Direct MHC Class Research Articles

Acute Pharmacologic Degradation of a Stable Antigen enhances its Direct Presentation on MHC class I Molecules

Bifunctional degraders, also referred to as proteolysis-targeting chimeras (PROTACs), are a recently developed class of small molecules. They were designed to specifically target endogenous proteins for ubiquitin/proteasome-dependent degradation and to thereby interfere with pathological mechanisms of diseases, including cancer. In this study, we hypothesized that this process of acute pharmacologic protein degradation might increase the direct MHC class I presentation of degraded targets. By studying this question, we contribute to the discussion about the origin of peptides feeding the MHC class I presentation pathway. Two scenarios have been postulated: peptides can either be derived from homeostatic turnover of mature proteins and/or from short-lived defective ribosomal products (DRiPs), but currently, it is still under debate to what ratio and efficiency both pathways contribute to the overall MHC class I presentation in a specific context. We therefore generated the intrinsically stable model antigen GFP-S8L-F12 that was susceptible to acute pharmacologic degradation via the previously described degradation tag (dTAG) system. Using different murine cell lines, we could show that the bifunctional molecule dTAG-7 induced rapid proteasome-dependent degradation of GFP-S8L-F12 and simultaneously increased its direct presentation on MHC class I molecules. Using the same model in a doxycycline-inducible setting, we could further show that stable, mature antigen was the major source of peptides presented, thereby arguing against a dominant role of DRiPs in our system. This study was, to our knowledge, the first to investigate targeted pharmacologic protein degradation in the context of antigen presentation and our data point towards future applications by strategically combining therapies using bifunctional degraders with their stimulating effect on direct MHC class I presentation.

MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.

Direct MHC Class I Antigen Presentation Regulation by Ribosomal Biogenesis Inhibitor CX-5461

Abstract Direct MHC class I antigen presentation relies on on-going protein synthesis by the antigen presenting cell. This is especially true for the presentation of peptides derived from Defective Ribosomal Products (DRiPs) which are inherently defective proteins rapidly degraded following their synthesis. Recently an inhibitor of RNA polymerase I, known as CX-5461, has been described which can inhibit ribosome biogenesis. Here we queried if interruption of ribosome biogenesis could impact direct antigen presentation using a reporter protein whose stability can be controlled post-translationally. We first determined a dose and time of treatment with CX-5461 which was not toxic to cells, but greatly decreased the levels of RNA within EL4 cells. Once an acceptable dose was determined, we examined presentation of a model peptide derived from the Shield-controlled recombinant antigenic protein (SCRAP) which is a fusion protein consisting of a destabilization domain, the SIINFEKL peptide, and a fluorescent reporter protein. SCRAP levels can be controlled through the addition of the small molecule Shield-1, which stabilizes SCRAP and prevents its degradation. After treatment with CX-5461, we found the levels of SCRAP protein partially decreased within the cell, indicating a loss in protein synthesis. Concurrently, presentation of the reporter peptide was diminished from both the DRiP and non-DRiP form of SCRAP. These data indicate that interruption of ribosome biogenesis has a minor impact on direct antigen presentation and protein synthesis.

Substrate-specific presentation of MHC class I-restricted antigens via autophagy pathway

The accumulation of protein aggregates is toxic and linked to different diseases such as neurodegenerative disorders, but the role of the immune system to target and destroy aggregate-carrying cells is still relatively unknown. Here we show a substrate-specific presentation of antigenic peptides to the direct MHC class I pathway via autophagy. We observed no difference in presentation of peptides derived from the viral EBNA1 protein following suppression of autophagy by knocking down Atg5 and Atg12. However, the same knock down treatment suppressed the presentation from ovalbumin. Fusing the aggregate-prone poly-glutamine (PolyQ) to the ovalbumin had no effect on antigen presentation via autophagy. Interestingly, fusing the EBNA1-derived gly-ala repeat (GAr) sequence to ovalbumin rendered the presentation Atg5/12 independent. We also demonstrate that the relative levels of protein expression did not affect autophagy-mediated antigen presentation. These data suggest a substrate-dependent presentation of antigenic peptides for the MHC class I pathway via autophagy and indicate that the GAr of the EBNA1 illustrates a novel virus-mediated mechanism for immune evasion of autophagy-dependent antigen presentation.

Targeted protein degradation via NEDD8 conjugation does not enhance direct MHC class I antigen presentation.

Abstract Knowledge of different pathways contributing to peptide generation for direct MHC class I antigen presentation is important to the field of immunotherapy. Here we investigated the role of the ubiquitin-like protein NEDD8 in antigen presentation. We show that proteins tagged N-terminally with NEDD8 undergo rapid degradation via the proteasomal, and autophagy-lysosomal pathways. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule K b , and compared the results to ovalbumin N-terminally modified with ubiquitin. While NEDD8-OVA was processed for degradation via the proteasomal and autophagosomal pathway Ub-OVA was only degraded via the proteasome. Peptide presentation from NEDD8-OVA was dependent on NEDD8-ultimate buster 1 (NUB1), while autophagy did not contribute peptides antigen presentation. Overall, we found that N-terminal Ub conjugation was far more effective at generating antigenic peptides than N-terminal NEDD8 conjugation. However, upon inhibition of NEDDYylation by treatment with MLN4924, we did detect a decrease in peptide presentation from a model antigen used specifically to track Defective Ribosomal Product (DRiP) presentation. These data demonstrate that while NEDD8 can cause the rapid degradation of substrates it is not as efficient as Ubiquitin for generating antigenic peptides. Our data provides a basis for understanding the role of NEDD8 in protein degradation and antigen presentation, especially in conditions of NEDD8 dysregulation, such as in some cancers and protein folding pathologies.

The Unfolded Protein Response reveals eIF2α phosphorylation as a critical factor for direct MHC class I antigen presentation.

Abstract Modifying direct Major Histocompatibility Class I (MHC I) antigen presentation is an attractive target for the treatment of various conditions including autoimmune diseases, chronic viral infections, and cancers. Disease perturbations alter the cellular environment, influencing the presentation of peptides to CD8+ T cells. The unfolded protein response (UPR) is a signaling pathway activated in response to unfolded proteins within the ER. Previous studies have established the relationship between the UPR and MHC I antigen presentation, but the exact mechanisms remained unknown. In this study, we utilized small inhibitory molecules to individual UPR pathway targets to investigate the effect on global MHC class I levels and specific peptide presentation. Preventing PERK pathway signaling and the resulting phosphorylation of eIF2α by GSK2656157 treatment did not change MHC I antigen presentation. However, treatment with salubrinal which has the opposite effect of GSK2656157, decreased both antigen presentation and overall cell-surface MHC I levels. Treatment with 4μ8C, an inhibitor of the IRE1α UPR activation pathway which prevents splicing of Xbp1 mRNA, also diminished MHC I antigen presentation. Unexpectedly, 4μ8C treatment led to an increase in eIF2α phosphorylation in addition to blocking IRE1α signaling. Given that salubrinal and 4μ8C increase phosphorylation levels of eIF2α while decreasing antigen presentation, we conclude that UPR signaling through PERK and the subsequent phosphorylation of eIF2α, results in a modest decrease in direct MHC I antigen presentation. These data suggest that the PERK pathway alters host antigen presentation centering on translational control by eIF2α.

The Unfolded Protein Response Reveals eIF2α Phosphorylation as a Critical Factor for Direct MHC Class I Antigen Presentation.

The ability to modulate direct MHC class I (MHC I) Ag presentation is a desirable goal for the treatment of a variety of conditions, including autoimmune diseases, chronic viral infections, and cancers. It is therefore necessary to understand how changes in the cellular environment alter the cells' ability to present peptides to T cells. The unfolded protein response (UPR) is a signaling pathway activated by the presence of excess unfolded proteins in the endoplasmic reticulum. Previous studies have indicated that chemical induction of the UPR decreases direct MHC I Ag presentation, but the precise mechanisms are unknown. In this study, we used a variety of small molecule modulators of different UPR signaling pathways to query which UPR signaling pathways can alter Ag presentation in both murine and human cells. When signaling through the PERK pathway, and subsequent eIF2α phosphorylation, was blocked by treatment with GSK2656157, MHC I Ag presentation remain unchanged, whereas treatment with salubrinal, which has the opposite effect of GSK2656157, decreases both Ag presentation and overall cell-surface MHC I levels. Treatment with 4μ8C, an inhibitor of the IRE1α UPR activation pathway that blocks splicing of Xbp1 mRNA, also diminished MHC I Ag presentation. However, 4μ8C treatment unexpectedly led to an increase in eIF2α phosphorylation in addition to blocking IRE1α signaling. Given that salubrinal and 4μ8C lead to eIF2α phosphorylation and similar decreases in Ag presentation, we conclude that UPR signaling through PERK, leading to eIF2α phosphorylation, results in a modest decrease in direct MHC I Ag presentation.

Targeted protein degradation via NEDD8 conjugation does not enhance direct MHC class I antigen presentation

Abstract MHC class I molecules present peptide antigens derived from proteins synthesized and degraded within the antigen presenting cell. An understanding the protein degradation pathways that contribute to peptide generation is necessary for developing next generation immunotherapies. We recently discovered that conjugation of the ubiquitin-like molecule NEDD8 to the N-terminus of a protein can result in the rapid degradation of the target protein by either the ubiquitin-proteasome system (UPS) and the autophagy pathway. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule Kb using a monoclonal antibody specific for the Kb-SIINFEKL complex. Like other constructs we have characterized, NEDDylated OVA was rapidly degraded by the UPS and autophagy pathways. While proteasome inhibition prevented presentation of SIINFEKL peptides, inhibiting autophagy did not affect antigen presentation. We then compared presentation of NEDDylated OVA to ubiquitin conjugated OVA and found that ubiquitin conjugation was far more effective at generating antigenic peptides. These data demonstrate that rapid degradation per se of an antigenic protein is not sufficient to enhance antigen presentation but rather the precise manner in which a protein is degraded governs the efficiency of peptide generation. Our findings are consistent with the idea that protein degradation is organized in some way to optimize generation the immunopeptidome for immunosurveillance.

Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

Inhibition of the Deubiquitinase Usp14 Diminishes Direct MHC Class I Antigen Presentation.

Infected or transformed cells must present peptides derived from endogenous proteins on MHC class I molecules to be recognized and targeted for elimination by Ag-specific cytotoxic T cells. In the first step of peptide generation, proteins are degraded by the proteasome. In this study, we investigated the role of the ubiquitin-specific protease 14 (Usp14), a proteasome-associated deubiquitinase, in direct Ag presentation using a ligand-stabilized model protein expressed as a self-antigen. Chemical inhibition of Usp14 diminished direct presentation of the model antigenic peptide, and the effect was especially pronounced when presentation was restricted to the defective ribosomal product (DRiP) form of the protein. Additionally, presentation specifically from DRiP Ags was diminished by expression of a catalytically inactive form of Usp14. Usp14 inhibition did not appreciably alter protein synthesis and only partially delayed protein degradation as measured by a slight increase in the half-life of the model protein when its degradation was induced. Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP-derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.

Acute Pharmacologic Degradation of a Stable Antigen Enhances Its Direct Presentation on MHC Class I Molecules.

Bifunctional degraders, also referred to as proteolysis-targeting chimeras (PROTACs), are a recently developed class of small molecules. They were designed to specifically target endogenous proteins for ubiquitin/proteasome-dependent degradation and to thereby interfere with pathological mechanisms of diseases, including cancer. In this study, we hypothesized that this process of acute pharmacologic protein degradation might increase the direct MHC class I presentation of degraded targets. By studying this question, we contribute to an ongoing discussion about the origin of peptides feeding the MHC class I presentation pathway. Two scenarios have been postulated: peptides can either be derived from homeostatic turnover of mature proteins and/or from short-lived defective ribosomal products (DRiPs), but currently, it is still unclear to what ratio and efficiency both pathways contribute to the overall MHC class I presentation. We therefore generated the intrinsically stable model antigen GFP-S8L-F12 that was susceptible to acute pharmacologic degradation via the previously described degradation tag (dTAG) system. Using different murine cell lines, we show here that the bifunctional molecule dTAG-7 induced rapid proteasome-dependent degradation of GFP-S8L-F12 and simultaneously increased its direct presentation on MHC class I molecules. Using the same model in a doxycycline-inducible setting, we could further show that stable, mature antigen was the major source of peptides presented, thereby excluding a dominant role of DRiPs in our system. This study is, to our knowledge, the first to investigate targeted pharmacologic protein degradation in the context of antigen presentation and our data point toward future applications by strategically combining therapies using bifunctional degraders with their stimulating effect on direct MHC class I presentation.

Cutting Edge: The Aging Immune System Reveals the Biological Impact of Direct Antigen Presentation on CD8 T Cell Responses.

The vertebrate immune system uses multiple, sometimes redundant, mechanisms to contain pathogenic microorganisms that are always evolving to evade host defenses. Thus, the cowpox virus (CPXV) uses genes encoding CPXV12 and CPXV203 to prevent direct MHC class I presentation of viral peptides by infected cells. However, CD8 T cells are effectively primed against CPXV by cross-presentation of viral Ags in young mice. Old mice accumulate defects in both CD8 T cell activation and cross-presentation. Using a double-deletion mutant (∆12∆203) of CPXV, we show that direct priming of CD8 T cells in old mice yields superior recall responses, establishing a key contribution of this mechanism to host antipoxvirus responses and enhancing our fundamental understanding of how viral manipulation of direct presentation impacts pathogenesis. This also provides a proof of principle that suboptimal CD8 T cell in old organisms can be optimized by manipulating Ag presentation, with implications for vaccine design.

Chlamydial infection and the efficiency of MHC class I self-antigen presentation in a lymphoblastoid cell line

Abstract The direct MHC class I antigen presentation pathway presents cytosolic proteins for immune surveillance. While this pathway is an attractive target for therapeutics, we must first understand the efficiency of antigen presentation. Previous studies have shown the presentation efficiency from pathogen-derived antigens ranges from 0.03–25%. The intracellular lifestyle of Chlamydia makes them an attractive model to study class I presentation. It is likely that Chlamydia have developed mechanisms to evade CD8+ T cells through manipulation of this pathway. Here, we investigate the impact of Chlamydial infection on a recombinant fusion protein expressed in a human lymphoblastoid cell line (JY). The fusion protein contains a destabilization domain, an antigenic peptide, and a reporter. Chlamydial infection led to decreased accumulation of the model protein and increased presentation of model protein-derived antigens. This enhanced self-peptide presentation was only observed when antigen presentation was restricted to the DRiP form of the protein. We also investigated the antigen presentation efficiency of different model recombinant proteins in JY cells. We calculated the antigen presentation efficiency to be 0.35%, much lower than was previously observed in murine cells with a similar protein. The skewed antigen presentation phenotype observed following Chlamydial infection was not statistically different from the efficiency observed in the absence of infection. These data suggest that Chlamydia have evolved a mechanism to skew the host antigen presentation machinery toward presentation of DRiPs, and that the efficiency of direct class I presentation may be mechanistically unique among common immune signaling pathways.

Calculating the efficiency of direct MHC class I antigen presentation of a model antigen in a lymphoblastoid cell line (APP5P.110)

Abstract The direct MHC class I antigen presentation pathway presents endogenous peptides at the cell surface, allowing the body to mount a CD8+ T cell response in order to eliminate diseased or damaged cells. In order to target this pathway for disease treatment, we must first understand the efficiency of antigen presentation, or the number of proteins that must be degraded in order to present one peptide-MHC complex at the cell surface. Previous studies have primarily examined the efficiency of proteins produced by infectious agents with values ranging from 0.03%-25. In this study, we investigated antigen presentation efficiency of different recombinant fusion proteins expressed in a human lymphoblastoid cell line expressed as self-proteins. These proteins are composed of a destabilization domain, an antigenic peptide that is presented on MHC class I HLA-A2 molecules, and a fluorescent reporter. We calculated the efficiency of antigen presentation in JY cells to be 0.35%. This value is considerably lower than what was previously observed with similar constructs in murine EL4 cells. We also found that treatment of the cells with either TLR agonists or IFNg, which have both been shown to increase MHC class I expression, did not alter the efficiency of antigen presentation. These findings suggest that the efficiency of MHC class I antigen presentation may be mechanistically distinct from common immune signaling pathways.

Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

Sensing of low-dose soluble antigen via Immunoglobulin E (106.1)

Abstract Dendritic cells (DCs) are the most potent antigen presenting cells and continuously encounter exogenous antigens at low dose. We show here that DCs use IgE-mediated antigen uptake to efficiently sense soluble antigen at a dose range that otherwise escapes immune detection. IgE-mediated antigen sampling via Fc-epsilon-RI simultaneously shuttles into the direct MHC class II-restricted presentation and the cross-presentation pathway resulting in the activation of Th2-type CD4+ T cells as well as cytotoxic CD8+ T cells (CTLs). Fc-epsilon-RI stabilizes at the cell surface of DCs after monovalent ligation with IgE. Antigen-mediated crosslinking of the receptor induces internalization into endo/lysosomal compartments. Absence of IL-12 production during IgE-mediated antigen uptake and presentation is critical for shifting the CD4 response towards Th2, but does not affect the generation of CTLs. Presence of IL-12 during IgE-mediated presentation redirects the immune response towards Th1 by suppressing the production of IL-4 and IL-13. Neither basophils nor mast cells can induce primary T cell responses after antigen sampling via IgE. These results establish a uniquely sensitive pathway for the detection of soluble exogenous antigen by DCs with high relevance for IgE-mediated immune responses in humans.

Viral infection sensitizes hepatocytes towards TNF-induced apoptosis

We identified a novel mechanism by which CD8 T cells selectively eliminate virus-infected hepatocytes without direct MHC class I recognition. This mechanism involves TNF secreted by CD8 T cells, which selectively induces death of infected hepatocytes. Triggering of the TNF receptor can induce both, pro-survival pathways via NFkB activation, and apoptotic pathways involving generation of ROS and caspase activation. The molecular mechanisms directing into one or the other pathway are still unknown. However, in virus-infected hepatocytes stimulation of the TNF receptor induces apoptotic pathways, whereas uninfected hepatocytes survive. Here, we investigate the molecular mechanism(s) underlying the sensitization of infected hepatocytes towards the apoptosis-inducing effect of TNF.

HIV Nef-mediated major histocompatibility complex class I down-modulation is independent of Arf6 activity.

HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.

Cutting edge: B cells promote CD8+ T cell activation in MRL-Fas(lpr) mice independently of MHC class I antigen presentation.

Spontaneous CD8+ T cell activation in MRL-Faslpr mice is B cell dependent. It is unclear whether this B-dependent activation is mediated by direct Ag presentation via MHC class I proteins (i.e., cross-presentation) or whether activation occurs by an indirect mechanism, e.g., via effects on CD4+ cells. To determine how CD8+ T cell activation is promoted by B cells, we created mixed bone marrow chimeras where direct MHC class I Ag presentation by B cells was abrogated while other leukocyte compartments could express MHC class I. Surprisingly, despite the absence of B cell class I-restricted Ag presentation, CD8+ T cell activation was intact in the chimeric mice. Therefore, the spontaneous B cell-dependent CD8+ T cell activation that occurs in systemic autoimmunity is not due to direct presentation by B cells to CD8+ T cells.

CREB Regulates MHC Class II Expression in a CIITA-Dependent Manner

The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.