Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.

Immunization with the amino-terminus region of dense granule protein 6 (GRA6) of Toxoplasma gondii activates CD8+ cytotoxic T cells capable of removing tissue cysts of the parasite through antigen presentation by human HLA-A2.1.

CD8+ T cells from HLA-A2.1-transgenic mice, but not wild-type mice, immunized with the amino-terminus region (aa 41-152) of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii secreted large amounts of perforin and granzyme B in response to GRA6Nt through antigen presentation by HLA-A2.1 in vitro. When those CD8+ T cells were transferred into chronically infected HLA-A2.1-expressing NSG mice deficient in T cells, cerebral cyst burden of the recipients of HLA-A2.1-transgenic T cells, but not of WT T cells, became significantly less than that of control mice with no cell transfer. Furthermore, the significant reduction of the cyst burden by a transfer of the HLA-A2.1-transgenic CD8+ immune T cells required an expression of HLA-A2.1 in the recipient NSG mice. Thus, antigen presentation of GRA6Nt by human HLA-A2.1is able to activate anti-cyst CD8+ T cells that eliminate T. gondii cysts through antigen presentation by human HLA-A2.1.

Chimeric HLA antibody receptor T cells for targeted therapy of antibody-mediated rejection in transplantation.

The presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal long-term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA-producing B cells. We have genetically engineered an HLA-A2-specific CHAR (A2-CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti-HLA-A2 antibody-expressing target cells. In addition, we have performed A2-CHAR-Tc cytotoxic assays in an immunodeficient mouse model. A2-CHAR expressing T cells could selectively eliminate HLA-A2 antibody-producing B cells in vitro. The cytotoxic capacity of A2-CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2-CHAR-T cells could identify and eradicate HLA-A2 antibody-producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody-producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation.

TCR T cells overexpressing c-Jun have better functionality with improved tumor infiltration and persistence in hepatocellular carcinoma.

Background: The overall 5-year survival rate of hepatocellular carcinoma (HCC), a major form of liver cancer, is merely 20%, underscoring the need for more effective therapies. We recently identified T cell receptors (TCR) specific for the HLA-A2/alpha fetoprotein amino acids 158-166 (AFP158) and showed that these TCR engineered T cells could control HCC xenografts in NSG mice. However, their efficacy was limited by poor expansion, loss of function, and short persistence of the TCR T cells. Here, we studied whether overexpression of c-Jun, a transcription factor required for T cell activation, in the TCR T cells could enhance their expansion, function, and persistence in HCC tumor models. Methods: Recombinant lentiviral vectors (lv), expressing either the HLA-A2/AFP158-specific TCR or both the TCR and c-Jun (TCR-JUN), were constructed and used to transduce primary human T cells to generate the TCR or TCR-JUN T cells, respectively. We compared the expansion, effector function, and exhaustion status of the TCR and TCR-JUN T cells in vitro after HCC tumor stimulation. Additionally, we studied the persistence and antitumor effects of the TCR and TCR-JUN T cells using the HCC xenografts in NSG mice. Results: We could effectively transduce primary human T cells to express both TCR and c-Jun. Compared to the HLA-A2/AFP158 TCR T cells, the TCR-JUN T cells have better expansion potential in culture, with enhanced functional capacity against HCC tumor cells. In addition, the TCR-JUN T cells were less apoptotic and more resistant to exhaustion after HepG2 tumor stimulation. In the HCC xenograft tumor model, c-Jun overexpression enhanced the TCR T cell expansion and increased the overall survival rate of the treated mice. Importantly, the TCR-JUN T cells were less exhausted in the tumor lesions and demonstrated enhanced tumor infiltration, functionality, and persistence. Conclusion: c-Jun overexpression can enhance the expansion, function, and persistence of the A2/AFP158 TCR engineered T cells. The c-Jun gene co-delivery has the potential to enhance the antitumor efficacy of AFP specific TCR T cells when treating patients with HCC.

Altered HLA-A2-restricted TP53 epitope induces specific CTL cytotoxicity against hepatocellular carcinoma.

High-frequency mutation of the TP53 tumor suppressor gene is observed in multiple human cancers, which promotes cancer progression. However, the mutated gene-encoded protein may serve as a tumor antigen to elicit tumor-specific immune responses. In this study, we detected widespread expression of shared TP53-Y220C neoantigen in hepatocellular carcinoma with low affinity and low stability of binding to HLA-A0201 molecules. We substituted the amino acid sequences VVPCEPPEV with VLPCEPPEV in the TP53-Y220C neoantigen to yield a TP53-Y220C (L2) neoantigen. This altered neoantigen was found to increase affinity and stability and induce more cytotoxic T lymphocytes (CTLs), indicating improvements in immunogenicity. In vitro assays showed the cytotoxicity of CTLs stimulated by both TP53-Y220C and TP53-Y220C (L2) neoantigens against multiple HLA-A0201-positive cancer cells expressing TP53-Y220C neoantigens; however, the TP53-Y220C (L2) neoantigen showed higher cytotoxicity than the TP53-Y220C neoantigen against cancer cells. More importantly, in vivo assays demonstrated greater inhibition of hepatocellular carcinoma cell proliferation by TP53-Y220C (L2) neoantigen-specific CTLs relative to TP53-Y220C neoantigen in zebrafish and nonobese diabetic/severe combined immune deficiency mouse models. The results of this study demonstrate enhanced immunogenicity of the shared TP53-Y220C (L2) neoantigen, which has the potential as dendritic cells or peptide vaccines for multiple cancers.

A class-mismatched TCR bypasses MHC restriction via an unorthodox but fully functional binding geometry.

MHC restriction, which describes the binding of TCRs from CD4+ T cells to class II MHC proteins and TCRs from CD8+ T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4+ T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3ß loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy.

Human MHC class I molecule, HLA-A2.1, mediates activation of CD8+ T cell IFN-γ production and the T cell-dependent protection against reactivation of cerebral Toxoplasma infection.

To examine whether the HLA-A2.1, one of the most common MHC class I molecules in humans, activates the protective immunity against reactivation of cerebral infection with Toxoplasma gondii, HLA-A2.1-transgenic and wild-type (WT) mice were infected and treated with sulfadiazine to establish chronic infection in their brains. One month after discontinuation of sulfadiazine, which initiates reactivation of the infection, mRNA levels for tachyzoite (the acute stage form)-specific SAG1 and numbers of the foci associated tachyzoites were significantly less in the brains of the HLA-A2.1-transgenic than WT mice. Greater numbers of IFN-γ-producing CD8+ T cells were detected in the spleens of infected transgenic than WT mice, and CD8+ T cells from the former produced markedly greater amounts of IFN-γ than the T cells from the latter in response to tachyzoite antigens in vitro. When their CD8+ T cells were systemically transferred to infected immunodeficient NSG mice expressing the HLA-A2.1, the CD8+ T cells from HLA-A2.1-transgenic mice inhibited reactivation of the cerebral infection in the recipients more efficiently than did the WT T cells. Furthermore, the inhibition of reactivation of the infection by CD8+ T cells from the transgenic mice was associated with increased cerebral expression of IFN-γ and effector molecules against tachyzoites in the recipients when compared to the WT CD8+ T cell recipients. Thus, the human HLA-A2.1 is able to effectively activate IFN-γ production of CD8+ T cells against T. gondii tachyzoites and confer a potent protection against reactivation of cerebral infection with this parasite through the CD8+ T cells activation.

H3.3K27M mutation is not a suitable target for immunotherapy in HLA-A2+ patients with diffuse midline glioma.

Diffuse midline glioma is the leading cause of solid cancer-related deaths in children with very limited treatment options. A majority of the tumors carry a point mutation in the histone 3 variant (H3.3) creating a potential HLA-A*02:01 binding epitope (H3.3K27M26-35). Here, we isolated an H3.3K27M-specific T cell receptor (TCR) from transgenic mice expressing a diverse human TCR repertoire. Despite a high functional avidity of H3.3K27M-specific T cells, we were not able to achieve recognition of cells naturally expressing the H3.3K27M mutation, even when overexpressed as a transgene. Similar results were obtained with T cells expressing the published TCR 1H5 against the same epitope. CRISPR/Cas9 editing was used to exclude interference by endogenous TCRs in donor T cells. Overall, our data provide strong evidence that the H3.3K27M mutation is not a suitable target for cancer immunotherapy, most likely due to insufficient epitope processing and/or amount to be recognized by HLA-A*02:01 restricted CD8+ T cells.

Identification of human MHC-I HPV18 E6/E7-specific CD8 + T cell epitopes and generation of an HPV18 E6/E7-expressing adenosquamous carcinoma in HLA-A2 transgenic mice.

BACKGROUND: Human Papillomavirus type 18 (HPV18) is a high-risk HPV that is commonly associated with cervical cancer. HPV18 oncogenes E6 and E7 are associated with the malignant transformation of cells, thus the identification of human leukocyte antigen (HLA)-restricted E6/E7 peptide-specific CD8 + T cell epitopes and the creation of a HPV18 E6/E7 expressing cervicovaginal tumor in HLA-A2 transgenic mice will be significant for vaccine development. METHODS: In the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice. RESULTS: Therapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A*0201/Dd) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice. CONCLUSIONS: We characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.

Molecular Characterization of MHC Class I Alpha 1 and 2 Domains in Asian Seabass (Lates calcarifer).

The Asian seabass is of importance both as a farmed and wild animal. With the emergence of infectious diseases, there is a need to understand and characterize the immune system. In humans, the highly polymorphic MHC class I (MHC-I) molecules play an important role in antigen presentation for the adaptive immune system. In the present study, we characterized a single MHC-I gene in Asian seabass (Lates calcarifer) by amplifying and sequencing the MHC-I alpha 1 and alpha 2 domains, followed by multi-sequence alignment analyses. The results indicated that the Asian seabass MHC-I α1 and α2 domain sequences showed an overall similarity within Asian seabass and retained the majority of the conserved binding residues of human leukocyte antigen-A2 (HLA-A2). Phylogenetic tree analysis revealed that the sequences belonged to the U lineage. Mapping the conserved binding residue positions on human HLA-A2 and grass carp crystal structure showed a high degree of similarity. In conclusion, the availability of MHC-I α1 and α2 sequences enhances the quality of MHC class I genetic information in Asian seabass, providing new tools to analyze fish immune responses to pathogen infections, and will be applicable in the study of the phylogeny and the evolution of antigen-specific receptors.

Enhanced In Vitro and In Vivo Potency of a T Cell Epitope in the Ebola Virus Glycoprotein Following Amino Acid Replacement at HLA-A*02:01 Binding Positions.

Studies on Ebola virus disease (EVD) survivors and clinical studies on Ebola virus (EBOV) vaccine candidates have pinpointed the importance of a strong antibody response in protection and survival from EBOV infection. However, little is known about the T cell responses to EBOV or EBOV vaccines. We used HLA-A*02:01 (HLA-A2) transgenic mice to study HLA-A2-specific T cell responses elicited following vaccination with EBOV glycoprotein (EBOV-GP) presented with three different systems: (i) recombinant protein (rEBOV-GP), (ii) vesicular stomatitis replication-competent recombinant virus (VSV-EBOV-GP), and (iii) modified vaccinia Ankara virus recombinant (MVA-EBOV-GP). T cells from immunized animals were analyzed using peptide pools representing the entire GP region and individual peptides. Regardless of the vaccine formulation, we identified a minimal 9mer epitope containing an HLA-A2 motif (FLDPATTS), which was confirmed through HLA-A2 binding affinity and immunization studies. Using binding prediction software, we identified substitutions surrounding position 9 (S9V, P10V, and Q11V) that predicted enhanced binding to the HLA-A2 molecule. This enhanced binding was confirmed through in vitro binding studies and enhanced potency was shown with in vivo immunization studies using the enhanced sequences and the wild-type sequence. Of note, in silico studies predicted the enhanced 9mer epitope carrying the S9V substitution as the best overall HLA-A2 epitope for the full-length EBOV-GP. These results suggest that EBOV-GP-S9V and EBOV-GP-P10V represent more potent in vivo immunogens. Identification and enhancement of EBOV-specific human HLA epitopes could lead to the development of tools and reagents to induce more robust T cell responses in human subjects. IMPORTANCE Vaccine efficacy and immunity to viral infection are often measured by neutralizing antibody titers. T cells are specialized subsets of immune cells with antiviral activity, but this response is variable and difficult to track. We showed that the HLA-A2-specific T cell response to the Ebola virus glycoprotein can be enhanced significantly by a single residue substitution designed to improve an epitope binding affinity to one of the most frequent MHC alleles in the human population. This strategy could be applied to improve T cell responses to Ebola vaccines designed to elicit antibodies and adapted to target MHC alleles of populations in regions where endemic infections, like Ebola virus disease, are still causing outbreaks with concerning pandemic potential.

Identification of CD8+ T-cell epitope from multiple myeloma-specific antigen AKAP4.

Multiple myeloma (MM) is a malignant plasma cell disorder affecting mainly the elderly population. Revolutionary progress in immunotherapy has been made recently, including monoclonal antibodies and chimeric antigen receptor T cell (CAR-T) therapies; however, the high relapse rate remains problematic. Therefore, combination therapies against different targets would be a reasonable strategy. In this study, we present a new X-chromosome encoded testis-cancer antigen (CTA) AKAP4 as a potential target for MM. AKAP4 is expressed in MM cell lines and MM primary malignant plasma cells. HLA-A*0201-restricted cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transduced with an adenovirus vector encoding the full-length AKAP4 gene were demonstrated to lyse AKAP4+ myeloma cells. Seven of the 12 candidate epitopes predicated by the BIMAS and SYFPEITH algorithms were able to bind HLA-A*0201 in the T2 binding assay, of which only two peptides were able to induce CTL cytotoxicity in the co-culture of peptide-loaded human mature dendritic cells and the autologous peripheral blood mononuclear cells (PBMCs) from the same HLA-A*0201 donor. The AKAP4 630-638 VLMLIQKLL was identified as the strongest CTL epitope by the human IFN-γ ELISPOT assay. Finally, the VLMLIQKLL-specific CTLs can lyse the HLA-A*0201+AKAP4+ myeloma cell line U266 in vitro, and inhibit tumor growth in the mice bearing U266 tumors in vivo. These results suggest that the VLMLIQKLL epitope could be used to develop cancer vaccine or T-cell receptor transgenic T cells (TCR-T) to kill myeloma cells.

Optimized Anchor-Modified Peptides Targeting Mutated RAS Are Promising Candidates for Immunotherapy.

RAS mutations occur in approximately 20% of all cancers and given their clonality, key role as driver mutation, association with poor prognosis and undruggability, they represent attractive targets for immunotherapy. We have identified immunogenic peptides derived from codon 12 mutant RAS (G12A, G12C, G12D, G12R, G12S and G12V), which bind to HLA-A*02:01 and HLA-A*03:01 and elicit strong peptide-specific CD8+ T cell responses, indicating that there is an effective CD8+ T-cell repertoire against these mutant RAS-derived peptides that can be mobilized. Alterations in anchor residues of these peptides enhanced their binding affinity to HLA-A*02:01 molecules and allowed generation of CD8+ T cells that responded to target cells pulsed with the anchor-modified and also with the original peptide. Cytotoxic T cells generated against these peptides specifically lysed tumor cells expressing mutant RAS. Vaccination of transgenic humanized HLA-A2/DR1 mice with a long peptide encompassing an anchor-modified 9-mer G12V epitope generated CD8+ T cells reactive to the original 9-mer and to a HLA-A*02:01-positive human cancer cell line harboring the G12V mutation. Our data provide strong evidence that mutant RAS can be targeted by immunotherapy.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma.

BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Structural patterns in class 1 major histocompatibility complex-restricted nonamer peptide binding to T-cell receptors.

The startling diversity in αß T-cell receptor (TCR) sequences and structures complicates molecular-level analyses of the specificity and sensitivity determining T-cell immunogenicity. A number of three-dimensional (3D) structures are now available of ternary complexes between TCRs and peptides: major histocompatibility complexes (pMHC). Here, to glean molecular-level insights we analyze structures of TCRs bound to human class I nonamer peptide-MHC complexes. Residues at peptide positions 4-8 are found to be particularly important for TCR binding. About 90% of the TCRs hydrogen bond with one or both of the peptide residues at positions 4 and 8 presented by MHC allele HLA-A2, and this number is still ~79% for peptides presented by other MHC alleles. Residue 8, which lies outside the previously-identified central peptide region, is crucial for TCR recognition of class I MHC-presented nonamer peptides. The statistics of the interactions also sheds light on the MHC residues important for TCR binding. The present analysis will aid in the structural modeling of TCR:pMHC complexes and has implications for the rational design of peptide-based vaccines and T-cell-based immunotherapies.

Cutting Edge: Unconventional CD8+ T Cell Recognition of a Naturally Occurring HLA-A*02:01-Restricted 20mer Epitope.

Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity.

Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19.

We report a Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2 RγcKO.NOD) for COVID-19 research. DRAGA mice express transgenically HLA-class I and class-II molecules in the mouse thymus to promote human T cell development and human B cell Ig-class switching. When infused with human hematopoietic stem cells from cord blood reconstitute a functional human immune system, as well as human epi/endothelial cells in lung and upper respiratory airways expressing the human ACE2 receptor for SARS-CoV-2. The DRAGA mice were able to sustain SARS-CoV-2 infection for at least 25 days. Infected mice showed replicating virus in the lungs, deteriorating clinical condition, and human-like lung immunopathology including human lymphocyte infiltrates, microthrombi and pulmonary sequelae. Among the intra-alveolar and peri-bronchiolar lymphocyte infiltrates, human lung-resident (CD103+) CD8+ and CD4+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, suggesting anti-viral cytotoxic activity. Infected mice also mounted human IgG antibody responses to SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathological mechanisms and testing the safety and efficacy of candidate vaccines and therapeutics.

A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo.

The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.