Downregulate Major Histocompatibility Complex Class Research Articles

In vitro cellular responses to Neospora caninum glycosylphosphatidylinositols depend on the host origin of antigen presenting cells

Neosporosis due to Neospora caninum causes abortions in farm animals such as cattle. No treatment and vaccine exist to fight this disease, responsible for considerable economic losses. It is thus important to better understand the immune responses occurring during the pathogenesis to control them in a global strategy against the parasite. In this context, we studied the roles of N. caninum glycosylphosphatidylinositols (GPIs), glycolipids defined as toxins in the related parasite Plasmodium falciparum. We demonstrated for the first time that GPIs could be excreted in the supernatant of N. caninum culture and trigger cell signalling through the Toll-like receptors 2 and 4. In addition, antibodies specific to N. caninum GPIs were detected in the serum of infected mice. As shown for other protozoan diseases, they could play a role in neutralizing GPIs. N. caninum GPIs were able to induce the production of tumour necrosis factor-α, interleukin(IL)-1β and IL-12 cytokines by murine macrophages and dendritic cells. Furthermore, GPIs significantly reduced expression of major histocompatibility complex (MHC) molecules of class I on murine dendritic cells. In contrast to murine cells, bovine blood mononuclear cells produced increased levels of IFN-γ and IL-10, but reduced levels of IL-12p40 in response to GPIs. On these bovine cells, GPI had the tendency to up-regulate MHC class I, but to down-regulate MHC class II. Altogether, these results suggest that N. caninum GPIs might differentially participate in the responses of antigen presenting cells induced by the whole parasite in mouse models of neosporosis and in the natural cattle host.

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients

ABSTRACTPurpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma.Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line).Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells’ cytotoxicity.Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.

PD-1/PD-L1 Blockade Therapy for Tumors with Downregulated MHC Class I Expression.

The therapy of different advanced-stage malignancies with monoclonal antibodies blocking programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) signaling has had an impressive long-lasting effect in a portion of patients, but in most cases, this therapy was not successful, or a secondary resistance developed. To enhance its efficacy in treated patients, predictive biomarkers are searched for and various combination treatments are intensively investigated. As the downregulation of major histocompatibility complex (MHC) class I molecules is one of the most frequent mechanisms of tumor escape from the host’s immunity, it should be considered in PD-1/PD-L1 checkpoint inhibition. The potential for the use of a PD-1/PD-L1 blockade in the treatment of tumors with aberrant MHC class I expression is discussed, and some strategies of combination therapy are suggested.

Emerging Major Histocompatibility Complex Class I-Related Functions of NLRC5.

Recent evidence demonstrates a key role for the nucleotide-binding oligomerization domain-like receptor (NLR) family member NLRC5 (NLR family, CARD domain containing protein 5) in the transcriptional regulation of major histocompatibility complex (MHC) class I and related genes. Detailed information on NLRC5 target genes in various cell types and conditions is emerging. Thanks to its analogy to CIITA (class II major MHC transactivator), a NLR family member known for over 20 years to be the master regulator of MHC class II gene transcription, also the molecular mechanisms underlying NLRC5 function are being rapidly unraveled. MHC class I molecules are crucial in regulating innate and adaptive cytotoxic responses. Whereas CD8+ T cells detect antigens presented on MHC class I molecules by infected or transformed cells, natural killer (NK) lymphocytes eliminate target cells with downregulated MHC class I expression. Data uncovering the relevance of NLRC5 in homeostasis and activity of these two lymphocyte subsets have been recently reported. Given the importance of CD8+ T and NK cells in controlling infection and cancer, it is not surprising that NLRC5 is also starting to emerge as a central player in these diseases. This chapter summarizes and discusses novel insights into the molecular mechanisms underlying NLRC5 activity and its relevance to pathological conditions. A thorough understanding of both aspects is essential to evaluate the clinical significance and therapeutic potential of NLRC5.

Using Human Cytomegalovirus Glycoproteins to Prevent Graft versus Host Disease through Downregulation of Major Histocompatibility Complex Class I and Class II: A Novel Approach

Graft Versus Host Disease (GVHD) is an immune-mediated disease occurs as a complication of allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Differences between donor and recipient Major Histocompatibility Complex (MHC) antigens initiate the disease. The immunocompetent cells of the donor recognize the cells of the immunocompromised host as non-self, thus commencing an immune response against them. The human Cytomegalovirus (hCMV) is a member of the herpesvirus family that has developed strategies to escape the immune surveillance and defense system via encoding a series of glycoproteins that down regulate host MHC antigens. The Unique Short (US) hCMV glycoproteins US2, US3, US6, US10 and US11 have shown variable capabilities to downregulate MHC class I and II. Theoretically, these capabilities could be utilized to downregulate the expression of host MHC antigens, thus inhibiting the allograft recognition and the subsequent immune response, which would prevent GVHD. In this systematic review, 620 literatures have been identified through a PubMed, Epistemonikos, and Google Scholar search. An inclusion criterion has been applied to these studies, of which 27 have been selected. This review found that the hCMV glycoproteins act as partner to downregulate MHC class I and class II, CMV glycoproteins regulate destruction of class I MHC molecules, and degrade MHC class II. The Preferred Reporting Items for Systematic Reviews (PRISMA) Statement has been used to increase the quality of the review, and thus a Population Intervention Comparison Outcome Study design (PICOS) model has been formulated. The findings of this research could be further studied and validated to offer an alternative approach to the current pharmacological preventive measures of GVHD, possibly without compromising patients’ immunity.

Mechanical stress downregulates MHC class I expression on human cancer cell membrane.

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm−1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

IK Induced by Coxsackievirus B3 Infection Transiently Downregulates Expression of MHC Class II Through Increasing cAMP

Major histocompatibility complex (MHC) class II expression is critical for the presentation of antigens in the immune response to viral infection. Consequently, some viruses regulate the MHC class II-mediated presentation of viral antigens as a mechanism of immune escape. In this study, we found that Coxsackievirus B3 (CVB3) infection transiently increased IK expression, which reduced the expression of MHC class II (I-A/I-E) on splenic B cells. Interestingly, CVB3-induced IK elevated cAMP, a downstream molecule of the G protein-coupled receptors, which inhibited MHC class II presentation on B cells. Transgenic mice expressing truncated IK showed lower expression of MHC class II on B cells than did wild-type mice after CVB3 infection. Taken together, these results imply that IK plays a role in downregulating MHC class II expression on B cells during CVB3 infection through the induction of cAMP.

Staying undetected

Downregulation of major histocompatibility complex (MHC) molecules is a well-recognized immune evasion mechanism used by several viruses. Cowpox virus (CPXV), for example, encodes the immune evasion protein CPXV203, which inhibits MHC class I trafficking by sequestering it in the endoplasmic reticulum (ER). However, CPXV203-deficient CPXV can still downregulate MHC class I surface expression, indicating that another mechanism must also be involved. Using distinct approaches, Byun et al. and Alzhanova et al. now show that the previously unknown cowpox protein CPXV12 mediates immune evasion by inhibiting effective MHC class I peptide biosynthesis.

Hsp90-mediated Assembly of the 26 S Proteasome Is Involved in Major Histocompatibility Complex Class I Antigen Processing

Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90alpha to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90alpha- and PA28alpha-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90alpha and PA28alpha, whereas two different epitopes were identified in the hsp90alpha- and PA28alpha-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.

A new strategy to understand how HIV infects women: identification of a window of vulnerability during the menstrual cycle

Although 85% of new HIV cases are due to sexual transmission from men to women little attention is being paid to the immune system in the female reproductive tract (FRT) and to how it meets the conflicting challenges of protecting from pathogens and permitting procreation. As a new approach we have tried to envision how HIV evades FRT mucosal immune protection and have been led to the unexpected conclusion that in a normal menstrual cycle there is a window of vulnerability (7-10 days following ovulation) in which the potential for viral infectivity in the FRT is enhanced. During that period aspects of the innate humoral and cell-mediated immune systems are suppressed by sex hormones to optimize conditions for procreation. Suppression occurs in the upper (Fallopian tubes uterus endocervix) and lower (ectocervix and vagina) FRT and coincides with the recruitment of potentially infectable cells and upregulation of coreceptors essential for viral uptake. Implications of these findings are that the entire FRT is a potential target for HIV infection immune cells and antibodies in blood are not surrogate markers for immune protection in the FRT and immune protection against HIV will require an understanding of the hormone-induced regulation of humoral cellmediated and innate immune systems throughout the FRT. (excerpt)

Cowpox Virus Exploits the Endoplasmic Reticulum Retention Pathway to Inhibit MHC Class I Transport to the Cell Surface

Major histocompatibility complex (MHC) class I molecules assemble with peptides in the ER lumen and are transported via Golgi to the plasma membrane for recognition by T cells. Inhibiting MHC assembly, transport, and surface expression are common viral strategies of evading immune recognition. Cowpox virus, a clinically relevant orthopoxvirus, downregulates MHC class I expression on infected cells. However, the viral protein(s) and mechanisms responsible are unknown. We identify CPXV203 as a cowpox virus protein that associates with fully assembled MHC class I molecules and blocks their transport through the Golgi. A C-terminal KTEL motif in CPXV203 closely resembles the canonical ER retention motif KDEL and is required for CPXV203 function, indicating that a physiologic pathway is exploited to retain MHC class I in the ER. This viral mechanism for MHC class I downregulation may explain virulence differences between clinical isolates of orthopoxviruses.

Chemotherapy Enhances CD8+ T Cell-mediated Antitumor Immunity Induced by Vaccination With Vaccinia Virus

The use of immunotherapy or chemotherapy alone is generally ineffective against well-established tumors. To overcome this intrinsic resistance against therapy for tumors, we have attempted to combine immunotherapy with chemotherapy. In this study, we tried to induce a rapid antitumor effect via chemoimmunotherapy using a vaccinia viral vaccine as an immunotherapeutic agent with anticancer agents including epigallocatechin-3-gallate (EGCG) and conventional anticancer drugs. Although a combination of vaccinia-mediated vaccination and chemotherapy led to a strong inhibition of tumor growth, monotherapy alone failed to completely cure tumors. In contrast, intravenous injection of cisplatin (CDDP) or cyclophosphamide (CTX) after vaccinia virus vaccination led to complete regression of the established tumors. Interestingly, anticancer drugs appear to augment the antitumor effect of the vaccinia virus-mediated immunotherapy. This effect is mainly associated with the enhanced tumor-specific CD8(+) T cell immune response induced by vaccinia virus, which was demonstrated by antibody depletion. However, anticancer drugs alone failed to induce a significant enhancement of the tumor-specific CD8(+) T cell immune response. Taken together, these results suggest that combining vaccinia virus-based immunotherapy with anticancer drugs is particularly effective against established tumors by increasing the tumor antigen-specific CD8(+) T cell immune response, which is primed by vaccinia virus-mediated vaccination.

Transcriptional regulation of major histocompatibility complex class I gene by insulin and IGF-I in FRTL-5 thyroid cells

Increased major histocompatibility complex (MHC) class I gene expression in nonimmune cell 'target tissues' involved in organ-specific diseases may be important in the pathogenesis of autoimmune diseases. This possibility in part evolves from studies of cultured thyrocytes where properties appear relevant to the development of thyroid autoimmune disease. In FRTL-5 rat thyroid cells in continuous culture, hormones and growth factors that regulate cell growth and function specifically decrease MHC class I gene expression. We hypothesized that this could reflect a mechanism to preserve self-tolerance and prevent autoimmune disease. The mechanisms of action of some of these hormones, namely TSH and hydrocortisone, have been already characterized. In this report, we show that IGF-I transcriptionally downregulates MHC class I gene expression and that its action is similar to that of insulin. The two hormones have a complex effect on the promoter of the MHC class I gene, PD1. In fact, they decrease the full promoter activity, but upregulate the activity of deleted mutants that have lost an upstream, tissue-specific regulatory region but still retain the enhancer A region. We show that insulin/IGF-I promotes the interactions of the p50/p65 subunits of NF-kappaB and AP-1 family members with these two regions, and that the tissue-specific region acts as a dominant silencer element on insulin/IGF-I regulation of promoter activity. These observations may be important to understand how MHC class I gene transcription is regulated in the cells.

Impact of Nef-Mediated Downregulation of Major Histocompatibility Complex Class I on Immune Response to Simian Immunodeficiency Virus

Functional activities that have been ascribed to the nef gene product of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) include CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, downregulation of other plasma membrane proteins, and lymphocyte activation. Monkeys were infected experimentally with SIV containing difficult-to-revert mutations in nef that selectively eliminated MHC downregulation but not these other activities. Monkeys infected with these mutant forms of SIV exhibited higher levels of CD8(+) T-cell responses 4 to 16 weeks postinfection than seen in monkeys infected with the parental wild-type virus. Furthermore, unusual compensatory mutations appeared by 16 to 32 weeks postinfection which restored some or all of the MHC-downregulating activity. These results indicate that nef does serve to limit the virus-specific CD8 cellular response of the host and that the ability to downregulate MHC class I contributes importantly to the totality of nef function.

Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors.

The accessory Nef protein allows human immunodeficiency virus type 1 (HIV-1) to persist at high levels and to cause AIDS in infected humans. The function of HIV-1 group M subtype B nef alleles has been extensively studied, and a variety of in vitro activities believed to be important for viral pathogenesis have been established. However, the function of nef alleles derived from naturally simian immunodeficiency virus (SIV)-infected chimpanzees, the original host of HIV-1, or from the HIV-1 N and O groups resulting from independent zoonotic transmissions remains to be investigated. In the present study we demonstrate that SIVcpz and HIV-1 group N or O nef alleles down-modulate CD4, CD28, and class I or II MHC molecules and up-regulate surface expression of the invariant chain (Ii) associated with immature major histocompatibility complex (MHC) class II. Furthermore, the ability of Nef to interact with the p21-activated kinase 2 was generally conserved. The functional activity of HIV-1 group N and O nef genes did not differ significantly from group M nef alleles. However, SIVcpz nef genes as a group showed a 1.8- and 2.0-fold-higher activity in modulating CD28 (P = 0.0002) and Ii (P = 0.016) surface expression, respectively, but were 1.7-fold less active in down-regulating MHC class II molecules (P = 0.006) compared to HIV-1 M nef genes. Our finding that primary SIVcpz nef alleles derived from naturally infected chimpanzees modulate the surface expression of various human cellular receptors involved in T-cell activation and antigen presentation suggests that functional nef genes helped the chimpanzee virus to persist efficiently in infected humans immediately after zoonotic transmission.

Collapse and Restoration of MHC Class-I-Dependent Immune Privilege: Exploiting the Human Hair Follicle as a Model

The collapse of major histocompatibility complex (MHC) class-I-dependent immune privilege can lead to autoimmune disease or fetal rejection. Pragmatic and instructive models are needed to clarify the as yet obscure controls of MHC class I down-regulation in situ, to dissect the principles of immune privilege generation, maintenance, and collapse as well as to develop more effective strategies for immune privilege restoration. Here, we propose that human scalp hair follicles, which are abundantly available and easily studied, are ideally suited for this purpose: interferon-gamma induces ectopic MHC class I expression in the constitutively MHC class-I-negative hair matrix epithelium of organ-cultured anagen hair bulbs, likely via interferon regulatory factor-1, along with up-regulation of the MHC class I pathway molecules beta(2)microglobulin and transporter associated with antigen processing (TAP-2). In the first report to identify natural immunomodulators capable of down-regulating MHC class I expression in situ in a normal, neuroectoderm-derived human tissue, we show that ectopic MHC class I expression in human anagen hair bulbs can be normalized by treatment with alpha-MSH, IGF-1, or TGF-beta1, all of which are locally generated, as well as by FK506. These agents are promising candidates for immune privilege restoration and for suppressing MHC class I expression where this is clinically desired (eg, in alopecia areata, multiple sclerosis, autoimmune uveitis, mumps orchitis, and fetal or allograft rejection).

Ectopic transplantation of equine invasive trophoblast.

A system for transplanting invasive equine trophoblast (i.e., chorionic girdle) to ectopic sites has been developed as a means to study the differentiation of this tissue and to assess maternal immune responses to the conceptus tissue in a site outside the uterus. Chorionic girdle was isolated from Day 33 to 34 conceptuses and surgically placed into the vulvar mucosa or subdermal skin of recipient mares. Biopsy specimens of the graft sites for immunohistochemical staining were taken at weekly or biweekly intervals after grafting. Serum samples were collected from each recipient and tested for antibody to donor major histocompatibility complex (MHC) class I antigens using the lymphocyte microcytotoxicity assay. Transplanted trophoblast cells expressed differentiation markers associated with invading chorionic girdle and endometrial cup cells. The transplanted trophoblast cells were also labeled by an antibody to eCG. Strong cellular and humoral immune responses to the transplanted tissue were mounted by the recipients, similar to those occurring during normal equine pregnancy. Despite these responses, the invasive trophoblast transplants survived for at least 28 days after grafting and downregulated MHC class I antigens, as do the mature endometrial cup cells in equine pregnancy. These findings suggest that invasive equine trophoblast has the capacity to differentiate fully in equine nonuterine tissues, and that it can evade maternal immune responses independent of the physiological state of pregnancy and in sites other than the uterus.

Retroviral Antibody Binding of the MHC Class II Molecule: A Biochemical Influence on CD4 T Cell Differentiation in HIV Infection?

Retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule has been documented in human immunodeficiency virus-1 (HIV-1)-infected patients. Interactions between the MHC Class II receptor and the T-cell receptor (TCR) are central to the immune response. Importantly, retroviral antibody possesses a much higher binding affinity for the MHC Class II receptor, when compared to the TCR. Experiments have manipulated a number of factors related to antigen-presenting cell (APC) interaction with differentiating T-cells. These studies have observed the effects of lowering antigen dose and reducing ligand density on precursor Th (T helper) cell differentiation. Studies have also examined the effect of downregulated MHC Class II receptors and co-stimulatory molecules on APC–Th cell interaction. In addition, the sequestration of antigens away from the Class II processing pathway has been studied. These investigations reveal a general trend that can determine whether a naive CD4 T-cell becomes a Th1 or Th2-like cell. If the experimental manipulation weakens the APC-Th cell interaction, a weak ligating TCR signal results. Consequently, a weak ligating TCR signal can influence precursor Th cells to become Th2-like cells. Retroviral antibody binding of MHC Class II receptors may mimic a number of experimental conditions responsible for creating a weak ligating TCR signal.

Association of Histone Deacetylase with COUP-TF in Tumorigenic Ad12-Transformed Cells and Its Potential Role in Shut-Off of MHC Class I Transcription

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor that represses transcription of many genes. In adenovirus type 12 (Ad12) transformed cells, a high level of binding activity of COUP-TF to the major histocompatibility complex (MHC) class I enhancer correlates with the down-regulation of class I transcription, which, in turn, contributes to tumorigenesis. The mechanism by which COUP-TF represses transcription has yet to be elucidated. Here we show that COUP-TF represses transcription through its association with histone deacetylase. This was demonstrated using reciprocal binding assays that determined that the interaction between COUP-TF and histone deacetylase requires the COUP-TF C-terminal repression domain. Moreover, a histone deacetylase enzymatic activity was found to be associated with COUP-TF in Ad12-transformed cells. Transfection experiments further revealed that exogenous histone deacetylase facilitates transcriptional repression by COUP-TF. Also, supershift assays suggest that the transcriptional corepressor N-CoR, which is known to associate with histone deacetylases, is a part of the COUP-TF complex bound to the MHC class I enhancer R2 site. Finally, we provide evidence that inhibition of histone deacetylases relieves the repression of MHC class I expression in Ad12-transformed cells. Taken together these results support the notion that deacetylation of histones, mediated through COUP-TF, serves to down-regulate MHC class I transcription in Ad12-transformed cells.

A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4

Nef, a ∼200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1–7]. Nef contains a globular core domain and two disordered segments – a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the β-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes.