte numbers. MHC class II expression in both lymphoid tissues of C mice varied synchronously across the preselected times of day; they were relatively high just prior to activity onset and later in this period. Comparatively, the diurnal protein profile was altered in both lymphoid tissues of SR: 1) the rise of MHC class II expression during the rest period occurred 4–5 hours earlier and 2) the cyclical pattern during the activity period was blunted and protein expression was maintained at relatively high levels. MHC class II expression was higher in the lymph nodes and lower in the spleen of SR than C, though these differences did not reach statistical significance. In SR; however, the average protein level was significantly higher in lymph nodes than spleen (376.0 + 184.9 vs 188.6 + 42.2, respectively; p = .002) and higher in the granulocytes relative to agranulocytes. Our findings provide empirical evidence of a constitutive diurnal expression pattern for MHC class II molecules that is prone to upregulation upon SR, namely in lymph nodes, and specifically expressed by granulocytes. We speculate that, in mice, chronic sleep deprivation would further dysregulate MHC class II expression that might result in aberrant T cell activation with probable immune-associated pathological diseases such as allergies, autoimmunity, and tumors.

Indoles derived from intestinal microbiota act via type I interferon signaling to limit graft-versus-host disease

AbstractThe intestinal microbiota in allogeneic bone marrow transplant (allo-BMT) recipients modulates graft-versus-host disease (GVHD), a systemic inflammatory state initiated by donor T cells that leads to colitis, a key determinant of GVHD severity. Indole or indole derivatives produced by tryptophan metabolism in the intestinal microbiota limit intestinal inflammation caused by diverse stressors, so we tested their capacity to protect against GVHD in murine major histocompatibility complex–mismatched models of allo-BMT. Indole effects were assessed by colonization of allo-BMT recipient mice with tryptophanase positive or negative strains of Escherichia coli, or, alternatively, by exogenous administration of indole-3-carboxaldehyde (ICA), an indole derivative. Treatment with ICA limited gut epithelial damage, reduced transepithelial bacterial translocation, and decreased inflammatory cytokine production, reducing GVHD pathology and GVHD mortality, but did not compromise donor T-cell-mediated graft-versus-leukemia responses. ICA treatment also led to recipient-strain-specific tolerance of engrafted T cells. Transcriptional profiling and gene ontology analysis indicated that ICA administration upregulated genes associated with the type I interferon (IFN1) response, which has been shown to protect against radiation-induced intestinal damage and reduce subsequent GVHD pathology. Accordingly, protective effects of ICA following radiation exposure were abrogated in mice lacking IFN1 signaling. Taken together, these data indicate that indole metabolites produced by the intestinal microbiota act via type I IFNs to limit intestinal inflammation and damage associated with myeloablative chemotherapy or radiation exposure and acute GVHD, but preserve antitumor responses, and may provide a therapeutic option for BMT patients at risk for GVHD.

Invariant Natural Killer T Cell Subsets Have Diverse Functions: iNKT2 and iNKT17 Protect from Graft-Versus-Host-Disease, Whereas iNKT1 Have Antitumor Potential

Invariant natural killer T (iNKT) cells are an interesting subpopulation of T cells that can potently inhibit graft-versus-host-disease (GVHD) through the production of Interleukin 4 (IL-4), while also carrying anti-tumor potential (Leveson-Gower et al. Blood. 2011;117:3220-9; Schneidawind et al. Blood. 2014;124:3320-8). Murine iNKT cells differentiate during thymic development into three distinct sublineages, named according to the classification of conventional T cells: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th-17 like iNKT (iNKT17) cells (Brennan et al. Nat Rev Immunol. 2013; 13:101-1). In this study we investigated the immune regulatory and anti-tumor potential of iNKT1, iNKT2 and iNKT17 cell subsets.Thymic iNKT1 cells, iNKT2 cells and iNKT17 cells were isolated from 8-10 week-old FVB/NJ mice by flow cytometry based on PBS-57-CD1d-Tetramer and a combination of cell surface molecules (iNKT1: ICOS- PD1- CD27+ CD24-; iNKT2: ICOS+ PD1+ CD27+ CD4+ CD24-; iNKT17: ICOS+ PD1+ CD4- CD27- CD24-) and purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORγT. RNA sequencing analysis determined that iNKT1 cells were the main subset expressing proinflammatory and cytotoxic genes, such as Interferon gamma (IFN-γ), Fas Ligand (FasL), Perforin, and Granzyme B (Gzmb), whereas IL-4 was expressed by iNKT2 cells and, at a lesser extent, by iNKT17 cells. To assess the immuno-regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)-mismatched bone marrow transplantation model. BALB/c (H-2Kd) recipients were lethally irradiated with 8.8 Gy; on the same day, 4 x 106 TCD-BM cells and 1 x 106 conventional CD4 and CD8 T cells (Tcon) from FVB/NJ (H-2kq) mice were injected intravenously. Additionally, 5 x 104 purified iNKT1, iNKT2 or iNKT17 cell subsets from FVB donors were injected. A significant survival benefit was observed when iNKT2 (p=0.017) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 x104 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. As iNKT1, iNKT2 and iNKT17 have a very different gene expression profile, we tested the ability of the sublineages to kill a B-cell lymphoma cell line transduced to express high levels of CD1d (A20-CD1d) in vitro and found that iNKT1 cells killed A20-CD1d cells significantly better than iNKT2 (p=0.006) or iNKT17 (p=0.0001) cells. These findings are in line with the sequencing data mentioned above, showing that iNKT1 cells express a more inflammatory phenotype.In summary, we demonstrate here that only iNKT2 and iNKT17 cells protect from GVHD, whereas iNKT1 cells have cytotoxic function. To our knowledge, this is the first study to show functional differences between the iNKT sublineages, suggesting that iNKT1, iNKT2 and iNKT17 cells have diverse functions. Therefore, these data provide new biological insights, which will be useful for developing iNKT cell-based cell therapy. DisclosuresChang:Spring Discovery: Membership on an entity's Board of Directors or advisory committees; Epinomics and Accent Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-β-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

A tissue-based draft map of the murine MHC class I immunopeptidome

The large array of peptides presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC class I immunopeptidome. Although the MHC class I immunopeptidome is ubiquitous in mammals and represents a critical component of the immune system, very little is known, in any species, about its composition across most tissues and organs in vivo. We applied mass spectrometry (MS) technologies to draft the first tissue-based atlas of the murine MHC class I immunopeptidome in health. Peptides were extracted from 19 normal tissues from C57BL/6 mice and prepared for MS injections, resulting in a total number of 28,448 high-confidence H2Db/Kb-associated peptides identified and annotated in the atlas. This atlas provides initial qualitative data to explore the tissue-specificity of the immunopeptidome and serves as a guide to identify potential tumor-associated antigens from various cancer models. Our data were shared via PRIDE (PXD008733), SysteMHC Atlas (SYSMHC00018) and SWATH Atlas. We anticipate that this unique dataset will be expanded in the future and will find wide applications in basic and translational immunology.

The Use of Reverse Vaccinology and Molecular Modeling Associated with Cell Proliferation Stimulation Approach to Select Promiscuous Epitopes from Schistosoma mansoni.

Schistosomiasis remains an important parasitic disease that affects millions of individuals worldwide. Despite the availability of chemotherapy, the occurrence of constant reinfection demonstrates the need for additional forms of intervention and the development of a vaccine represents a relevant strategy to control this disease. With the advent of genomics and bioinformatics, new strategies to search for vaccine targets have been proposed, as the reverse vaccinology. In this work, computational analyses of Schistosoma mansoni membrane proteins were performed to predict epitopes with high affinity for different human leukocyte antigen (HLA)-DRB1. Ten epitopes were selected and along with murine major histocompatibility complex (MHC) class II molecule had their three-dimensional structures optimized. Epitope interactions were evaluated against murine MHC class II molecule through molecular docking, electrostatic potential, and molecular volume. The epitope Sm141290 and Sm050890 stood out in most of the molecular modeling analyses. Cellular proliferation assay was performed to evaluate the ability of these epitopes to bind to murine MHC II molecules and stimulate CD4+ T cells showing that the same epitopes were able to significantly stimulate cell proliferation. This work showed an important strategy of peptide selection for epitope-based vaccine design, achieved by in silico analyses that can precede in vivo and in vitro experiments, avoiding excessive experimentation.

Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

High-resolution analysis of the murine MHC class II immunopeptidome.

The reliable identification of peptides bound to major histocompatibility complex (MHC) class II is fundamental for the study of the host immune response against pathogens and the pathogenesis of autoimmune conditions. Here, we describe an improved methodology combining immuno-affinity enrichment of MHC class II complexes, optimized elution conditions and quadrupole Orbitrap mass spectrometry-based characterization of the immunopeptidome. The methodology allowed the identification of over 1000 peptides with 1% false discovery rate from 10(8) murine A20 lymphoma cells. The study revealed the I-A(d) -specific motif in high resolution after multisequence alignment. The methodology was generally applied to the purification of MHC class II from cell lines and murine spleens. We identified 2963 peptides from BALB/c and 2712 from C57BL/6 mouse spleens. The identification of peptides bound to MHC class II in vitro and in vivo will facilitate the characterization of T-cell specificities, as well as the development of biotherapeutics and vaccines.