Bio X Cell

Clone	Catalog #	Category
28-14-8S	BE0451

About InVivoMAb anti-mouse MHC Class I (H-2Db)

The 28-14-8S monoclonal antibody reacts with the α3 domains of H-2Db of the MHC class I alloantigen expressed on nucleated cells from mice of the H-2Db haplotype. The 28-14-8S antibody shows weak cross-reactivity with Ld antigen, but it doesn’t cross-react with Dd. H-2Db is a member of the MHC subgroup, and it is characterized by a hydrophobic ridge in the binding cleft. The H2 class I molecules are made up of three parts: a heavy chain that is 42 kDa, a 12-kDa protein called β2-microglobulin, and a short peptide made up of 8 to 11 amino acids. The endoplasmic reticulum carries out this assembly, but the stable trimeric complexes translocate to cell surfaces. H-2Db is a class I molecule that is able to reach the cell surface even in the absence of either β2 microglobulin or TAP-provided peptides. Interestingly, despite being incomplete and unstable, this molecule is still able to exhibit some functional capacities, in that it can induce and be recognized by allogeneic and self-restricted cytotoxic T lymphocytes. The H-2Db plays a critical role in antigen presentation to T cells expressing CD3/TCR and CD8 proteins. Like other MHC antibodies, the 28-14-8S clone finds its application in various immunoassays involving MHC-peptide complexes. It is a useful tool for immunopeptidomics research, making it easier to identify and describe neoantigens using HPLC and mass spectrometry.

InVivoMAb anti-mouse MHC Class I (H-2Db) Specifications

Isotype	Mouse IgG2a, κ
Recommended Isotype Control(s)	InVivoMAb mouse IgG2a isotype control, unknown specificity
Recommended Dilution Buffer	InVivoPure pH 7.0 Dilution Buffer
Immunogen	C3H.SW mouse splenocytes
Reported Applications	MHC-I Immunopeptidomics in vitro blocking of MHC-I Immunocapture of peptide-MHC class - I complexes Immunoprecipitation Flow cytometry Immunohistochemistry (frozen) ELISA
Formulation	PBS, pH 7.0 Contains no stabilizers or preservatives
Endotoxin	<2EU/mg (<0.002EU/μg) Determined by LAL gel clotting assay
Purity	>95% Determined by SDS-PAGE
Sterility	0.2 μm filtered
Production	Purified from cell culture supernatant in an animal-free facility
Purification	Protein G
Molecular Weight	150 kDa
Storage	The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.

Application References

InVivoMAb anti-mouse MHC Class I (H-2Db) (CLONE: 28-14-8S)

Tokita S, Fusagawa M, Matsumoto S, Mariya T, Umemoto M, Hirohashi Y, Hata F, Saito T, Kanaseki T, Torigoe T (2024). "Identification of immunogenic HLA class I and II neoantigens using surrogate immunopeptidomes" Sci Adv 10(38):eado6491. PubMed

Neoantigens arising from somatic mutations are tumor specific and induce antitumor host T cell responses. However, their sequences are individual specific and need to be identified for each patient for therapeutic applications. Here, we present a proteogenomic approach for neoantigen identification, named Neoantigen Selection using a Surrogate Immunopeptidome (NESSIE). This approach uses an autologous wild-type immunopeptidome as a surrogate for the tumor immunopeptidome and allows human leukocyte antigen (HLA)-agnostic identification of both HLA class I (HLA-I) and HLA class II (HLA-II) neoantigens. We demonstrate the direct identification of highly immunogenic HLA-I and HLA-II neoantigens using NESSIE in patients with colorectal cancer and endometrial cancer. Fresh or frozen tumor samples are not required for analysis, making it applicable to many patients in clinical settings. We also demonstrate tumor prevention by vaccination with selected neoantigens in a preclinical mouse model. This approach may benefit personalized T cell-mediated immunotherapies.

Larouche JD, Laumont CM, Trofimov A, Vincent K, Hesnard L, Brochu S, Côté C, Humeau JF, Bonneil É, Lanoix J, Durette C, Gendron P, Laverdure JP, Richie ER, Lemieux S, Thibault P, Perreault C (2024). "Transposable elements regulate thymus development and function" Elife . PubMed

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/β. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

Iorgulescu JB, Ruthen N, Ahn R, Panagioti E, Gokhale PC, Neagu M, Speranza MC, Eschle BK, Soroko KM, Piranlioglu R, Datta M, Krishnan S, Yates KB, Baker GJ, Jain RK, Suvà ML, Neuberg D, White FM, Chiocca EA, Freeman GJ, Sharpe AH, Wu CJ, Reardon DA (2023). "Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model" Front Immunol . PubMed

Background: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy. Methods: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics. Results: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants. Conclusion: Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

Hoover AR, Kaabinejadian S, Krawic JR, Sun XH, Naqash AR, Yin Q, Yang X, Christopher Garcia K, Davis MM, Hildebrand WH, Chen WR (2022). "Localized ablative immunotherapy drives de novo CD8⁺ T-cell responses to poorly immunogenic tumors" J Immunother Cancer 10(10):e004973. PubMed

Background: Localized ablative immunotherapies hold great promise in stimulating antitumor immunity to treat metastatic and poorly immunogenic tumors. Tumor ablation is well known to release tumor antigens and danger-associated molecular patterns to stimulate T-cell immunity, but its immune stimulating effect is limited, particularly against metastatic tumors. Methods: In this study, we combined photothermal therapy with a potent immune stimulant, N-dihydrogalactochitosan, to create a local ablative immunotherapy which we refer to as laser immunotherapy (LIT). Mice bearing B16-F10 tumors were treated with LIT when the tumors reached 0.5 cm³ and were monitored for survival, T-cell activation, and the ability to resist tumor rechallenge. Results: We found that LIT stimulated a stronger and more consistent antitumor T-cell response to the immunologically 'cold' B16-F10 melanoma tumors and conferred a long-term antitumor memory on tumor rechallenge. Furthermore, we discovered that LIT generated de novo CD8⁺ T-cell responses that strongly correlated with animal survival and tumor rejection. Conclusion: In summary, our findings demonstrate that LIT enhances the activation of T cells and drives de novo antitumor T-cell responses. The data presented herein suggests that localized ablative immunotherapies have great potential to synergize with immune checkpoint therapies to enhance its efficacy, resulting in improved antitumor immunity.

Lin LCW, Croft SN, Croft NP, Wong YC, Smith SA, Tang SS, Purcell AW, Tscharke DC (2021). "Direct Priming of CD8⁺ T Cells Persists in the Face of Cowpox Virus Inhibitors of Antigen Presentation" J Virol 95(10):e00186-21. PubMed

Vaccinia virus (VACV) was the vaccine used to eradicate smallpox and is being repurposed as a vaccine vector. CD8⁺ T cells are key anti-viral mediators, but require priming to become effector or memory cells. Priming requires an interaction with dendritic cells that are either infected (direct priming), or that have acquired virus proteins but remain uninfected (cross priming). To investigate CD8⁺ T cell priming pathways for VACV, we engineered the virus to express CPXV12 and CPXV203, two inhibitors of antigen presentation encoded by cowpox virus. These intracellular proteins would be expected to block direct but not cross priming. The inhibitors had diverse impacts on the size of anti-VACV CD8⁺ T cell responses across epitopes and by different infection routes in mice, superficially suggesting variable use of direct and cross priming. However, when we then tested a form of antigen that requires direct priming, we found surprisingly that CD8⁺ T cell responses were not diminished by co-expression with CPXV12 and CPXV203. We then directly quantified the impact of CPXV12 and CPXV203 on viral antigen presentation using mass spectrometry, which revealed strong, but incomplete inhibition of antigen presentation by the CPXV proteins. Therefore, direct priming of CD8⁺ T cells by poxviruses is robust enough to withstand highly potent viral inhibitors of antigen presentation. This is a reminder of the limits of viral immune evasion and shows that viral inhibitors of antigen presentation cannot be assumed to dissect cleanly direct and cross priming of anti-viral CD8⁺ T cells.ImportanceCD8⁺ T cells are key to anti-viral immunity, so it is important to understand how they are activated. Many viruses have proteins that protect infected cells from T cell attack by interfering with the process that allows virus infection to be recognised by CD8⁺ T cells. It is thought that these proteins would also stop infected cells from activating T cells in the first place. However, we show here that this is not the case for two very powerful inhibitory proteins from cowpox virus. This demonstrates the flexibility and robustness of immune processes that turn on the immune responses required to fight infection.

Barbeau DJ, Cartwright HN, Harmon JR, Spengler JR, Spiropoulou CF, Sidney J, Sette A, McElroy AK (2021). "Identification and Characterization of Rift Valley Fever Virus-Specific T Cells Reveals a Dependence on CD40/CD40L Interactions for Prevention of Encephalitis" J Virol 95(23):e0150621. PubMed

Rift Valley fever virus (RVFV) is an arbovirus found throughout Africa. It causes disease that is typically mild and self-limiting; however, some infected individuals experience severe manifestations, including hepatitis, encephalitis, or even death. Reports of RVFV encephalitis are notable among immunosuppressed individuals, suggesting a role for adaptive immunity in preventing this severe complication. This phenomenon has been modeled in C57BL/6 mice depleted of CD4 T cells prior to infection with DelNSs RVFV (RVFV containing a deletion of nonstructural protein NSs), resulting in late-onset encephalitis accompanied by high levels of viral RNA in the brain in 30% of animals. In this study, we sought to define the specific type(s) of CD4 T cells that mediate protection from RVFV encephalitis. The viral epitopes targeted by CD4 and CD8 T cells were defined in C57BL/6 mice, and tetramers for both CD4 and CD8 T cells were generated. RVFV-specific CD8 T cells were expanded and of a cytotoxic and proliferating phenotype in the liver following infection. RVFV-specific CD4 T cells were identified in the liver and spleen following infection and phenotyped as largely Th1 or Tfh subtypes. Knockout mice lacking various aspects of pathways important in Th1 and Tfh development and function were used to demonstrate that T-bet, CD40, CD40L, and major histocompatibility complex class II (MHC-II) mediated protection from RVFV encephalitis, while gamma interferon (IFN-γ) and interleukin-12 (IL-12) were dispensable. Virus-specific antibody responses correlated with protection from encephalitis in all mouse strains, suggesting that Tfh/B cell interactions modulate clinical outcome in this model. IMPORTANCE The prevention of RVFV encephalitis requires intact adaptive immunity. In this study, we developed reagents to detect RVFV-specific T cells and provide evidence for Tfh cells and CD40/CD40L interactions as critical mediators of this protection.

Nagaoka K, Sun C, Kobayashi Y, Kanaseki T, Tokita S, Komatsu T, Maejima K, Futami J, Nomura S, Udaka K, Nakagawa H, Torigoe T, Kakimi K (2021). "Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy" Cancers (Basel) 14(1):106. PubMed

To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8⁺ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

Ito K, Kanaseki T, Tokita S, Torigoe T, Hirasawa N, Ogasawara K (2021). "Palladium-Induced Temporal Internalization of MHC Class I Contributes to T Cell-Mediated Antigenicity" Front Immunol . PubMed

Palladium (Pd) is a widely used metal and extremely important biomaterial for the reconstruction of occlusions during dental restorations. However, metallic biomaterials can cause serious allergic reactions, such as Pd-related oral mucositis seen in dentistry. Metal allergy is categorized as a type IV allergy and we demonstrated that CD8 T cells play an important role in Pd allergy previously. As TCR of CD8 T cells recognizes MHC class I/peptide complex, the antigen specificity to this complex seems to be generated during Pd allergy. However, it remains unknown if Pd affects the MHC class I/peptide complex. In this study, we investigated the behavior of the MHC class I/peptide complex in response to Pd treatment. We found that PdCl₂ treatment altered peptide presentation on MHC class I and that co-culture with Pd-treated DC2.4 cells induced activation of Pd-responsive TCR-expressing T cell line. Furthermore, PdCl₂ treatment induced temporal MHC class I internalization and inhibition of membrane movement suppressed Pd-induced T cell-mediated antigenicity. These data suggest that Pd-induced MHC class I internalization is critical for generation of antigenicity through a mechanism including differential peptide loading on MHC class I, which results in Pd allergy.

Wei P, Jordan KR, Buhrman JD, Lei J, Deng H, Marrack P, Dai S, Kappler JW, Slansky JE, Yin L (2021). "Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer" Proc Natl Acad Sci U S A 118(23):e2100588118. PubMed

Tumors frequently express unmutated self-tumor-associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA-specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I-TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA-specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

Son ET, Faridi P, Paul-Heng M, Leong ML, English K, Ramarathinam SH, Braun A, Dudek NL, Alexander IE, Lisowski L, Bertolino P, Bowen DG, Purcell AW, Mifsud NA, Sharland AF (2021). "The self-peptide repertoire plays a critical role in transplant tolerance induction" J Clin Invest 131(21):e146771. PubMed

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

DeVette CI, Gundlapalli H, Lai SA, McMurtrey CP, Hoover AR, Gurung HR, Chen WR, Welm AL, Hildebrand WH (2019). "A pipeline for identification and validation of tumor-specific antigens in a mouse model of metastatic breast cancer" Oncoimmunology 9(1):1685300. PubMed

Cancer immunotherapy continues to make headway as a treatment for advanced stage tumors, revealing an urgent need to understand the fundamentals of anti-tumor immune responses. Noteworthy is a scarcity of data pertaining to the breadth and specificity of tumor-specific T cell responses in metastatic breast cancer. Autochthonous transgenic models of breast cancer display spontaneous metastasis in the FVB/NJ mouse strain, yet a lack of knowledge regarding tumor-bound MHC/peptide immune epitopes in this mouse model limits the characterization of tumor-specific T cell responses, and the mechanisms that regulate T cell responses in the metastatic setting. We recently generated the NetH2pan prediction tool for murine class I MHC ligands by building an FVB/NJ H-2q ligand database and combining it with public information from six other murine MHC alleles. Here, we deployed NetH2pan in combination with an advanced proteomics workflow to identify immunogenic T cell epitopes in the MMTV-PyMT transgenic model for metastatic breast cancer. Five unique MHC I/PyMT epitopes were identified. These tumor-specific epitopes were confirmed to be presented by the class I MHC of primary MMTV-PyMT tumors and their T cell immunogenicity was validated. Vaccination using a DNA construct encoding a truncated PyMT protein generated CD8 + T cell responses to these MHC class I/peptide complexes and prevented tumor development. In sum, we have established an MHC-ligand discovery pipeline in FVB/NJ mice, identified and tracked H-2D^q/PyMT neoantigen-specific T cells, and developed a vaccine that prevents tumor development in this metastatic model of breast cancer.

Wu T, Guan J, Handel A, Tscharke DC, Sidney J, Sette A, Wakim LM, Sng XYX, Thomas PG, Croft NP, Purcell AW, La Gruta NL (2019). "Quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza CTL responses" Nat Commun 10(1):2846. PubMed

The magnitude of T cell responses to infection is a function of the naïve T cell repertoire combined with the context and duration of antigen presentation. Using mass spectrometry, we identify and quantify 21 class 1 MHC-restricted influenza A virus (IAV)-peptides following either direct or cross-presentation. All these peptides, including seven novel epitopes, elicit T cell responses in infected C57BL/6 mice. Directly presented IAV epitopes maintain their relative abundance across distinct cell types and reveal a broad range of epitope abundances. In contrast, cross-presented epitopes are more uniform in abundance. We observe a clear disparity in the abundance of the two key immunodominant IAV antigens, wherein direct infection drives optimal nucleoprotein (NP)_366-374 presentation, while cross-presentation is optimal for acid polymerase (PA)_224-233 presentation. The study demonstrates how assessment of epitope abundance in both modes of antigen presentation is necessary to fully understand the immunogenicity and response magnitude to T cell epitopes.

Covre A, Coral S, Nicolay H, Parisi G, Fazio C, Colizzi F, Fratta E, Di Giacomo AM, Sigalotti L, Natali PG, Maio M (2015). "Antitumor activity of epigenetic immunomodulation combined with CTLA-4 blockade in syngeneic mouse models" Oncoimmunology 4(8):e1019978. PubMed

The multifaceted immunomodulatory activity of DNA hypomethylating agents improves immunogenicity and immune recognition of neoplastic cells; thus, we predicted they could be utilized to design new immunotherapeutic combinations in cancer. Testing this hypothesis, the antitumor efficacy of the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) combined with the anti-CTLA-4 monoclonal antibody (mAb) 9H10 in syngeneic transplantable murine models was investigated. Murine mammary carcinoma TS/A or mesothelioma AB1 cells were injected in BALB/c, athymic nude, and SCID/Beige mice that were treated with 5-AZA-CdR, mAb 9H10, or their combination. Tumor volumes were captured at different time-points; molecular and immunohistochemical assays investigated changes in neoplastic and normal tissues. A significant antitumor effect of 5-AZA-CdR combined with mAb 9H10 was found: compared to controls, a 77% (p < 0.01), 54% (p < 0.01) and 33% (p = 0.2) decrease in TS/A tumor growth was induced by 5-AZA-CdR combined with mAb 9H10, 5-AZA-CdR or mAb 9H10, respectively. These antitumor activities were confirmed utilizing the AB1 model. 5-AZA-CdR-based regimens induced a promoter-demethylation-sustained tumor expression of cancer testis antigens. MHC class I expression was up-regulated by 5-AZA-CdR. Antitumor efficacy of 5-AZA-CdR in athymic nude and SCID/Beige mice was not increased by mAb 9H10. In BALB/c mice, combined treatment induced the highest tumor infiltration by CD3⁺ lymphocytes, which included both CD8⁺ and CD4⁺ T cells; no such infiltrates were observed in normal tissues. This significant immune-related antitumor activity of 5-AZA-CdR combined with CTLA-4 blockade, demonstrated in highly aggressive mouse tumor models, provides a strong scientific rationale to implement epigenetically-based immunotherapies in cancer patients.

Chen L, Reyes-Vargas E, Dai H, Escobar H, Rudd B, Fairbanks J, Ho A, Cusick MF, Kumánovics A, Delgado J, He X, Jensen PE (2014). "Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity" J Immunol 193(3):1427-39. PubMed

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP(+) and TAP(-) cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

Trujillo JA, Gras S, Twist KA, Croft NP, Channappanavar R, Rossjohn J, Purcell AW, Perlman S (2014). "Structural and functional correlates of enhanced antiviral immunity generated by heteroclitic CD8 T cell epitopes" J Immunol 192(11):5245-56. PubMed

Peptides that bind poorly to MHC class I molecules often elicit low-functional avidity T cell responses. Peptide modification by altering the anchor residue facilitates increased binding affinity and may elicit T cells with increased functional avidity toward the native epitope ("heteroclitic"). This augmented MHC binding is likely to increase the half-life and surface density of the heteroclitic complex, but precisely how this enhanced T cell response occurs in vivo is not known. Furthermore, the ideal heteroclitic epitope will elicit T cell responses that completely cross-react with the native epitope, maximizing protection and minimizing undesirable off-target effects. Such epitopes have been difficult to identify. In this study, using mice infected with a murine coronavirus that encodes epitopes that elicit high (S510, CSLWNGPHL)- and low (S598, RCQIFANI)-functional avidity responses, we show that increased expression of peptide S598 but not S510 generated T cells with enhanced functional avidity. Thus, immune responses can be augmented toward T cell epitopes with low functional avidity by increasing Ag density. We also identified a heteroclitic epitope (RCVIFANI) that elicited a T cell response with nearly complete cross-reactivity with native epitope and demonstrated increased MHC/peptide abundance compared with native S598. Structural and thermal melt analyses indicated that the Q600V substitution enhanced stability of the peptide/MHC complex without greatly altering the antigenic surface, resulting in highly cross-reactive T cell responses. Our data highlight that increased peptide/MHC complex display contributes to heteroclitic epitope efficacy and describe parameters for maximizing immune responses that cross-react with the native epitope.

Washburn LR, Zekzer D, Eitan S, Lu Y, Dang H, Middleton B, Evans CJ, Tian J, Kaufman DL (2011). "A potential role for shed soluble major histocompatibility class I molecules as modulators of neurite outgrowth" PLoS One 6(3):e18439. PubMed

The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCI's actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed "NSE-Db") whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off it's transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptor's signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCI's biological activities in the nervous system.

Escande-Beillard N, Washburn L, Zekzer D, Wu ZP, Eitan S, Ivkovic S, Lu Y, Dang H, Middleton B, Bilousova TV, Yoshimura Y, Evans CJ, Joyce S, Tian J, Kaufman DL (2010). "Neurons preferentially respond to self-MHC class I allele products regardless of peptide presented" J Immunol 184(2):816-23. PubMed

Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination.

Li XL, Liu YY, Knight D, Odaka Y, Mathis JM, Shi R, Glass J, Zhang QJ (2009). "Effect of B7.1 costimulation on T-cell based immunity against TAP-negative cancer can be facilitated by TAP1 expression" PLoS One 4(7):e6385. PubMed

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP(-)) or TAP1-transfected (TAP1(+)) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP(-) and TAP1(+) B7.1 expressing CMT.64 cells depending on the doses of gamma-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1(+) cells rejected TAP(-) tumors, only high dose immunization with B7.1-expressing TAP(-) cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP(-) tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of gamma-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP(-) cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.

Riond J, Rodriguez S, Nicolau ML, al Saati T, Gairin JE (2009). "In vivo major histocompatibility complex class I (MHCI) expression on MHCIlow tumor cells is regulated by gammadelta T and NK cells during the early steps of tumor growth" Cancer Immun . PubMed

Cell surface expression of MHC class I molecules by tumor cells is determinant in the interplay between tumor cells and the immune system. Nevertheless, the mechanisms which regulate MHCI expression on tumor cells are not clear. We previously showed that immune innate cells from the spleen can regulate MHCI expression on MHCI(low) tumor cells. Here, using the murine model of B16 melanoma, we demonstrate that the MHCI status of tumor cells in vivo is regulated by the microenvironment. In subcutaneous grafts, induction of MHCI molecules on tumor cells is concomitant to the recruitment of lymphocytes and relies on an IFNgamma-mediated mechanism. gammadelta T and NK cells are essential to this regulation. A small proportion of tumor-infiltrating NK cells and gammadelta T cells were found to produce IFNgamma, suggesting a possible direct participation to the MHCI increase on the tumor cells upon tumor cell recognition. Depletion of gammadelta T cells increases the tumor growth rate, confirming their anti-tumoral role in our model. Taken together, our results demonstrate that in vivo, NK and gammadelta T cells play a dual role during the early growth of MHCI(low) tumor cells. In addition to controlling the growth of tumor cells, they contribute to modifying the immunogenic profile of residual tumor cells.

Jordan KR, Crawford F, Kappler JW, Slansky JE (2009). "Vaccination of mice with baculovirus-infected insect cells expressing antigenic proteins" Curr Protoc Immunol . PubMed

Methods to induce antigen-specific immune responses in mice using insect cells infected with recombinant baculoviruses are described in this unit. Although this vaccine strategy has been used to generate both antibody and T cell responses, it has been more thoroughly characterized for the peptide-specific cytotoxic T cell responses. Nonspecific responses to the vaccine vehicle are controlled for by vaccinating with insect cells infected with baculoviruses encoding irrelevant antigens or no antigen. The baculovirus-infected insect cells alone are an effective immune adjuvant to elicit antigen-specific T cells. Overall, immune responses generated using this approach are similar to those generated by more conventional vaccine strategies.

Maciel M, Kellathur SN, Chikhlikar P, Dhalia R, Sidney J, Sette A, August TJ, Marques ET (2008). "Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model" Virology 378(1):105-17. PubMed

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice.

Kanaseki T, Blanchard N, Hammer GE, Gonzalez F, Shastri N (2006). "ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum" Immunity 25(5):795-806. PubMed

The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.

Dominiecki ME, Beatty GL, Pan ZK, Neeson P, Paterson Y (2005). "Tumor sensitivity to IFN-gamma is required for successful antigen-specific immunotherapy of a transplantable mouse tumor model for HPV-transformed tumors" Cancer Immunol Immunother 54(5):477-88. PubMed

Purpose: Many human tumors lose responsiveness to IFN-gamma, providing a possible mechanism for the tumor to avoid immune recognition and destruction. Here we investigate the importance of tumor responsiveness to IFN-gamma in the successful immunotherapy of TC1 tumors that were immortalized with human papillomavirus proteins E6 and E7. Methods: To investigate the role of IFN-gamma in vivo, we constructed a variant of TC1, TC1.mugR, that is unresponsive to IFN-gamma due to overexpression of a dominant negative IFN-gamma receptor. Results: Using recombinant Listeria monocytogenes that express HPV-16 E7 (Lm-LLO-E7) to stimulate an antitumor response, we demonstrate that sensitivity to IFN-gamma is required for therapeutic efficacy in that Lm-LLO-E7 induces regression of TC1 tumors but not TC1.mugR. In addition, we show that tumor sensitivity to IFN-gamma is not required for inhibition of tumor angiogenesis by Lm-LLO-E7 or for trafficking of CD4+ and CD8+ T cells to the tumor. However, it is required for penetration of lymphocytes into the tumor mass in vivo. Conclusions: Our findings identify a role for IFN-gamma in immunity to TC1 tumors and show that loss of tumor responsiveness to IFN-gamma poses a challenge to antigen-based immunotherapy.

Wells AD, Rai SK, Salvato MS, Band H, Malkovsky M (1997). "Restoration of MHC class I surface expression and endogenous antigen presentation by a molecular chaperone" Scand J Immunol 45(6):605-12. PubMed

Presentation of cytosolic peptides in the context of major histocompatibility complex (MHC) class I antigen is crucial for immune recognition of virus-infected and malignant cells. This process, which is often defective in cancer cells, involves a series of cellular events which may be facilitated by heat shock proteins (molecular chaperones). To address the influence of chaperone function on the presentation of cytosolic peptides, we have utilized B16 melanoma cells (H-2b). These tumour cells are resistant to lysis by MHC class I-restricted cytotoxic T lymphocytes (CTL), due to a very low level of surface MHC expression. The authors found that stably transfected clones of B16 expressing a heterologous heat shock protein (Hsp65) exhibit significantly increased levels of MHC class I antigens on their surface, and are effectively lysed by alloreactive CTL. These MHC class I molecules can form functional MHC-peptide complexes which are recognized by virus-specific CTL. Moreover, mice immunized with Hsp65-expressing tumour cells, but not with control-transfected tumour cells, display a significantly increased resistance to a subsequent challenge with live, wild-type B16. Together, these results indicate that the suitable expression of a molecular chaperone can overcome a defect in MHC class I expression and antigen presentation, and suggest a novel approach to cancer immunotherapy.

Claesson MH, Endel B, Ulrik J, Pedersen LO, Skov S, Buus S (1994). "Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants" Scand J Immunol 40(2):257-64. PubMed

Beta-2 microglobulin (beta 2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2b, H-2d, as well as H-2k cells by FACS analysis. The MoAbs stained to two beta 2m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous beta 2m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous beta 2m suggesting that xenogeneic beta 2m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26 degrees C, however, staining was reduced five-fold when RMA-S cells were cultured at 37 degrees C. In total, these observations suggest that the MoAbs recognize conformational, presumably beta 2m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epitopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.

Hengel H, Lucin P, Jonjić S, Ruppert T, Koszinowski UH (1994). "Restoration of cytomegalovirus antigen presentation by gamma interferon combats viral escape" J Virol 68(1):289-97. PubMed

An immediate-early protein of murine cytomegalovirus (MCMV), pp89, elicits an immunodominant and protective major histocompatibility complex (MHC) class I Ld-restricted CD8+ T-lymphocyte response. Remarkably, presentation of the naturally processed peptide of pp89, the nonapeptide YPHFMPTNL, is abolished during permissive MCMV infection in vitro. This defect in pp89 presentation is due to the expression of MCMV early gene functions that specifically block the transport of peptide-charged MHC class I complexes to the cell surface (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). Here, we demonstrate that MCMV-specific CD8+ T lymphocytes can reconstitute pp89 presentation in a parakrine fashion. The lymphocytes mediate the restoration of antigen presentation by MCMV-infected cells by releasing gamma interferon (IFN-gamma). IFN-gamma has no effect on synthesis and stability of the viral antigen pp89 nor does it interfere with the expression of viral early genes and their inhibitory effect on MHC class I molecular maturation. IFN-gamma results in a 25-fold increase in the synthesis of MHC class I molecules and a similar increase in the abundance of pp89-derived peptide. Many of the MHC molecules remain retained by the viral effect, but a surplus of MHC molecules escapes the effect and provides the effective surface presentation of the peptide. Adoptive cell transfer studies demonstrate the IFN-gamma dependence of CD8+ T-lymphocyte function in vivo. Altogether, these data reconcile the paradoxical findings of an impaired pp89 presentation in vitro in parallel with pp89-specific CD8+ T-cell protection in vivo. The results also imply a role of IFN-gamma in the T-lymphocyte-mediated control of cytomegalovirus infection. The known propensity of cytomegalovirus to cause serious disease in the immunocompromised host is discussed in the light of these findings.

del Val M, Hengel H, Häcker H, Hartlaub U, Ruppert T, Lucin P, Koszinowski UH (1992). "Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment" J Exp Med 176(3):729-38. PubMed

Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule Ld of a peptide derived from MCMV IE protein pp89 (Reddehase, M.J., J. B. Rothbard, and U.H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide 168YPHFMPTNL176 (del Val, M., H.-J. Schlicht, T. Ruppert, M.J. Reddehase, and U.H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Münch, M.J. Reddehase, and U.H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of beta-galactosidase to the same control and excluded antigen specificity. The Ld-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/cis-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC class I heavy chain, beta 2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment.

Levy RB, Shearer GM (1983). "Cell-mediated lympholytic responses against autologous cells modified with haptenic sulfhydryl reagents. V. H-2Ld self products are recognized by anti-AED-specific cytotoxic T cells" J Immunol 130(4):1506-11. PubMed

The present studies were designed to examine the restriction elements involved in the H-2d haplotype CTL response against AED-self. BALB/c mice were immunized by in vivo administration of syngeneic BALB/c spleen cells conjugated with the sulfhydryl reactive haptenic reagent I-AEDANS. The cytotoxic T cell response generated by subsequent in vitro stimulation with AED-self was found to contain H-2K and H-2D region-specific components. In contrast to the predominant H-2D hapten-self CTL responses induced by amino-reactive haptenic reagents like TNBS and FITC, the AED-self response was predominantly directed against H-2K self products. Antibody inhibition analysis demonstrated that the H-2D region component contained Ld-AED-self CTL, as well as Dd-AED-self CTL. However, although both Ld- and Dd-restricted components could be identified shortly (less than 1 wk) after priming, the H-2D region AED-self CTL response shifted to a single restricting molecule, namely Dd, 1 wk and later after immunization. The results of this study provide the first demonstration that the H-2L locus encodes a cell surface product that can function as a restricting molecule for hapten-self-specific CTL. The shift from Ld and Dd AED-specific CTL to Dd only suggests the existence of a highly specific regulatory mechanism directed against antigen (i.e., hapten) plus self.

Ozato K, Sachs DH (1981). "Monoclonal antibodies to mouse MHC antigens. III. Hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression" J Immunol 126(1):317-21. PubMed

Eleven hybridoma antibodies directed against mouse major histocompatibility complex products of the H-2b haplotype have been produced and characterized. Of 7 antibodies reacting to H-2Kb and/or H-2Db antigens, all cross-reacted with other H-2 antigens, and 5 exhibited no correspondence with a known H-2 specificity established in the H-2 chart. Four anti-Iab antibodies all reacted with antigens encoded by the I-A subregion. Some of these antibodies showed no cross-reaction with other haplotypes, indicating reactions to private specificities of the I-Ab antigen. In addition, these anti-Ia antibodies appeared to be capable of distinguishing fine determinant differences, which corresponding alloantisera failed to reveal. A high frequency of hybridomas secreting IgM antibodies was found after fusions of spleen cells obtained from C3H anti-C3H.SW immunized mice, in contrast to the dominance of IgG hybridomas produced previously by fusions of spleen cells from mice immunized in the reverse direction. An isotype analysis of conventional cytotoxic alloantisera from the same strain combinations was therefore performed. The same correlation with respect to isotype expression was found, indicating that hybridoma antibodies reflect normal antibody responses and suggesting H-2-linked control of this expression.