MHC Haplotype Research Articles

The 18th International HLA & Immunogenetics workshop project report: Creating fully representative MHC reference haplotypes.

A fundamental endeavor of the International Histocompatibility and Immunogenetics Workshop (IHIW) was assembling a collection of DNA samples homozygous through the MHC genomic region. This collection proved invaluable for assay development in the histocompatibility and immunogenetics field, for generating the human reference genome, and furthered our understanding of MHC diversity. Defined by their HLA‐A, ‐B, ‐C and ‐DRB1 alleles, the combined frequency of the haplotypes from these individuals is ∼20% in Europe. Thus, a significant proportion of MHC haplotypes, both common and rare throughout the world, and including many associated with disease, are not yet represented. In this workshop component, we are collecting the next generation of MHC‐homozygous samples, to expand, diversify and modernize this critical community resource that has been foundational to the field. We asked laboratories worldwide to identify samples homozygous through all HLA class I and/or HLA class II genes, or through whole‐genome SNP genotyping or sequencing, to have extensive homozygosity tracts within the MHC region. The focus is non‐Europeans or those having HLA haplotypes less common in Europeans. Through this effort, we have obtained samples from 537 individuals representing 294 distinct haplotypes, as determined by their HLA class I and II alleles, and an additional 50 haplotypes distinct in HLA class I or II alleles. Although we have expanded the diversity, many populations remain underrepresented, particularly from Africa, and we encourage further participation. The data will serve as a resource for investigators seeking to characterise variation across the MHC genomic region for disease and population studies. [Correction added on [24 July 2024], after first online publication: the Abstract was inadvertently remove and has been reinstated in this version.]

B cell MHC haplotype affects follicular inclusion, germinal center participation and plasma cell differentiation in a mouse model of lupus.

MHC class II molecules are essential for appropriate immune responses against pathogens but are also implicated in pathological responses in autoimmune diseases and transplant rejection. Previous studies have shed light on the systemic contributions of MHC haplotypes to the development and severity of autoimmune diseases. In this study, we addressed the B cell intrinsic MHC haplotype impact on follicular inclusion, germinal center (GC) participation and plasma cell (PC) differentiation in the context of systemic lupus erythematosus (SLE). We leveraged the 564Igi mouse model which harbors a B cell receptor knock-in from an autoreactive B cell clone recognizing ribonuclear components, including double-stranded DNA (dsDNA). This model recapitulates the central hallmarks of the early stages of SLE. We compared 564Igi heterozygous offspring on either H2b/b, H2b/d, or H2d/d background. This revealed significantly higher germinal center (GC) B cell levels in the spleens of H2b/b and H2b/d as compared to H2d/d (p<0.0001) mice. In agreement with this, anti-dsDNA-antibody levels were higher in H2b/b and H2b/d than in H2d/d (p<0.0001), with H2b/b also being higher compared to H2b/d (p<0.01). Specifically, these differences held true both for autoantibodies derived from the knock-in clone and from wild-type (WT) derived clones. In mixed chimeras where 564Igi H2b/b, H2b/d and H2d/d cells competed head-to-head in the same environment, we observed a significantly higher inclusion of H2b/b cells in GC and PC compartments relative to their representation in the B cell repertoire, compared to H2b/d and H2d/d cells. Furthermore, in mixed chimeras in which WT H2b/b and WT H2d/d cells competed for inclusion in GCs associated with an epitope spreading process, H2b/b cells participated to a greater extent and contributed more robustly to the PC compartment. Finally, immature WT H2b/b cells had a higher baseline of BCRs with an autoreactive idiotype and were subject to more stringent negative selection at the transitional stage. Taken together, our findings demonstrate that B cell intrinsic MHC haplotype governs their capacity for participation in the autoreactive response at multiple levels: follicular inclusion, GC participation, and PC output. These findings pinpoint B cells as central contributors to precipitation of autoimmunity.

Deletion of Vβ3+CD4+ T cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T cells.

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vβ3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vβ3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV replication in M3+ Mauritian cynomolgus macaques initiated on ART two weeks post-infection.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Both C57BL/KsJ (H2d haplotype) and CB10-H2 (H2b haplotype) mice are highly susceptible to congenital toxoplasmosis

Congenital toxoplasmosis may cause abortion, neonatal death, or foetal abnormalities. Despite little information from human studies, a genetic influence over congenital disease was demonstrated and, host genome have been implicated to resistance/susceptibility to Toxoplasma gondii infection in both human and mice. It was previously shown that BALB/c mice (H2d) were more resistant to congenital toxoplasmosis than C57BL/6 mice (H2b). However, it is unclear whether these differences are attributable to the MHC haplotype or to other components of the mouse's genetic background. Therefore, in this work, we intend to address this question by investigating the pregnancy outcome in H2d -congenic C57BL/6 mice (C57BL/KsJ-H2d) and H2b-congenic BALB/c mice (CB10-H2-H2b). For this, animals were infected by intragastric route on the first day of pregnancy and examined on days 8 (8dP/8dI) or 18 (18dP/18dI) of gestation and infection. The pregnancy outcome, parasite burden, systemic cytokine profile and antibody response to infection were evaluated. Infected mice showed adverse pregnancy outcomes, in parallel low parasite detection in the uterus/placenta, being that the C57BL/KsJ showed the worst results in relation to CB10-H2 mice. Both mouse lineages showed an increase in IFN-γ and TNF levels systemically on 8dP/8dI and on 18dP/18dI, and C57BL/KsJ showed an increase in IL-6 levels in both gestation/infection periods. Additionally, C57BL/KsJ showed 7- and 7-fold increase in IL-6, 4- and 2.5-fold increase in IFN-γ and, 6- and 4-fold increase in TNF production on 8dP/8dI and 18dP/18dI, respectively in association with 1.5-fold decrease in TGF-β levels on 8dP/8dI compared to CB10-H2 mice. In conclusion, the high IFN-γ and TNF serum levels observed in C57BL/KsJ (H2d) and CB10-H2 (H2b) mice were involved in the poor pregnancy outcomes in congenital toxoplasmosis. In addition, the higher IFN‐γ, IL‐6 and TNF levels detected in C57BL/KsJ in relation to CB10‐H2 mice on 8dP/8dI seem to be related to the genetic background of C57BL/6J mice that may have contributed to the worse pregnancy outcome in this mouse lineage.

Mate genetic similarity affects mating behaviour but not maternal investment in mice

Maternal investment can affect the survival and development of offspring. Here we experimentally investigated in mice, whether females alter implantation rates and pup survival after embryo transfer depending on the genetic similarity with their vasectomised mating partner. We selected the MHC genotype and genetic background of males and paired females either with males that shared the same MHC haplotype and genetic background (CBA/J inbred males, isogenic group), that shared half of the MHC haplotype and genetic background (B6CBAF1 hybrid males, semi-isogenic group), or that had a different MHC haplotype and genetic background (C57BL/6N inbred males, allogenic group). We performed 304 pairings, resulting in 81 vaginal plugs, which confirmed mating. Plug rates were significantly higher in the semi-isogenic group (36.9%) compared to the isogenic group (19.5%), but not the allogenic group (26%). We found no difference in the number of implantation sites, the number of born or surviving pups until weaning, or litter weight or sex ratio between groups. Even though we found a mating bias, we found no difference in maternal investment under laboratory conditions. At least under pathogen-free conditions our study does not provide any evidence for differential maternal investment when females could increase offspring genetic diversity or heterozygosity.

QuickSwitch ™: an MHC tetramer platform for making fast and reliable custom tetramers for T cell staining

Abstract The use of MHC tetramers to identify antigen specific T cells has become widespread among immunologists since the late 90s. Tetramers have become so ubiquitous that many companies and research facilities can ship and deliver some of the most commonly used tetramers within a matter of days. But when it comes to the uncommon tetramers or tetramers with antigenic peptides that have only recently been identified, it may be quite a challenge to find the needed tetramer. In this case, most researchers are left with trying to produce their own tetramer or having another facility do it for them, which can become a lengthy process. In an effort to help those researchers, we have developed the QuickSwitch ™MHC tetramer. This MHC tetramer comes loaded with a place-holder peptide (or exiting peptide) that can be exchanged for most well-binding peptides of a given MHC haplotype. This way, researchers are able to make a tetramer with any peptide they have available in a matter of hours. More importantly, our MHC tetramer platform comes with a means to monitor how much of the available peptide has exchanged with the exiting peptide and quantify the MHC occupancy. This allows the user to not only determine if the peptide in question can make a viable MHC tetramer, but also identify the strong and weak binding peptides for that MHC haplotype. This piece of information can be invaluable in future studies, in particular for vaccine development. MBL International

Improving thymus implantation for congenital athymia with interleukin-7.

Thymus implantation is a recently FDA-approved therapy for congenital athymia. Patients receiving thymus implantation develop a functional but incomplete T cell compartment. Our objective was to develop a mouse model to study clinical thymus implantation in congenital athymia and to optimise implantation procedures to maximise T cell education and expansion of naïve T cells. Using Foxn1 nu athymic mice as recipients, we tested MHC-matched and -mismatched donor thymi that were implanted as fresh tissue or cultured to remove donor Tcells. We first implanted thymus under the kidney capsule and then optimised intramuscular implantation. Using competitive adoptive transfer assays, we investigated whether the failure of newly developed T cells to expand into a complete T cell compartment was because of intrinsic deficits or whether there were deficits in engaging MHC molecules in the periphery. Finally, we tested whether recombinant IL-7 would promote the expansion of host naïve T cells educated by the implanted thymus. We determined that thymus implants in Foxn1 nu athymic mice mimic many aspects of clinical thymus implants in patients with congenital athymia. When we implanted cultured, MHC-mismatched donor thymus into Foxn1 nu athymic mice, mice developed a limited T cell compartment with notably underdeveloped naïve populations and overrepresented memory-like T cells. Newly generated T cells were predominantly educated by MHC molecules expressed by the donor thymus, thus potentially undergoing another round of selection once in the peripheral circulation. Using competitive adoptive transfer assays, we compared expansion rates of T cells educated on donor thymus versus T cells educated during typical thymopoiesis in MHC-matched and -mismatched environments. Once in the circulation, regardless of the MHC haplotypes, T cells educated on a donor thymus underwent abnormal expansion with initially more robust proliferation coupled with greater cell death, resembling IL-7 independent spontaneous expansion. Treating implanted mice with recombinant interleukin (IL-7) promoted homeostatic expansion that improved T cell development, expanded the T cell receptor repertoire, and normalised the naïve T cell compartment. We conclude that implanting cultured thymus into the muscle of Foxn1 nu athymic mice is an appropriate system to study thymus implantation for congenital athymia and immunodeficiencies. T cells are educated by the donor thymus, yet naïve T cells have deficits in expansion. IL-7 greatly improves T cell development after thymus implantation and may offer a novel strategy to improve outcomes of clinical thymus implantation.

Diversity and distribution of MHC class I and II alleles in chicken populations worldwide

Almost everything known about antigen processing and presentation outside of mammals is from work on chickens. In contrast to mammals, unusually strong genetic associations exist between MHC haplotype and responses to infection in chickens. These associations, at least in part, seem to be due to the expression of a single classical class I and II molecule in most tissues. Unlike in mammals, chicken TAP and tapasin genes are highly polymorphic. Furthermore, peptide translocation specificity is very similar to the peptide binding specificity of the dominantly expressed MHCI molecule in each haplotype. These genes appear to be co-evolving – a process made possible by strong linkage disequilibrium across the ‘minimal essential MHC’. Similar co-evolution with the chaperone DM has been proposed for the single dominantly expressed class II gene. This study aimed to begin the most complete and exhaustive analysis of global MHC population genetics outside of humans. Once haplotypes have been defined by their class I and II alleles, interesting haplotypes would be selected for more detailed studies. Illumina MiSeq was used with a novel double-barcoding system to perform multiplexed amplicon se- quencing of exons 2 and 3 from all classical class I and class II loci. Samples were obtained from all major commercial breeders, ‘fancy’ breeds and free-range local chickens from Ethiopia, Nigeria, Tan- zania and Ghana. Our results show that, globally, there is huge diversity in chicken MHC alleles, most of which has never previously been reported from studies on experimental lines. Commercial flocks, however, have extremely low diversity, suggesting that artificial selection and population bottlenecks have had a drastic effect on the immune phenotypes of commercial flocks. These populations of commercial birds are also enriched in haplotypes with apparently promiscuous class I alleles that bind a wide variety of peptides and confer resistance to many common infectious diseases.

Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus.

The salivary glands are a crucial site of replication for human cytomegalovirus (HCMV) and its murine counterpart, murine cytomegalovirus (MCMV). Studies of MCMV often involve the use of BALB/c strain mice, but most in vitro assays are carried out in the NIH 3T3 cell line, which is derived from Swiss Albino mice. This report describes a BALB/c-derived mouse salivary gland cell line immortalized using the SV40 large T antigen. Cells stained positive for PDGFR1 and negative for E-cadherin and PECAM-1, indicating mesenchymal origin. This cell line, which has been named murine salivary gland mesenchymal (mSGM), shows promise as a tool for ex vivo immunological assays due to its MHC haplotype match with the BALB/c mouse strain. In addition, plaque assays using mSGM rather than NIH 3T3 cells are significantly more sensitive for detecting low concentrations of MCMV particles. Finally, it is demonstrated that mSGM cells express all 3 BALB/c MHC class I isotypes and are susceptible to T cell-mediated ex vivo cytotoxicity assays, leading to many possible uses in immunological studies of MCMV.

Major Histocompatibility Complex Variation and Haplotype Associated Survival in Response to Experimental Infection of Two Bd-GPL Strains Along a Latitudinal Gradient

While both innate and adaptive immune system mechanisms have been implicated in resistance against the chytrid fungus Batrachochytrium dendrobatidis (Bd), studies on the role of specific MHC haplotypes on Bd infection are rare. Here, we studied variation in MHC Class IIB loci in the common toad Bufo bufo along a latitudinal gradient across Sweden. In general, Swedish toad populations had few MHC Class IIB haplotypes and MHC diversity declined from south (13 haplotypes) to the north (four haplotypes). The low diversity may compromise the ability of northern populations to fight emerging disease, such as Bd. In a laboratory experiment, we infected newly metamorphosed toads with two strains of the Global Pandemic Lineage of the fungus (Bd-GPL) and compared survival with sham controls. Bd-infected toads had lower survival compared to controls. Moreover, survival was dependent on the Bd-strain and northern toads had lower Bd-mediated survival than southern individuals. MHC diversity was lower in northern toads. All northern experimental animals were monomorphic for a single MHC haplotype, whereas we found seven different haplotypes in southern experimental animals. In southern toads, survival was dependent on both Bd-strain and MHC haplotype suggesting differential infection dynamics depending on both Bd-strain and host immune system characteristics.

Caecal microbiota composition of experimental inbred MHC-B lines infected with IBV differs according to genetics and vaccination

Interactions between the gut microbiota and the immune system may be involved in vaccine and infection responses. In the present study, we studied the interactions between caecal microbiota composition and parameters describing the immune response in six experimental inbred chicken lines harboring different MHC haplotypes. Animals were challenge-infected with the infectious bronchitis virus (IBV), and half of them were previously vaccinated against this pathogen. We explored to what extent the gut microbiota composition and the genetic line could be related to the immune response, evaluated through flow cytometry. To do so, we characterized the caecal bacterial communities with a 16S rRNA gene amplicon sequencing approach performed one week after the IBV infectious challenge. We observed significant effects of both the vaccination and the genetic line on the microbiota after the challenge infection with IBV, with a lower bacterial richness in vaccinated chickens. We also observed dissimilar caecal community profiles among the different lines, and between the vaccinated and non-vaccinated animals. The effect of vaccination was similar in all the lines, with a reduced abundance of OTU from the Ruminococcacea UCG-014 and Faecalibacterium genera, and an increased abundance of OTU from the Eisenbergiella genus. The main association between the caecal microbiota and the immune phenotypes involved TCRϒδ expression on TCRϒδ+ T cells. This phenotype was negatively associated with OTU from the Escherichia-Shigella genus that were also less abundant in the lines with the highest responses to the vaccine. We proved that the caecal microbiota composition is associated with the IBV vaccine response level in inbred chicken lines, and that the TCRϒδ+ T cells (judged by TCRϒδ expression) may be an important component involved in this interaction, especially with bacteria from the Escherichia-Shigella genus. We hypothesized that bacteria from the Escherichia-Shigella genus increased the systemic level of bacterial lipid antigens, which subsequently mitigated poultry γδ T cells.

Abstract 5199: Tumor-specific antibody response induced by novel immunomodulator, IFx-Hu2.0, reflects extent of epitope spreading to multiple tumor epitopes

Abstract While the role of T cells in cancer immunotherapy has been well studied and is relatively clear and well defined, there are few studies elucidating the more complicated involvement of B cells in anti-cancer processes. Preclinical data showed that IFx-Hu2.0 primes both innate and adaptive immune responses through the expression of Emm55, a bacterial protein. Recognition by antigen presenting cells (APC) is followed by interantigenic epitope spreading to tumor antigens expressed in transfected tumor cells. The current study was designed to determine the extent of epitope spreading through quantitating the IgM and IgG responses to known melanoma antigens. Plasma samples (pre-and post-intralesional injection of IFx-Hu2.0) from seven patients with unresectable stage III-IV cutaneous melanoma enrolled in a first-in-human, phase 1 open-label trial (NCT03655756) were analyzed and compared using PEPperCHIP® Melanoma Antigen Microarrays with 21 melanoma-associated antigens converted into linear 15 amino acid peptides with a peptide-peptide overlap of 13 amino acids. The resulting array contained 4,125 different peptides printed in duplicate along with multiple control peptides. The arrays were incubated with plasma samples [collected immediately prior to and four weeks (N=6) or two weeks (N=1) post treatment and three weeks post injection #2 (N=1)] at three dilutions, followed by staining with secondary and control antibodies. Readout with a LI-COR Odyssey Imaging System was followed by quantification of spot intensities and peptide annotation with PepSlide® Analyzer. For generation of IgM/IgG intensity ratios, a minimal baseline intensity of 200 fluorescence units was set. Changes in both IgM and IgG epitope recognition to melanoma-associated antigens was detected in all patients. Baseline and IFx-Hu2.0-induced antibody response profiles for all individuals were heterogeneous, with no dominant common antibody response, pointing to the unpredictability of specific epitope recognition and a relative independence from MHC haplotype requirements due to epitope spreading. Notwithstanding the signals from adjacent peptides, high numbers of newly recognized melanoma epitopes were observed for all patients and all plasma samples. These data suggest enhanced crosstalk between innate and adaptive immune cells, epitope presentation by APC to T and B cells and epitope spreading to multiple previously unrecognized tumor epitopes. IgM and IgG levels were maintained at four weeks post IFx-Hu2.0 administration suggesting continued primary recognition and maturation of effector immune response. B cells represent an important proportion of infiltrating lymphocytes in melanoma and are associated with good prognosis in most cancer types. Studies are underway to determine the relative contribution of B cell activity to anti-tumor effects seen with IFx-Hu2.0. Citation Format: Patricia Lawman, Michael Shamblott, Ashraf Dehlawi, James Bianco. Tumor-specific antibody response induced by novel immunomodulator, IFx-Hu2.0, reflects extent of epitope spreading to multiple tumor epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5199.

Tolerance of a Vascularized Composite Allograft Achieved in MHC Class-I-mismatch Swine via Mixed Chimerism.

BackgroundVascularized composite allografts (VCAs) allow reconstruction of devastating injuries and amputations, yet require lifelong immunosuppression that is associated with significant morbidity. Induction of immune tolerance of VCAs would permit widespread use of these procedures. VCAs are acquired from deceased donors most likely to be fully-MHC-mismatched (in contrast to living-related renal transplant donor-recipient pairs matched at one MHC haplotype). After achieving VCA tolerance in a swine model equivalent to clinical living-related renal transplants (single-haplotype MHC mismatches: e.g., “mother-daughter”/haploidentical), we tested our protocol in MHC class I, class II, and fully-MHC-mismatched pairs. Although class II mismatched swine demonstrated similar results as the haploidentical scenario (stable mixed chimerism and tolerance), our protocol failed to prevent rejection of class I and full mismatch VCAs. Here, we describe a new adapted conditioning protocol that successfully achieved tolerance across MHC class-I-mismatch barriers in swine.MethodsSwine were treated with non-myeloablative total body and thymic irradiation two days prior to infusion of bone marrow cells from an MHC class I-mismatched donor. They also received a short-term treatment with CTLA4-Ig (Belatacept®) and anti-IL6R mAb (Tociluzimab®) and were transplanted with an osteomyocutaneous VCA from the same donor.ResultsStable mixed chimerism and tolerance of MHC class-I-mismatched VCAs was achieved in 3 recipients. Allograft tolerance was associated with a sustained lack of anti-donor T cell response and a concomitant expansion of double negative CD4-CD8- T cells producing IL-10.ConclusionsThis study demonstrates the first successful mixed chimerism-induced VCA tolerance in a large animal model across a MHC class-I-mismatch. Future studies aimed at fully-mismatched donor-recipient pairs are under investigation with this protocol.

Defining the cooperation between T cell responses to MHC-I and MHC-II melanoma neoantigens towards developing effective personalized cancer immunotherapies.

Abstract Immune checkpoint therapy (ICT) (e.g. anti-CTLA-4, anti-PD-1) enables durable T-cell dependent anti-tumor immunity in patients with solid tumors. Since not all patients respond to ICT, this work aims at developing a more in-depth understanding of T-cell responses to MHC class I (MHC-I) and MHC class II (MHC-II) tumor antigens that occur as a consequence of aberrant expression of non-mutant antigens or driver and passenger mutations that form tumor neoantigens. We used a poorly immunogenic Brafv600e Pten−/−Cdkn2a−/− YUMM1.7 (Y1.7) murine melanoma line that with a paucity of endogenous neoantigens that is unresponsive to ICT, and introduced model neoantigens previously identified in a murine sarcoma tumor line expressing the same MHC haplotype as the Y1.7 line. The MHC-I neoantigen used was formed by a G1254V point mutation in the Laminin alpha subunit-4 (Lama4) and the MHC-II neoantigen was formed by an A710T point mutation in Integrin beta-1 (Itgb1). Anti-CTLA-4-treated mice bearing the parental Y1.7 melanoma displayed progressive tumor growth, whereas the modified Y1.7 line expressing mLama4 and mItgb1 (Y1.7.mLama4.mItgb1) was rendered sensitive to ICT. Preliminary data showed that anti-CTLA-4 treated Y1.7.mLama4.mItgb1 tumors reject in mice in a CD4+ T cell and CD8+ T cell-dependent manner. Additionally, intratumoral mLama4-specific CD8+ T cells were detected in anti-CTLA-4-treated Y1.7.mLama4.mItgb1 melanoma bearing mice. Overall this model will allow us to better understand T cell mediated anti-tumor responses to both MHC-I and MHC-II neoantigens, as well as shared, non-mutant melanoma antigens (e.g. Pmel/GP100 and Trp2), which may facilitate development of improved neoantigen-based personalized immunotherapies.

Genetic analysis of CNS autoimmunity using the diversity of the Collaborative Cross reveals unique phenotypes and mechanisms

Abstract Multiple Sclerosis (MS) is a complex disease with remarkable heterogeneity in disease course and progression, the genetic basis of which remains obscure. Here we leveraged the Collaborative Cross (CC) - a highly genetically diverse mouse strain panel - and myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE), to model genetics of MS disease course. 33 CC strains were selected based on compatible MHC haplotypes (H2b and H2g7), which captured a wide spectrum of distinct EAE phenotypes, compared with typical chronic EAE in C57BL/6 mice. CC028 mice exhibited severe and rapidly progressing disease. In contrast, several strains, including CC011 and CC040, were highly resistant to EAE. Sex differences in EAE course were observed in 4 strains, including CC042. Remitting-relapsing EAE was observed in 4 strains, including CC002. In addition to classical EAE clinical signs (ascending paralysis), we identified two strains, CC004 and CC083 that exhibited high incidence of axial rotary (AR)-EAE, including profound ataxia, head tilt, and axial rotation. Preliminary quantitative trait locus (QTL) analysis revealed a distinct linkage pattern in each sex for classical EAE severity, with several emerging peaks on chromosome (Chr) 2, 8, 12, 18, and X in females, Chr10 in males, and Chr4 in both sexes. QTL analysis of AR-EAE severity revealed a narrow interval on Chr18 (39.2–41.0Mb) passing suggestive linkage significance at the 90% confidence threshold. Experiments are ongoing to determine the immunopathologic basis of distinct genetically controlled EAE phenotypes in CC strains of interest. Supported by grants from NIH (R21 AI145306-01) and the National MS Society (RG-1901-33309)

IMMU-47. THE ANTIGENIC PROFILE OF MURINE AND HUMAN MEDULLOBLASTOMA

Abstract BACKGROUND Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel immunotherapies. We performed a multi-faceted computer algorithm, the Open Reading Frame Antigen Network (O.R.A.N.), on medulloblastoma transcription profiles and predicted antigens across a broad array of antigen classes. METHODS Patient-specific HLA haplotypes were called via customized Optitype and Phlat algorithms. Preclinical models- sonic hedgehog driven (Ptch1) and Group 3 MYC-driven (NSC) medulloblastoma were derived from C57BL6 murine strain with known MHC haplotypes. Only expressed mutations such as single nucleotide variations, small indels, gene fusions, and personalized TAAs were used for antigenic epitope predictions. Patient-specific or murine tumor associated antigens (TAA) were selected only if expressed &amp;gt;1 transcript per million (TPM) in tumor and the standardized expression across a human tissue database (29 organs or sub-regions, n=9,141) or a mouse normal tissue database (ENCODE, n=99) was below 1 TPM, respectively. TAA sequences were passed through eight MHC class I and four MHC class II affinity algorithms. All epitopes were screened against a customized human or murine proteomic library to guarantee that epitopes were not shared by other expressed isoforms or genes. Immune deconvolution with single cell RNASeq integration was leveraged for teasing out medulloblastoma immunologic landscape. RESULTS MB patients harbor MHC-I restricted 1.9 SNV, 0.1 Indel, 0.5 gene fusions and MHC-II restricted 2.5 SNV, 0.1 Indel and 0.5 gene fusion. 79.4% patients have at least 1 neoantigen. 88.2% patients have at least one immunogenic TAA. Importantly, cancer testis antigens and previously unappreciated neurodevelopmental antigens were found expressed across all medulloblastoma subgroups. We predicted 6 neoantigens and 14 TAAs for murine NSC tumor and 19 neoantigens and 13 TAAs for Ptch1 tumor. CONCULSION: Using a custom antigen prediction pipeline, we identified potential human and murine tumor rejection antigens with important implications for development of medulloblastoma cellular therapies.

MHC haplotype and B cell autoimmunity: Correlation with pathogenic IgG autoantibody subclasses and Fc glycosylation patterns.

Genome-wide association studies (GWAS) have identified many genes that are associated with the development of certain autoimmune disorders, but the MHC haplotypes still represent the most prevalent genetic risk factor for many autoimmune diseases. The mechanisms by which MHC-associated genetic susceptibility translates into B cell autoimmunity and the development of autoimmune diseases are complex. There is increasing evidence that the MHC haplotype modulates autoreactive B cell responses in multiple ways. Instead of merely inhibiting the production of IgG autoantibodies and mediating complete immunological tolerance, the non-permitting MHC haplotypes seem to facilitate the production of IgG autoantibodies exhibiting Fc glycosylation patterns that are associated with reduced pathogenicity and a protective cytokine profile of T follicular helper (Tfh) cells. Here, we discuss mechanisms linking MHC haplotypes to the production of pathogenic IgG autoantibodies, which could be relevant for the development of improved diagnosis, particularly in the context of individual medicine.

Genetic effects of long-term captive breeding on the endangered pygmy hog.

Long-term captive populations often accumulate genetic changes that are detrimental to their survival in the wild. Periodic genetic evaluation of captive populations is thus necessary to identify deleterious changes and minimize their impact through planned breeding. Pygmy hog (Porcula salvania) is an endangered species with a small population inhabiting the tall sub-Himalayan grasslands of Assam, India. A conservation breeding program of pygmy hog from six founders has produced a multi-generational captive population destined for reintroduction into the wild. However, the impact of conservation breeding on its genetic diversity remained undocumented. Here, we evaluate temporal genetic changes in 39 pygmy hogs from eight consecutive generations of a captive population using genome-wide SNPs, mitochondrial genomes, and MHC sequences, and explore the relationship between genetic diversity and reproductive success. We find that pygmy hog harbors a very low genome-wide heterozygosity (H) compared to other members of the Suidae family. However, within the captive population we find excess heterozygosity and a significant increase in H from the wild-caught founders to the individuals in subsequent generations due to the selective pairing strategy. The MHC and mitochondrial nucleotide diversities were lower in captive generations compared to the founders with a high prevalence of low-frequency MHC haplotypes and more unique mitochondrial genomes. Further, even though no signs of genetic inbreeding were observed from the estimates of individual inbreeding coefficient F and between individuals (FIS) in each generation, the kinship coefficient showed a slightly increasing trend in the recent generations, due to a relatively smaller non-random sample size compared to the entire captive population. Surprisingly, male pygmy hogs that had higher heterozygosity also showed lower breeding success. We briefly discuss the implications of our findings in the context of breeding management and recommend steps to minimize the genetic effects of long-term captive breeding.

Abstract 1628: Engineered toxin bodies targeting PD-L1 to alter tumor immunophenotypes and deliver broad antigenic diversity and patient coverage

Abstract Targeting PD-L1 has shown clinical efficacy in multiple solid tumor indications. Currently approved PD-L1 targeted approaches rely on monoclonal antibodies which sterically inhibit PD-L1 and prevent PD-1 mediated checkpoint activity. While these molecules have shown great activity in the clinic, the need for pre-existing tumor specific immunity and immune infiltration precludes responses in some patients and leads to resistance in others. Therefore, there remains a need for new modalities and treatment paradigms in these indications. Molecular Templates has developed MT-6402, an engineered toxin body (ETB) targeting PD-L1, as a single agent immunotoxin designed to overcome the challenges of current PD-L1 targeting approaches by 1) directly depleting PD-L1 positive tumor cells or immunosuppressive immune cells displaying PD-L1 in the tumor microenvironment and 2) delivery of an HLA: A*02 restricted viral peptide to alter the tumor immunophenotype for recruitment of CMV-restricted CTLs to target the tumor for depletion (antigen seeding). MT-6402 is slated for clinical development in 2021. Here we describe the preclinical characterization of several ETB candidates derived from MT-6402 delivering antigenic peptides restricted to the most prevalent MHC haplotypes in the U.S. population to broaden the patient population suitable for antigen seeding. ETBs were engineered with the ability to deliver viral peptides across a range of HLA restriction, including HLA: A*01, HLA: A*03, and HLA: A*24. ETBs were screened and benchmarked against MT-6402 and candidates were identified that retain comparable specificity, selectivity, and potency. Alteration of peptide antigen did not change the specificity or selectivity of ETBs which retained similar PD-L1 binding profiles to MT-6402. Binding profiles correlated to targeted potency and ETBs with varied HLA restricted peptides were found to target tumor and immune cells for depletion with similar potency to MT-6402. ETBs delivered an antigen seeding response in a PD-L1 dependent manner and only in conditions in which tumor cell and CTLs shared a matched HLA to the delivered antigenic peptide specificity. Preclinical assessment of the in vivo efficacy and safety profile of candidates is ongoing and further development is slated for 2021. Citation Format: Joseph D. Dekker, Swati Khanna, Elizabeth Saputra, Wenzhao Dong, Lindsey Aschenbach, Lilia A. Rabia, Garrett L. Cornelison, Michaela Sousares, Jay Zhao, Garrett L. Robinson, Betty Chang, Hilario J. Ramos. Engineered toxin bodies targeting PD-L1 to alter tumor immunophenotypes and deliver broad antigenic diversity and patient coverage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1628.