Human Papillomavirus Type 16 E7 Oncoprotein Expressed in Peripheral Epithelium Tolerizes E7-Directed Cytotoxic T-Lymphocyte Precursors Restricted through Human (and Mouse) Major Histocompatibility Complex Class I Alleles

Mice which coexpress human papillomavirus type 16 E7 and HLA A2.1 in peripheral squamous epithelium and thymic cortical epithelium are tolerant at the cytotoxic T-lymphocyte (CTL) level to E7 epitopes restricted through HLA A*0201 and H-2^b (T. Doan, M. Chambers, M. Street, G. J. Fernando, K. Herd, P. Lambert, and R. Tindle, Virology 244:352–364, 1998). Here we used bone marrow-reconstituted radiation chimeras to distinguish whether E7-directed CTL tolerance was mediated peripherally by E7 expression in skin or centrally by E7 expression in thymus. In chimeric mice expressing E7 in skin and reconstituted with E7-naïve bone marrow and E7-naïve thymus, CTL responses to vaccine-administered E7 epitopes were not restored, i.e., the mice remained tolerant. In contrast, chimeric mice not expressing E7 in skin and reconstituted with E7-naïve bone marrow and E7-expressing thymus had full E7-directed CTL responses. These results demonstrate that E7 protein expression in peripheral squamous epithelium is sufficient to tolerize the E7-directed CTL precursor repertoire. The data have implications for E7-mediated tumorigenesis and for the development of E7-based immunotherapeutic strategies, since peripheral immunological tolerance of tumor-associated antigens may create a barrier to effective immunotherapy.The E7 oncoprotein of human papillomavirus type 16 (HPV16) is a tumor-specific antigen when expressed in HPV16-associated cervical epithelial tumors, to which immunomanipulative strategies are being directed, both experimentally (see, for example, references 7, 9, and 29) and in E7-based therapeutic vaccine clinical trials (5). We recently reported studies with mice expressing HPV16 E7 protein, driven from the keratin 14 (K14) promoter, in basal epithelium of skin and in the thymic cortex (8). We showed that immunization-induced cytotoxic T-lymphocyte (CTL) responses to each of three CTL epitopes in the E7 protein restricted through two major histocompatibility complex (MHC) class 1 haplotypes were down-regulated in these E7-transgenic mice compared with non-E7 syngeneic control mice. However, in these studies we did not determine whether the down-regulation (i.e., tolerance) was induced centrally by E7 expressed in the thymus or peripherally by E7 expressed in epithelium. In the present study, we distinguish between these two possibilities by specific immunization of bone marrow-reconstituted thymus-transplanted chimeric E7 transgenic mice. We report that chimeric mice expressing the E7 transgene in peripheral epithelium but not in the thymus showed E7-specific down-regulated CTL responses to each of two E7 CTL epitopes restricted through a human and a mouse MHC class I allele, respectively, when compared with sham chimeric but non-E7 control mice. In contrast, chimeric mice expressing the E7 transgene in thymus, but not peripheral epithelium, showed E7-directed CTL responses indistinguishable from those of non-E7 control mice. Thus, we show that the expression of E7 in peripheral squamous epithelium is sufficient to induce and maintain a state of tolerance against E7.

E7-directed bone marrow-derived precursor CTLs (pCTLs) are not tolerized in mice expressing E7 in thymus but not in skin.

(K14E7 × A2.1K^b)F₁ mice (designated KA mice) were derived by crossing male K14.HPV16E7(+/+) mice (16), which express an HPV16 E7 transgene perinatally and throughout life in skin and thymic cortical epithelium, with female HLA A2.1K^b(+/+) mice (30). (FVB × A2.1K^b)F₁ mice (designated FA) are syngeneic but do not possess the E7 transgene. KA (E7⁺) and FA (E7⁻) mice are on an H-2^b background. To inquire whether pCTLs from E7-transgenic mice were tolerized on E7-expressing thymus, we constructed thymus-transplanted radiation chimeras as described elsewhere (8) from immunologically depleted FA (E7⁻) mice reconstituted with KA (E7⁺) bone marrow cells. In half of the mice, [designated KA→FA(FA) mice], the bone marrow-derived T-cell precursors were made to mature through a thymus implant from an FA (E7⁻) donor mouse; in the other half of the mice [designated KA→FA(KA) mice], the bone marrow cells were made to mature through an E7-expressing KA thymus implant (Fig. ​(Fig.1,1, panel I). KA→FA(FA) mice, KA→FA(KA) mice, and control FA (E7⁻) and KA (E7⁺) mice were immunized for CTL response induction with a mix of peptides containing ⁸²LLMGTLGIV⁹⁰ (an HLA A*0201-restricted E7 CTL epitope [24]), ⁴⁹RAHYNIVTF⁵⁷ (an H-2D^b-restricted E7 CTL epitope [9]), and ⁵⁸GILGFVFTL⁶⁶ (an HLA A*0201-restricted influenza virus matrix CTL epitope [13]). Control mice underwent surgical procedures but without receiving cell and/or organ transplants (sham). KA (E7⁺) mice showed the previously documented (8) down-regulated CTL response to the E7 epitopes (but not to the irrelevant influenza virus matrix epitope) compared to FA (E7⁻) mice (Fig. ​(Fig.1,1, panels IIC and IID). In contrast, KA→FA(KA) mice exhibited E7 (and influenza virus matrix)-directed CTL responses of the same magnitude as those of KA→FA(FA) mice and FA (E7⁻) mice (Fig. ​(Fig.1,1, panels IIA to IIC). These data indicate that E7-directed pCTLs from E7 transgenic mice which mature through an E7-expressing thymus, and emerge into a non-E7-expressing peripheral epithelial environment, are not tolerized. Open in a separate windowFIG. 1(I) Derivation of KA→FA(FA) and KA→FA(KA) chimeric mice from immunologically ablated FA (E7⁻) mice. (II) CTL responses of splenocytes from chimeric mice and sham control FA (E7⁻) and KA (E7⁺) mice (three per group) immunized with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Immunizations were given in Quil A adjuvant and tetanus toxoid as described elsewhere (8). Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells (8) pulsed with individual peptides as indicated. EL4.A2 cells are susceptible to specific CTL lysis through both HLA A*0201 and H-2^b restriction elements. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.To eliminate the possibility that bone marrow-derived precursors from KA (E7⁺) mice had somehow previously encountered E7 protein before transfer to recipient mice, thereby influencing their immunological status in the above-described experiment, we asked whether bone marrow-derived pCTLs from FA (E7⁻) mice would be tolerized during maturation in an E7-expressing thymus. We constructed chimeras from immunologically ablated FA (E7⁻) mice by reconstitution with FA (E7⁻) bone marrow cells. In half the mice [designated FA→FA(KA) mice], the bone marrow cells were made to mature through an E7-expressing KA thymus implant. In the other half of the mice [designated FA→FA(FA) mice], the bone marrow cells were made to mature through a non-E7-expressing FA thymus implant (Fig. ​(Fig.2,2, panel I). FA→FA(KA) mice, FA→FA(FA) mice, and control FA (E7⁻) and KA (E7⁺) mice were immunized for CTL induction with a mix of peptides containing the HLA A*0201-restricted and H-2^b-restricted E7 CTL epitopes and influenza virus matrix CTL epitope. FA→FA(KA) mice exhibited E7-directed CTL responses of the same magnitude as those of FA→FA(FA) mice and FA (E7⁻) mice, while KA (E7⁺) mice exhibited the expected down-regulated E7-directed (but not down-regulated influenza virus matrix-directed) CTL responses (Fig. ​(Fig.2,2, panel II). These data indicated that E7-naïve bone marrow-derived pCTLs which mature through an E7-expressing thymus and emerge into a non-E7 peripheral epithelial environment are not tolerized to E7. Open in a separate windowFIG. 2(I) Derivation of FA→FA(FA) and FA→FA(KA) chimeric mice from immunologically ablated FA (E7⁻) mice. (II) CTL responses of splenocytes from chimeric mice and sham control mice (three per group) immunized with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells pulsed with individual peptides as indicated. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.

Bone marrow-derived pCTLs are specifically tolerized in mice expressing E7 in skin but not in thymus.

To inquire whether E7-directed pCTLs were tolerized in mice expressing E7 in skin but not in thymus, we constructed chimeric mice in which bone marrow-derived precursors were made to mature through a non-E7-expressing thymus and to emerge into an E7-expressing peripheral epithelial environment. In a first experiment, immunologically ablated KA (E7⁺) mice were reconstituted with KA (E7⁺) bone marrow cells which were made to mature through a thymus implanted from an FA (E7⁻) mouse. The recipient mice, designated KA→KA(FA) (Fig. ​(Fig.3,3, panel I), and control FA (E7⁻) and KA (E7⁺) mice were immunized for CTL response induction with a mix of peptides containing the HLA A*0201-restricted and H-2^b-restricted E7 CTL epitopes and the influenza virus matrix CTL epitope. In KA→KA(FA) mice, E7-directed CTL responses to both E7 epitopes were down-regulated to the level seen in control KA (E7⁺) mice (Fig. ​(Fig.3,3, panel IIB), while control FA (E7⁻) mice showed the expected high responses to both E7 CTL epitopes. Open in a separate windowFIG. 3(I) Derivation of KA→KA(FA) chimeric mice from immunologically ablated KA (E7⁺) mice. (II) CTL responses of splenocytes from chimeric mice and sham control mice (three per group) immunized with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells pulsed with individual peptides as indicated. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.In a second experiment, immunologically ablated KA (E7⁺) mice were reconstituted with bone marrow from FA (E7⁻) mice, which was made to mature through a non-E7-expressing FA thymus. These mice, designated FA→KA(FA) mice (Fig. ​(Fig.4,4, panel I), were immunized for CTL response induction with a mix of peptides containing the HLA A*0201-restricted and the H-2^b-restricted E7 CTL epitopes and the influenza virus matrix CTL epitope. As with KA→KA(FA) mice in the previous experiment, E7-directed CTL responses to both E7 epitopes were down-regulated as in KA (E7⁺) controls and in contrast to FA (E7⁻) controls, while the influenza virus matrix response confirmed adequate reconstitution. Open in a separate windowFIG. 4(I) Derivation of FA→KA(FA) chimeric mice from immunologically ablated KA (E7⁺) mice. (II) CTL responses of splenocytes from chimeric mice and sham control mice (three per group) immunized once with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells pulsed with individual peptides as indicated. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.The results from these two experiments indicate that bone marrow-derived E7-directed pCTLs which mature through a non-E7-expressing thymus and emerge into an E7-expressing epithelial environment are specifically tolerized to E7.We have previously reported pCTL tolerance to epitopes of the HPV16 E7 oncoprotein in KA mice expressing a K14 promoter-driven E7 transgene perinatally and throughout life in the thymus and in basal and/or suprabasal cells of peripheral epithelium (8). In the present experiments, we demonstrate that the E7-directed pCTL repertoire is tolerized in mice expressing E7 in peripheral epithelium in the absence of thymic expression. Conversely, the repertoire is not tolerized in mice expressing E7 in the thymus, in the absence of E7 expression in peripheral epithelium. These data indicate that expression of E7 in peripheral epithelium, and not the thymus, is sufficient to induce and maintain a state of pCTL tolerance to E7. In the thymus, the K14 promoter directs transgene expression to the cortical epithelial compartment (19), which, in other mouse models, has been shown to contribute to the shaping of the T-cell repertoire by positive rather than negative selection (for example, see reference 20). Melero et al. (21) observed no functional down-regulation of the CTL responses induced by immunization with E7 peptide epitope RAHYNIVTF in H-2^b mice expressing HPV16 E7 from a K14 promoter and concluded that the mice remain immunologically ignorant of this epitope. This result contrasts with ours. Together, they provide further examples of T-cell tolerance to peripheral antigens in some systems (see, for example, references 1, 2, and 23) and T-cell ignorance in others (see, for example, references 14, 17, and 25). While determinants of immunological outcome of peripheral antigen expression are clearly complex (22), the level of expression (as well as timing and site of expression) can determine whether an antigen induces tolerance or is ignored by naïve T cells. This consideration may explain the difference between the results of Melero et al. and ours. The effect of the level of E7 expression on peripheral tolerance induction is under investigation in our laboratory.Specific CTL tolerance has implications for E7-mediated tumorigenesis. Nascent E7-expressing tumor cells will escape surveillance where little or no positive priming of cognate pCTLs by endogenous E7 occurs. Additionally, specific CTL tolerance which inhibits the generation of an immunization-induced CTL response will detract from effective immunotherapy (26). We have previously reported that (K14.E7 × C57)F₁ mice fail to control a challenge with an E7-expressing tumor following immunization with E7 CTL epitope RAHYNIVTF, whereas in immunized non-E7-transgenic control mice the tumors did not become established (12). Failure to control the tumor was correlated with a lack of an inducible RAHYNIVTF-directed CTL response in E7-transgenic mice, in contrast to non-E7-transgenic control mice, where a powerful CTL response was observed. In further experiments, multiple immunization of KA mice with E7 CTL epitopes or whole E7 protein failed to arrest the development of E7-associated endogenous tumors (8), again being correlated with a lack of E7-directed CTL responses.The current therapeutic vaccine strategy for HPV16-associated cervical carcinoma targets the E7 tumor-specific antigen by CTL induction (5, 28). The possibility arises that chronic expression of E7 in transformed cervical epithelial cells during the life of the tumor functionally tolerizes E7-directed pCTLs.Ongoing experiments in our laboratory will distinguish between presentation of E7 to pCTLs directly by keratinocytes and cross presentation of exogenously acquired E7 by bone marrow-derived professional antigen-presenting cells. Presentation of antigen by either of these routes can be tolerogenic (3, 6, 15, 27). Additionally, we will determine whether loss of functional E7-directed CTLs results from pCTL deletion (4, 11) or anergy (10, 25). Peripheral tolerance of tissue-specific antigen depends, at least in some cases, on the generation of regulatory CD4⁺ cells (see, for example, reference 18). That E7-directed CTL tolerance in KA (E7⁺) mice reflects an impairment of cognate CD4⁺ help is unlikely in view of our finding that (K14.E7 × C57)F₁ mice immunized with full-length E7 and displaying E7-specific pCTL tolerance showed concomitant enhanced E7-directed CD4⁺ T-helper responses (12).The data reported in the present study demonstrate the induction of peripheral tolerance in E7-directed pCTLs by HPV16 E7 expressed in squamous epithelial cells, in the context of human (and mouse) MHC class 1 haplotypes. There are direct implications for the development and progression of cervical cancers which express E7 in transformed squamous epithelium and for the design of E7-based immunotherapeutic strategies for cervical cancer. In the broader context, there are implications for CTL response induction to any foreign or aberrant protein expressed constitutively in squamous epithelial cells as a result of infection, tumorigenesis, or appearance of autoantigen.     

Immunization with a Single Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Recognition Epitope of Herpes Simplex Virus Type 2 Confers Protective Immunity

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8⁺ cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2K^b-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8⁺ T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8⁺ CTL in HSV vaccine design.

Both humoral and cell-mediated components of the immune response are involved in controlling herpes simplex virus (HSV) infection (51, 61). Studies of humans and of mice have implicated a role for both CD8⁺ (6, 25, 32, 33, 47, 65–67) and CD4⁺ (27, 37–39, 52, 53) T-lymphocyte subsets in mediating protection against HSV infection. For example, CD8⁺ T cells have been shown to be important in limiting replication of HSV in the footpad (6) and colonization of the spinal dorsal root ganglia (6, 66). In contrast, other studies using a zosteriform model of infection have primarily indicated a role for CD4⁺ T cells in the clearance of HSV (37–39). Both CD4⁺ and CD8⁺ (56, 72, 74–76) HSV-specific T lymphocytes have been detected in humans seropositive for HSV. However, the contribution of each subset in the control of HSV infection has not been clearly defined. This illustrates the controversy regarding the relative roles of each subset in the resolution of HSV infection.To address the role of the CD8⁺ T-cell subset in providing acquired immunity to HSV infection, we examined the protection afforded by HSV-specific, CD8⁺ cytotoxic T lymphocytes (CTL) directed to a single CTL recognition epitope. In previous studies by others, immunization with single CTL epitopes has been effective in controlling viral pathogens including lymphocytic choriomeningitis virus (14, 54, 62, 73), murine cytomegalovirus (15), influenza virus (55), and Sendai virus (28). Although HSV-encoded CTL recognition epitopes have been identified by their ability to serve as targets for HSV-specific CTL (3, 8, 24, 64), the ability of CTL directed to these individual epitopes to confer protection against HSV infection has not been determined. We have designed two separate vaccination strategies which permit the exclusive induction of a single HSV epitope-specific, CD8⁺ T-lymphocyte response and have evaluated the ability of this response to confer protective immunity to HSV infection.Hanke et al. (24) broadly identified an immunodominant, H-2K^b-restricted epitope within HSV glycoprotein B (gB). The minimal amino acid sequence of this epitope, gB498-505 (SSIEFARL), was demonstrated by Bonneau et al. (8), using synthetic peptides and an epitope-specific CTL clone. The amino acid sequence, SSIEFARL, is identical in both HSV type 1 (HSV-1) (gB498-505) and HSV-2 (gB496-503) (11). CTL specific for gB498-505 are readily induced by immunization with synthetic peptide (8), a cell line expressing gB498-505 in the context of simian virus 40 (SV40) T antigen (5), and a recombinant viral vector expressing this epitope in the context of a cellular protein (19). In the present study, two recombinant vaccinia viruses (rVV-ES-gB498-505 and rVV-gB498-505) and a recombinant influenza virus (WSN/NA/gB) were generated to express a single HSV-encoded epitope, HSV-1 gB498-505, and were characterized for the ability to induce a potent, HSV-specific CTL response upon mucosal immunization. To determine the protection afforded by immunization with each of the individual recombinant viruses, we used a lethal model of HSV-2 encephalitis. Our findings suggest that the induction of a CTL response directed against a single HSV-specific CTL recognition epitope is sufficient to confer significant protective immunity to HSV infection.     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

A Mechanistic Basis for the Co-evolution of Chicken Tapasin and Major Histocompatibility Complex Class I (MHC I) Proteins

MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical MHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed MHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo.     

小鼠组织相容性抗原的提取与纯化

以建立方便、大量纯化组织相容性抗原的方法为目的。用０．５％Ｔｒｉｔｏｎ／Ｔｒｉｓ抽提小鼠组织相容性抗原（Ｈ－２）抗原,利用抗Ｈ－２抗原抗体制备的亲和柱,特异性结合Ｈ－２抗原,再用０．５％ＤＯＣ、０．６５ＭＮａＣｌ洗脱结合Ｈ－２抗原。结果显示：电泳显示纯化物为４５ｋｄ（重链）,１２ｋｄ（轻链）两条带,纯化物具有明显的血清学及生物学活性;这种亲和层析法可大量纯化组织相容性抗原,用于器官移植研究及组织相容性抗原的免疫功能研究。     

Target HCV NS3 CD4+ Th1 Epitope to Major Histocompatibility Complex Class II Pathway

A hepatitis C virus (HCV) plasmid vaccine was constructed, based on class II-associated invariant chain peptide (CLIP) substitution which endogenously targets HCV non-structure protein 3 (NS3) CD4⁺ T helper 1(Th1) epitope (1248AA-1261AA) to major histocompatibility complex (MHC) class II antigen. The in vitro expression results demonstrated that the vaccine was expressed efficiently in COS-7 cell line. The expressed protein could co-localize in endo-membrane system with BALB/c mouse MHC class II molecule I-A^d. The recombinant invariant chain molecule could aggregate with BALB/c mouse I-A^d molecule and form the theoretical nonomer structure in the COS-7 cell line. The assembled molecules migrate to the cell surface by exocytosis. This has implications for HCV vaccine development.     

Contribution of Lysine 11-linked Ubiquitination to MIR2-mediated Major Histocompatibility Complex Class I Internalization

The polyubiquitin chain is generated by the sequential addition of ubiquitin moieties to target molecules, a reaction between specific lysine residues that is catalyzed by E3 ubiquitin ligase. The Lys⁴⁸-linked and Lys⁶³-linked polyubiquitin chains are well established inducers of proteasome-dependent degradation and signal transduction, respectively. The concept has recently emerged that polyubiquitin chain-mediated regulation is even more complex because various types of atypical polyubiquitin chains have been discovered in vivo. Here, we demonstrate that a novel complex ubiquitin chain functions as an internalization signal for major histocompatibility complex class I (MHC I) membrane proteins in vivo. Using a tetracycline-inducible expression system and quantitative mass spectrometry, we show that the polyubiquitin chain generated by the viral E3 ubiquitin ligase of Kaposi sarcoma-associated herpesvirus, MIR2, is a Lys¹¹ and Lys⁶³ mixed-linkage chain. This novel ubiquitin chain can function as an internalization signal for MHC I through its association with epsin1, an adaptor molecule containing ubiquitin-interacting motifs.     

Distortion of the Major Histocompatibility Complex Class I Binding Groove to Accommodate an Insulin-derived 10-Mer Peptide

The non-obese diabetic mouse model of type 1 diabetes continues to be an important tool for delineating the role of T-cell-mediated destruction of pancreatic β-cells. However, little is known about the molecular mechanisms that enable this disease pathway. We show that insulin reactivity by a CD8⁺ T-cell clone, known to induce type 1 diabetes, is characterized by weak T-cell antigen receptor binding to a relatively unstable peptide-MHC. The structure of the native 9- and 10-mer insulin epitopes demonstrated that peptide residues 7 and 8 form a prominent solvent-exposed bulge that could potentially be the main focus of T-cell receptor binding. The C terminus of the peptide governed peptide-MHC stability. Unexpectedly, we further demonstrate a novel mode of flexible peptide presentation in which the MHC peptide-binding groove is able to “open the back door” to accommodate extra C-terminal peptide residues.     

Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules

Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α₁-antitrypsin.     

Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells

Nef-specific CD8⁺ T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef_165–173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T₁₇₀I, unexpectedly and uniquely degraded Nef''s major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

Characterisation of Major Histocompatibility Complex Class I in the Australian Cane Toad,Rhinella marina

The Major Histocompatibility Complex (MHC) class I is a highly variable gene family that encodes cell-surface receptors vital for recognition of intracellular pathogens and initiation of immune responses. The MHC class I has yet to be characterised in bufonid toads (Order: Anura; Suborder: Neobatrachia; Family: Bufonidae), a large and diverse family of anurans. Here we describe the characterisation of a classical MHC class I gene in the Australian cane toad, Rhinella marina. From 25 individuals sampled from the Australian population, we found only 3 alleles at this classical class I locus. We also found large number of class I alpha 1 alleles, implying an expansion of class I loci in this species. The low classical class I genetic diversity is likely the result of repeated bottleneck events, which arose as a result of the cane toad''s complex history of introductions as a biocontrol agent and its subsequent invasion across Australia.     

Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.     

Mixed-Haplotype and Mixed-Isotype Major Histocompatibility Complex Class II Molecules Detected by Murine T-Cell Clones and Their Possible Involvement in Autoimmunity

The recognition of antigen by T lymphocytes (T cells) is restricted by Class I or Class II major histocompatibility complex (MHC) gene products, the phenomenon called “MHC restriction.” MHC restriction is speculated to be one of the major elements for the association of disease susceptibility to MHC haplotypes. Clones of T cells have been shown to be powerful tools for the analysis of such restriction specificity. In this report, I describe unique mixed-isotype Aβ^d/Eα^drestriction molecules detected by T-cell clones in (B6Eα^d× BALB/c)F1 transgenic mice. The restriction specificity of these clones was confirmed by anti-Class II mAb blocking experiments. The ability of spleen cells from Aβ^dand Eα^ddouble transgenic B6 (B6Aβ^dEα^d) mice that express Aβ^d/Eα^dmolecules to present KLH to these clones supported the existence of such unique specificity. I also describe autoreactive as well as KLH-reactive T-cell clones restricted by mixed-haplotype Aβz/Aα^dClass II molecules derived from (NZB × NZW)F1 (B/WF1) mice. The restriction specificity was demonstrated by mAb blocking experiments and by experiments using Class II gene-transfected antigen-presenting cells. It is possible that such unique mixed-isotype and mixed-haplotype Class II molecules are critically involved in autoimmunity. In addition, the detailed methodology for establishing T-cell clones currently employed in my laboratory is described.     

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.Herpesviruses are well known to interfere with major histocompatibility complex class I (MHC-I) molecules in order to ensure evasion from immune recognition. A majority of evidence so far indicates that they target MHC-I maturation events and MHC-I trafficking in the secretory pathway (33), although evidence exists suggesting that herpesviruses could also interfere with MHC-I functions in endosomal pathways (8). Murine cytomegalovirus (MCMV), a member of the herpesvirus family, dedicates a substantial part of its genome to encoding nonessential genes for the modulation of cellular functions (40), including MHC-I trafficking in the secretory pathway (24, 27, 45, 48, 49, 52). All known immune evasion functions encoded by MCMV are based on a direct interaction of viral gene products with MHC-I complexes in the secretory pathway. The egress of nascent MHC-I complexes to the cell surface of MCMV-infected cells is abolished as a consequence of their retention in the endoplasmic reticulum (ER)-cis-Golgi intermediate compartment (ERGIC) by the m152 gene product (10, 19, 24, 52, 56) as well as redirection of those that escape into the Golgi compartment toward late endosomes (LEs) for degradation by the m06 MCMV gene product (45). These effects are opposed by gp34, a product of the MCMV m04 gene, which associates with MHC-I complexes and reaches the cell surface (24, 27).The loss of MHC-I from the cell surface is an expected consequence of the activity of m152 and m06, which act in the secretory pathway. The level of cell surface MHC-I is substantially reduced at later times of infection (10, 19, 24, 48, 52), and cells stably transfected with either the m152 or m06 gene do not display MHC-I at the cell surface (20, 24). If the loss of MHC-I from the cell surface is a consequence of the prevented egress from the secretory pathway, then the cell surface loss should follow the kinetics of the constitutive internalization of MHC-I complexes in the endosomal pathway. Given that the constitutive internalization is the net result of cell surface supply from the secretory pathway, endocytic uptake, and endocytic recycling, it is a slow process that occurs in normal fibroblasts at a rate of ∼6 to 8% per hour (36). Therefore, the effect of MCMV immunoevasins on cell surface MHC-I should be expected at later times of infection. However, several reports demonstrated that the level of MHC-I surface expression was already reduced in the early phase of infection (10, 45, 48, 52). Thus, it would be reasonable to expect that MCMV contributes with a function that causes the accelerated retrieval of cell surface-resident MHC-I complexes.In this report we demonstrate that MCMV perturbs endosomal trafficking very early in infection by acting on distal parts of early endosome (EE) route and affecting the trafficking of both clathrin-dependent and clathrin-independent cargoes. Clathrin-dependent cargo does not share primary endocytic carriers with MHC-I proteins (12, 14), which enter the cell via the nonclathrin Arf6-associated endocytic carriers (12, 14, 41, 42, 53), but they meet in the proximal part of the common early endocytic route and redirect to distal endocytic carriers around the cell center (12, 14). The perturbation of the distal part of the EE route has dramatic consequences on MHC-I, since it supplements the viral mechanisms that act in the secretory pathway. The net result of this perturbation is a complete loss of MHC-I molecules from the cell surface.     

Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes

Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three αβ dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting αβ dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A^b αβ dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A^b class II molecules in lysosomes. In leupeptin, I-A^b dimers formed stable complexes with a 10-kD NH₂-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A^b dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of αβ dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.The initiation of most immune responses requires antigen recognition by helper T lymphocytes. The antigen receptors on T cells can only recognize antigens as small peptides bound to major histocompatibility complex (MHC)¹ class II molecules at the surface of antigen presenting cells (Cresswell, 1994; Germain, 1994). The complexes between class II molecules and antigenic peptides are formed intracellularly somewhere along the endocytic pathway (Germain, 1994; Wolf and Ploegh, 1995). This process requires the internalization of protein antigen and its delivery to a site suitable for the generation of antigenic peptides. In addition, the peptides must be generated within, or transferred to, a site to which newly synthesized MHC class II molecules are delivered and rendered competent for peptide binding (Davidson et al., 1991).Invariant (Ii) chain plays a central role in controlling the intracellular transport of MHC class II (Cresswell, 1996). In the ER, Ii chain is synthesized as a trimer that complexes with three αβ dimers of MHC class II (Roche et al., 1991). Its NH₂-terminal cytoplasmic domain contains a wellknown targeting signal that directs class II–Ii chain complexes to endosomes after exit from the TGN (Bakke and Dobberstein, 1990; Lotteau et al., 1990; Neefjes et al., 1990; Odorizzi et al., 1994; Pieters et al., 1993). Once in endosomes, Ii chain is subjected to proteolysis by acid hydrolases (Roche and Cresswell, 1991). Degradation occurs in a stepwise fashion, resulting in the appearance of class II– bound NH₂-terminal intermediates containing the Ii chain cytoplasmic domain, membrane anchor, and parts of its luminal domain (Newcomb and Cresswell, 1993). The intermediates accumulate in the presence of protease inhibitors that interfere with Ii chain processing such as the serinecysteine protease inhibitor leupeptin, treatment with which can also block the transport of at least some class II haplotypes to the cell surface (Amigorena et al., 1995; Blum and Cresswell, 1988; Neefjes and Ploegh, 1992). How leupeptin inhibits surface appearance is unknown.In human cells, Ii chain degradation intermediates include a 21–22-kD fragment (designated LIP [leupeptininducible peptide]) and a 10–12-kD fragment (designated SLIP [small leupeptin-inducible peptide]) (Blum and Cresswell, 1988; Maric et al., 1994). In murine cells, only a 10– 12-kD fragment has been identified (Ii-p10) (Amigorena et al., 1995). Ii-p10 remains as a trimer associated with three αβ dimers and blocks the binding of antigenic peptides (Amigorena et al., 1995; Morton et al., 1995). It is thus likely that Ii-p10 includes a luminal region of Ii chain (designated CLIP) known to occupy the peptide binding groove of αβ dimers. Cleavage of Ii-p10 by a leupeptinsensitive protease causes its dissociation from αβ dimers, while leaving CLIP in the peptide binding groove. The removal of CLIP is favored at acidic pH but is additionally catalyzed by a second MHC gene product, HLA-DM (Sloan et al., 1995; Denzin and Cresswell, 1995; Karlsson et al., 1994; Roche, 1995). In mutant cells lacking HLA-DM, there is defective loading of antigenic peptides and the appearance of CLIP-αβ dimers on the plasma membrane (Mellins et al., 1994; Riberdy et al., 1992).The precise site(s) where these events occur remains unclear. In A20 B cells, a specialized population of endosome-like vesicles designated CIIV (for class II vesicles) represents a site through which a majority of newly synthesized class II molecules pass en route to the cell surface and a place where antigenic peptides bind αβ dimers of the I-A^d haplotype (Amigorena et al., 1994, 1995; Barnes and Mitchell, 1995). CIIV are physically distinct from the bulk of endosomes and lysosomes and contain at least some HLA-DM (Pierre et al., 1996). Despite the fact that most of the αβ dimers reaching CIIV are newly synthesized, CIIV contain little or no intact Ii chain (Amigorena et al., 1995). Thus, Ii chain–αβ complexes first may be delivered to endosomes where Ii chain is cleaved before being delivered to CIIV. That peptide loading can occur in CIIV has been demonstrated by experiments showing that leupeptin causes CIIV to transiently accumulate Ii-p10– containing complexes, which can then bind peptide (Amigorena et al., 1995).In human Epstein-Barr virus–transformed B lymphoblasts, most class II molecules have been localized to structures collectively designated MIIC (for MHC class II compartment) (Peters et al., 1991; Tulp et al., 1994; West et al., 1994). MIICs differ from CIIVs in that the latter contain endosomal but not lysosomal markers, while MIICs have most or all of the features of lysosomes (Peters et al., 1991, 1995; Pierre et al., 1996). Interestingly, the distribution of class II between endosomal (CIIV) and lysosomal (MIIC) compartments varies widely among cell types. Since lysosomes are classically defined as terminal degradative organelles (Kornfeld and Mellman, 1989), such variations may reflect differences in the rates at which class II is turned over in different cell types. On the other hand, MIICs also contain the bulk of HLA-DM and can host the loading of antigenic peptides onto class II molecules (Sanderson et al., 1994). The extent to which these complexes escape degradation and reach the cell surface is unclear. Nor is it at all clear how different cell types regulate the intracellular distribution of class II molecules between early and late endocytic compartments.We now show that murine A20 cells expressing endogenous I-A^d and transfected I-A^b normally localize little class II in lysosomes. Selective lysosomal accumulation of I-A^b αβ dimers can be induced after leupeptin treatment. Interestingly, I-A^b dimers, but not I-A^d dimers, are induced by leupeptin to form stable complexes with Ii-p10. Upon removal of the inhibitor, the Ii-p10 was removed and class II molecules were slowly transported from lysosomes to the cell surface. Thus, the rate of dissociation of Ii chain intermediates can regulate whether newly synthesized class II molecules are transported to the plasma membrane or to lysosomes.     

MICA/B蛋白在不同宫颈病变组织中的表达及意义

目的:探讨主要组织相容性复合物I类相关蛋白A/B(MICA/B)在不同宫颈病变组织及宫颈细胞系中的表达及定位。方法:采用免疫组织化学SP法检测宫颈炎症组织、高级别鳞状上皮内病变(high-grade squamous intraepithelial lesions, HSIL)及宫颈鳞癌(cervical squamous cell carcinoma, CSCC)组织中MICA/B蛋白的表达情况。采用免疫荧光化学与激光共聚焦显微术结合的方法研究3种宫颈癌细胞系C33a(HPV-)、Siha(HPV16+)、Hela(HPV18+)及正常宫颈上皮细胞系H8中MICA/B的表达和定位。结果:MICA/B蛋白主要表达定位于细胞浆,部分细胞核,在宫颈鳞癌组织中阳性表达率(83.3%、81.8%)高于宫颈炎症组织(39.3%、44.0%),差异具有统计学意义(均有P0.001);MICA蛋白在HSIL组织的阳性表达率(81.8%)高于宫颈炎症组织(39.3%),差异具有统计学意义(P=0.002);与分化程度、临床分期、淋巴结转移等临床病理参数之间比较无统计学差异(P0.05)。结论:MICA蛋白随着宫颈组织病变的加重阳性表达率逐渐增高,MICB蛋白在宫颈癌组织的表达高于宫颈炎症组织。提示MICA/B蛋白可为宫颈癌的诊断及靶向治疗提供新方向。