Vaccine-Induced,Pseudorabies Virus-Specific,Extrathymic CD4+CD8+ Memory T-Helper Cells in Swine

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4⁺CD8⁺ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4⁺CD8⁺ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4⁺CD8⁺ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4⁺CD8⁺ memory T lymphocytes and showed that CD4⁺CD8⁺ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4⁺CD8⁺ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, is the causative agent of Aujeszky’s disease. This disease is lethal to young pigs and causes important economic losses (52). Therefore, vaccination of pigs is practiced in many countries.Several humoral immune system effector mechanisms are involved in the protection of pigs from PRV infection. Virus-neutralizing antibodies, antibodies mediating antibody-dependent cell-mediated cytotoxicity, and antibodies mediating complement-mediated lysis of PRV-infected target cells have been demonstrated (22, 23, 53, 54). The main targets of this humoral immune response were shown to be the viral glycoproteins (3, 45), and passive immunization with monoclonal antibodies (MAbs) against gB, gC, and gD protects pigs from a lethal challenge (20, 49).The protection conferred through cell-mediated immunity is poorly understood. An increase in major histocompatibility complex (MHC)-unrestricted cell-mediated cytotoxicity against uninfected and PRV-infected cells has been detected after infection or vaccination of pigs with PRV (16, 53, 54), and specific cellular immune responses to PRV infections could be demonstrated by stimulation of proliferation and lymphokine secretion of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as well as by the detection of PRV-specific cytotoxic lymphocytes (21, 56).There are some difficulties in defining more precisely the impact of cell-mediated immune effector mechanisms to protection from PRV-infection and their interplay with the observed humoral immune response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and mice. The pig has a substantial number of CD4⁻CD8⁻ T lymphocytes in the peripheral blood (4, 6, 12, 36, 39). In young animals, this subpopulation of T lymphocytes comprises up to 60% of the T lymphocytes and contains mainly γδ T lymphocytes. The pig is also the only species so far known to contain a substantial number of resting extrathymic CD4⁺CD8⁺ T lymphocytes (28, 36, 39). This T-lymphocyte population shows morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that CD4⁺CD8⁺ T lymphocytes comprise memory T cells which proliferate upon stimulation with recall antigen (43, 55). Since the observed proliferative response was shown to be MHC class II-restricted, it was speculated that the porcine CD4⁺CD8⁺ T-cell subset contains memory T-helper lymphocytes (43). However, the ability of these T lymphocytes to secrete cytokines or to provide help to B cells has so far not been demonstrated.To gain a better understanding of immune effector mechanisms conferring protection from PRV infection, the function of these unusual extrathymic T-lymphocyte subsets has to be elucidated. In the present study, we identified two T-cell epitopes on glycoprotein gC which are primed during vaccination of d/d haplotype inbred pigs (41) against PRV and demonstrated that MHC class II-restricted, peripheral CD4⁺CD8⁺ memory T lymphocytes are the responding T lymphocytes. We were further able to show that PRV-specific, extrathymic CD4⁺CD8⁺ T lymphocytes are able to secrete cytokines and have the capacity to stimulate the secretion of PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results demonstrate that porcine CD4⁺CD8⁺ T lymphocytes can function as memory T-helper cells and can direct humoral anti-PRV memory responses.     

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.     

Cytotoxic T-Lymphocyte Precursor Frequencies in BALB/c Mice after Acute Respiratory Syncytial Virus (RSV) Infection or Immunization with a Formalin-Inactivated RSV Vaccine

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.Respiratory syncytial virus (RSV) infection in infants and young children often results in lower respiratory tract disease and is a high priority for vaccine development (1, 2). Attempts to develop an effective live, inactivated, or subunit vaccine have been unsuccessful (24, 25, 28). Early efforts at vaccinating young children with a formalin-inactivated RSV (FI-RSV) vaccine failed to protect the children from naturally acquired infection and actually enhanced lower respiratory tract disease upon later virus infection (2, 15, 24, 25). This enhanced disease has created concern about the safety of any nonlive RSV vaccine and, consequently, understanding the pathogenesis of FI-RSV-induced enhanced disease is critically important to vaccine development. Studies with BALB/c mice suggest that induction of memory T cells producing Th2-like cytokines, as a result of FI-RSV vaccination, may be key to the pathogenesis of enhanced disease (6, 16, 28, 32, 40). Th2-like cytokine mRNA has been demonstrated in cells from lung tissue or bronchoalveolar lavage (BAL) specimens after RSV challenge of FI-RSV-immunized mice (17, 32, 40). In addition, in vivo studies using antibody (Ab) blockade showed that the enhanced histopathology in FI-RSV-immune mice challenged with live virus could be eliminated by using anti-interleukin-4 (IL-4) and anti-IL-10 Abs but not anti-IL-12 Abs (6). Recent evidence suggests that CD8⁺ T lymphocytes may be important in directing the type of inflammatory response to RSV in challenge of G glycoprotein-sensitized mice (21, 31).One aspect of the FI-RSV immune response that has not been well characterized is the cytotoxic T-lymphocyte (CTL) response. There is limited information on major histocompatibility complex (MHC) class I-restricted CTLs after FI-RSV immunization (29), while the information about the CTL response after live-RSV infection has been well documented. Several studies have shown class I-restricted CTLs to kill predominantly target cells expressing the M, N, or F RSV protein (5, 7, 9, 26, 29, 41). The role of CTLs in the immune response to RSV is well illustrated by in vivo depletion studies with BALB/c mice (8, 18, 30). These studies suggest that both CD4⁺ (class II) and CD8⁺ lymphocytes are important for clearing RSV and that both contribute to the inflammatory response associated with infection. A vaccinia virus construct expressing RSV membrane-associated, nonglycosylated protein M2 has been affiliated with short-term protection in the BALB/c mouse (7). This protein does not induce neutralizing Abs, and therefore, protection likely is mediated by CTLs. Passive transfer of CD8⁺ T lymphocytes has been associated with both clearance of the virus and enhanced histopathology (1).In this report, we describe studies of CTL precursor (CTLp) frequencies in both live-RSV-infected and FI-RSV-immunized mice for MHC class I- and class II-restricted target cells. These studies demonstrate clear differences in the CTLp response between RSV and FI-RSV immunizations and provide additional approaches to identifying potential FI-RSV-induced enhanced disease mechanisms.     

An Important Role for Major Histocompatibility Complex Class I-Restricted T Cells,and a Limited Role for Gamma Interferon,in Protection of Mice against Lethal Herpes Simplex Virus Infection

Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-γ) has been proposed. To address the roles of MHC class I and of IFN-γ, C57BL/6 mice deficient in MHC class I expression (β2 microglobulin knockout [β2KO] mice), in IFN-γ expression (IFN-γKO mice), or in both (IFN-γKO/β2KO mice) were infected with HSV by footpad inoculation. β2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-γ appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-γ.

Two gene products of herpes simplex virus (HSV) block presentation of viral proteins by class I major histocompatibility complex (MHC) molecules: the viral host shutoff protein (vhs), which is present in the viral particle, and the immediate-early protein ICP47 (1, 14, 41, 42). Through the sequential action of these proteins, antigen presentation by MHC class I is inhibited early in the viral replication cycle. ICP47 binds to human transporter associated with antigen-processing proteins (TAP), thereby inhibiting peptide loading on MHC class I and recognition by HSV-specific, MHC class I-restricted, CD8⁺ T cells (1, 14, 42, 43). This effect is greatest in nonhematopoietic cells in which the abundance of MHC class I and TAP are lower than in antigen-presenting cells (41). As a consequence, HSV is more likely to impair recognition of infected target cells in the tissues than to block the generation of antigen-specific CD8⁺ T cells. Consistent with this, recent studies indicate that HSV antigen-specific CD8⁺ cytotoxic-T-lymphocyte (CTL) precursors can be readily detected in the blood and cutaneous lesions of HSV-infected individuals (16, 31, 32). However, NK cells and HSV antigen-specific CD4⁺ T cells are detected earlier than antigen-specific CD8⁺ T cells in lesions of humans with recurrent HSV-2 disease (16). This finding has led to the proposal that gamma interferon (IFN-γ) produced by infiltrating NK and CD4⁺ T cells overrides the inhibitory effects of HSV on TAP function and MHC class I expression (22, 41), thereby allowing the eradication of virus by CD8⁺ T cells, whose numbers increase in lesions around the time of viral clearance (16, 31). In patients with AIDS, a lower frequency in the blood of HSV antigen-specific CD8⁺ CTL precursors is associated with more frequent and severe recurrences of genital disease (32). These correlative data suggest that CD8⁺ T cells may play an important role in the clearance of HSV in humans, at least from mucocutaneous lesions.ICP47 inhibits murine TAP poorly (1, 42), which may explain the greater ease with which anti-HSV CD8⁺ CTLs have been detected in mice than in humans (3, 8, 28, 34, 35). Despite the weak interaction of ICP47 with murine TAP, results of a recent study (12) suggested that ICP47 impairs CD8⁺ T-cell-dependent viral clearance from the nervous system: CD8⁺ T cells protected susceptible BALB/c or A/J mice from lethal, nervous system infection with an HSV mutant lacking ICP47 but did not appear to protect against infection with wild-type HSV or to contribute to clearance of either virus from the eye. These findings are consistent with data suggesting that CD8⁺ T cells limit persistence of HSV in the spinal ganglia and decrease spread to the central nervous system (35, 36). However, other studies have concluded that CD4⁺ T cells but not CD8⁺ T cells play the critical role in viral clearance and protection from lethal primary infection with wild-type HSV (20, 23, 24) or that either CD4⁺ or CD8⁺ T cells are sufficient for protection (26, 37). Since the effects of ICP47 are likely to be greater in humans than in mice, if MHC class I-restricted CD8⁺ T cells do not play an important role in protection of mice from lethal, neuroinvasive infection due to wild-type HSV, an important role in humans would be unlikely.The mechanisms by which T cells may limit the spread of infection in the nervous system are not clearly understood. Studies by Simmons and colleagues suggested that CD8⁺ T cells may lyse infected Schwann cells or satellite cells but that they probably do not lyse infected neurons (31, 32). They and others have proposed that CD8⁺ T cells protect neurons through the production of cytokines, in particular IFN-γ (35, 36). IFN-γ contributes to the clearance of HSV from mucocutaneous sites (4, 24, 25, 37, 44). However, the role of IFN-γ in protection from lethal, neuroinvasive infection is uncertain and may vary with the strain of mice, method used to inhibit IFN-γ function, and route of inoculation (4, 5, 24, 37, 44). IFN-γ is produced in the ganglia of mice with acute or latent HSV infection (5, 13, 19). Both CD4⁺ and CD8⁺ T cells (and NK cells) produce IFN-γ, but CD4⁺ T cells appear to be the predominant source of IFN-γ following intravaginal infection with HSV (24, 25). Thus, it is possible that the disparity in results regarding the relative importance of CD4⁺ and CD8⁺ T cells in protection from lethal, neuroinvasive HSV infection reflects their redundant roles in production of this cytokine or that IFN-γ and CD8⁺ T cells contribute independently to control of infection in the nervous system.To address in parallel the contributions of MHC class I-restricted T cells and of IFN-γ to protection of mice from HSV, MHC class I and CD8⁺ T-cell-deficient β2 microglobulin knockout (β2KO) mice, IFN-γ knockout (IFN-γKO) mice, and mice deficient in both MHC class I and IFN-γ expression (IFN-γKO/β2KO) were studied. The results indicated that loss of MHC class I expression in β2KO mice substantially increased their susceptibility to HSV, whereas the loss of IFN-γ expression had a much more limited effect. These findings indicate that MHC class I-restricted T cells play an important role in protection against neuroinvasive HSV infection in mice and that they do so largely by mechanisms other than the production of IFN-γ. Though MHC class I expression is more severely impaired in β2KO mice than in human cells infected with wild-type HSV, these findings support the notion that inhibition of MHC class I expression is an important factor in the virulence of this virus.     

Deficient Peptide Loading and MHC Class II Endosomal Sorting in a Human Genetic Immunodeficiency Disease: the Chediak-Higashi Syndrome

The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus–transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1–2 μm), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules.Major histocompatibility complex (MHC)¹ class II molecules are composed of an αβ dimer that associates in the ER with a third membrane molecule, the invariant chain (Ii; 33, 24). The αβ−Ii chain complexes are transported via the Golgi apparatus to the endocytic pathway, directed by a signal localized in the cytoplasmic tail of Ii chain (7, 41). Ii chain is then degraded (12), and upon complete removal of the remaining Ii fragments (60), antigenic peptides are loaded onto class II molecules under the control of HLA-DM (65, 22).Ii chain cleavage and antigen processing to fitting peptides occurs in endosomal and/or lysosomal compartments (24). Depending on the species origin of the cell, cell types, or even on the maturation status in the case of dendritic cells, accumulation of MHC class II molecules may occur in different endocytic compartments (43, 51). In human Epstein Barr virus–transformed B (EBV-B) cells, HLA-DR molecules accumulate in lysosomal compartments named MHC class II compartments (MIICs; 49). In murine splenic lipopolysaccharide-activated B cells (18) as well as in macrophages and human melanoma cells (30, 52), MHC class II is found all along the endocytic pathway, from early endosomes to lysosomes. In contrast, A20 murine B lymphoma cells accumulate MHC class II molecules in endosomal compartments, the class II vesicles (2, 4), whereas few class II molecules are found in conventional endosomes and lysosomes. However, upon inhibition of Ii chain degradation, class II molecules redistribute into lysosomal compartments (14).Recent results from the laboratory of H. Geuze (50, 35) showed that the distribution of MHC class II molecules in EBV-B cells is not as restricted as initially envisioned. Indeed, HLA-DR accumulates in two types of compartments: (a) in endosomes containing multiple internal vesicles that are reached by fluid phase markers after 20–30 min of internalization and contain some Ii chain (multivesicular late endosomes); and (b) in vesicles containing internal membranes organized in onion-like structures that accumulate fluid phase markers only after 60 min and contain no Ii chain (multilaminar lysosomal compartments). Both types of compartments also contain Lamp1/2, CD63, and HLA-DM.The functional relevance of this heterogeneity of endocytic MHC class II–containing compartments is still unclear, and the precise role of multivesicular and multilaminar endosomes in MHC class II transport and Ii chain degradation is not known. Moreover, it has recently been shown that the antigenic peptides generated in endosomal and lysosomal compartments might not be the same (30). In addition, we have recently shown that antigen internalization through different membrane receptors that may deliver antigens to particular endocytic compartments results in presentation of different antigenic peptides (3).To evaluate the role of this heterogeneity of endocytic compartments in MHC class II transport and function, we examined EBV-B cells of patients suffering from a rare genetic immunodeficiency disease, the Chediak-Higashi Syndrome (CHS), which affects the morphology and function of endocytic compartments. CHS results from mutations in a gene encoding a large cytosolic protein called lysosomal trafficking regulator (LYST), which displays limited sequence homology to a regulatory subunit of the yeast phosphatidyl-inositol-3 kinase (PI3K), VPS15 (9, 45). LYST also includes several WD40 and HEAT/ARM domains, a domain of limited homology to stathmin, as well as a unique domain that has been called BEACH (9, 8, 10, 45).Despite having identified several subdomains in the CHS protein, the precise function of the protein is not known. We do know, however, that mutations in this gene result in immunological disorders and susceptibility to multiple childhood infections. The lysosomal compartments in all cell types of CHS patients are enlarged, reaching over 1 μm/vesicle (70). In hematopoietic cells, including T lymphocytes, NK cells, and granulocytes, cytotoxicity is defective, most likely because of a defect in regulated secretion (61, 29, 6). In nonhematopoietic cells such as melanocytes and kidney cells, enlarged lysosomal morphology and defects in lysosomal enzyme secretion have been reported (15). It is yet unclear whether the defect in the secretory function of lysosomes in hematopoietic cells is a consequence or a cause of the abnormal lysosomal morphology. It is also possible that both phenotypes arise from a unique upstream defect in the endocytic pathway.Here we show that antigen presentation and MHC class II intracellular transport are affected in EBV-B cells from CHS patients. Surprisingly, only lysosomal multilaminar MHC class II–containing compartments are enlarged, while multivesicular late endosomes displayed normal size and morphology. However, a severe reduction in the staining of multivesicular endosomes for MHC class II, Lamp 1/2, CD63, CD82, and β-hexosaminidase was observed, suggesting that transport of these markers from the TGN and/or early endosomes into late endosomes is affected. Missorting of resident lysosomal proteins to the plasma membrane and early endosomes was also observed, as well as a striking redistribution of the cation-dependent mannose-6-phosphate receptor (CD-MPR) into giant multilaminar lysosomes. In addition, we showed that LYST partially colocalizes with microtubules, which have previously been shown to play a critical role in transport from early to late endosomes (19). Together, these results show severe missorting of membrane proteins along the endocytic pathway of CHS cells, and suggest that LYST may be directly involved in microtubule-dependent transport into late endocytic compartments.     

Efficient Class II Major Histocompatibility Complex Presentation of Endogenously Synthesized Hepatitis C Virus Core Protein by Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Lines to CD4+ T Cells

The induction of an efficient CD4⁺ T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4⁺ T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10⁴ transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.Hepatitis C virus (HCV) has been known as a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. Like the other members in the family Flaviviridae, HCV contains a single, positive-strand RNA genome with a single long open reading frame (ORF) coding for a polyprotein precursor of about 3,000 amino acids (aa) (12). HCV infection is frequently persistent in the majority of patients and is closely associated with the later development of liver cirrhosis and hepatocellular carcinoma (3, 12, 16, 32). The effective control of HCV infection has been limited by the high frequency of viral genetic heterogeneity (7), the low rate of response to alpha interferon (46), and inadequate production of protective immunity (44, 45). These features strongly suggest that there is a great need to establish a new, highly effective therapy.CD4⁺ T cells are considered to play a central role in the generation of protective immunity against infections, because they can provide help to B cells for antibody production (42) and to cytotoxic precursor T cells for their maturation to effectors (21). Some CD4⁺ T cells may also act as cytotoxic effectors (30). It has been recognized that CD4⁺ T-cell response to HCV antigens is important for determining the clinical course of HCV infection (17, 37). Generally, T-cell proliferation is more frequent and stronger in patients with a benign course (6, 17, 20, 33, 37) that is accompanied by the normalization of serum alanine aminotransferase and, in some cases, the clearance of viral RNA (17, 37). In contrast, patients who have a poor T-cell response tend to develop persistent infection (17, 37). These findings support the hypothesis that a sufficient CD4⁺ T-lymphocyte response is critical for limiting HCV infection.Activation of T lymphocytes depends on the recognition of processed viral peptides, but not native antigens, in the context of major histocompatibility complex (MHC) molecules that are presented by antigen-presenting cells (APCs) (56). The B cell is an important professional APC, and its role in mediating antigen-specific immune response has been described extensively (11). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells are frequently used as APCs in in vitro analyses for antigen processing and presentation to T cells. These cells are characterized by high-level expression of class I and class II MHC molecules, along with accessory molecules such as ICAM-1, B7/BB1, and LFA-3, known to be important costimulatory molecules for T-cell activation (9, 15, 24). Importantly, transfected EBV-immortalized B cells expressing a tumor antigen have been shown to be capable of eliciting both T-helper and cytotoxic-T-lymphocyte (CTL) responses following in vivo inoculation (40). Nevertheless, dendritic cells have been shown to be critical for initiating responses by naive T cells (53), and in some situations presentation by B cells has been suggested to be toleragenic (35). To date, the role of B cells in processing and presenting HCV antigens has not been studied in detail and the mechanisms underlying T-cell–B-cell interaction are still being worked out.In the present study, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) established from HCV-infected patients were transfected with an expression vector coding for the whole structural region and part of the NS2 region of the HCV genome. The capacity of transfected B-LCL APCs for presenting intracellularly synthesized peptides was assessed by in vitro induction of the HCV-specific lymphoproliferative response of autologous T-cell lines. Our results indicated that core protein was properly expressed and efficiently presented by B-LCL APCs to CD4⁺ T cells. We demonstrated that the endogenous core peptides were presented through the class II MHC pathway and that they need B7/BB1 for providing costimulatory signals.     

Targeting a Polyepitope Protein Incorporating Multiple Class II-Restricted Viral Epitopes to the Secretory/Endocytic Pathway Facilitates Immune Recognition by CD4+ Cytotoxic T Lymphocytes: a Novel Approach to Vaccine Design

The role of CD4⁺ and CD8⁺ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4⁺- and CD8⁺-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8⁺- or CD4⁺-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4⁺-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4⁺ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.There is now increasing evidence to suggest that both CD4⁺ and CD8⁺ T cells are critical for the generation of an effective immune response against intracellular pathogens. Although both CD4⁺ and CD8⁺ T cells recognize nonnative forms of the antigen in association with major histocompatibility complex (MHC) molecules, the presentation of antigen to these two types of T lymphocytes occurs through distinct pathways (24). In fact, the disparity in antigen presentation to these T cells is not due to processing differences but rather reflects the differences in the capacities of class I and class II molecules to bind antigenic determinants in an intracellular compartment. Indeed, earlier studies have shown that for processing and interaction with MHC class II molecules, antigen expressed de novo needs to be targeted to an endosomal or lysosomal compartment (5). There are two major pathways by which antigens are targeted to these compartments. The traditional pathway involves the phagocytosis or endocytosis of exogenous antigens, followed by degradation by acid proteases in the endosomal or lysosomal compartments (3, 8, 26, 41). On the other hand, class II-restricted presentation of endogenously synthesized proteins mainly involves membrane antigens which are thought to enter the endosomal or lysosomal pathway by internalization from the cell surface (11). Although, in certain experimental systems, cytoplasmic and nuclear proteins may also enter this endogenous pathway, generally these proteins are targets for the class I processing pathway (9, 14, 20, 27).One of the major limitations of the epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs (2, 35, 38). Based on these observations, we reasoned that a molecular approach that directly routes these epitopes into the MHC class II pathway, such as the endocytic or lysosomal compartments, might facilitate endogenous presentation to CD4⁺ T cells. The lysosome-associated membrane protein (LAMP-1) and the invariant chain (Ii) are transmembrane proteins which are localized predominantly in the lysosomes and endosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting signals that mediate their translocation to the specific compartments. We therefore designed a chimeric polyepitope construct capable of encoding multiple class II-restricted CTL epitopes from Epstein-Barr virus (EBV) and influenza virus linked to the cytoplasmic and/or transmembrane domains of LAMP-1 and the Ii protein, with the aim of targeting the epitopes to the endosomal and lysosomal compartments. The data presented in this study clearly demonstrate that if the endogenously synthesized polyepitope protein is targeted to the endocytic/secretory pathway, processing and presentation of all the epitopes are dramatically enhanced. More importantly, minimal epitope sequences, without any natural flanking sequences, were adequate for efficient stimulation of the virus-specific memory CTL response, a result that has important implications for epitope-based vaccine design.     

Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4⁺ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4⁺ T cell epitopes relevant in health and disease.Human Leukocyte Antigen (HLA)¹ class II molecules on professional antigen presenting cells such as dendritic cells (DC) expose peptide fragments derived from exogenous and endogenous proteins to be screened by CD4⁺ T cells (1, 2). The activation and recruitment of CD4⁺ T cells recognizing disease-related peptide antigens is critical for the development of efficient antipathogen or antitumor immunity. Furthermore, the presentation of self-peptides and their interaction with CD4⁺ T cells is essential to maintain immunological tolerance and homeostasis (3). Knowledge of the nature of HLA class II-presented peptides on DC is of great importance to understand the rules of antigen processing and peptide binding motifs (4), whereas the identity of disease-related antigens may provide new knowledge on immunogenicity and leads for the development of vaccines and immunotherapy (5, 6).Mass spectrometry (MS) has proven effective for the analysis HLA class II-presented peptides (4, 7, 8). MS-based ligandome studies have demonstrated that HLA class II molecules predominantly present peptides derived from exogenous proteins that entered the cells by endocytosis and endogenous proteins that are associated with the endo-lysosomal compartments (4). Yet proteins residing in the cytosol, nucleus or mitochondria can also be presented by HLA class II molecules, primarily through autophagy (9–11). Multiple studies have mapped the HLA class II ligandome of antigen presenting cells in the context of infectious pathogens (12), autoimmune diseases (13–17) or cancer (14, 18, 19), or those that are essential for self-tolerance in the human thymus (3, 20). Notwithstanding these efforts, and certainly not in line with the extensive knowledge on the HLA class I ligandome (21), the nature of the HLA class II-presented peptide repertoire and particular its relationship to the cellular source proteome remains poorly understood.To advance our knowledge on the HLA-DR ligandome on activated DC without having to deal with limitations in cell yield from peripheral human blood (12, 21, 22) or tissue isolates (3), we explored the use of MUTZ-3 cells. This cell line has been used as a model of human monocyte-derived DCs. MUTZ-3 cells can be matured to act as antigen presenting cells and express then high levels of HLA class II molecules, and can be propagated in vitro to large cell densities (23–25). We also evaluated the performance of complementary and hybrid MS fragmentation techniques electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD) (26), and higher-energy collision dissociation (HCD) to sequence and identify the HLA class II ligandome. Together this workflow allowed for the identification of an unprecedented large set of about 14 thousand unique peptide sequences presented by DC derived HLA-DR molecules, providing an in-depth view of the complexity of the HLA class II ligandome, revealing underlying features of antigen processing and surface-presentation to CD4⁺ T cells.     

Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule L^d were transplanted into the L^d gene deletion mutant BALB/c-H-2^dm2, an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of L^d-positive donor-derived and L^d-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against L^d. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by L^d within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.Cytomegaloviruses (CMV) are kept under tight immune control (for reviews, see references 22 and 23). As a consequence, acute CMV infection is resolved rapidly and does not result in disease unless the host is immunologically immature or immunocompromised. Bone marrow (BM) transplantation (BMT) as a therapy of hematological malignancies is associated with a transient immunodeficiency. Accordingly, during the period of immunocompromise, transmission of donor-type CMV with the transplant as well as recurrence of CMV from latency established within the organs of the transplantation recipient both entail a risk for destructive virus replication in tissues resulting in multiple-organ CMV disease (16). In BMT recipients, CMV-induced interstitial pneumonia is a frequent and endangering manifestation of CMV disease (11, 27). However, CMV infection does not inevitably result in fatal disease. It appears that CD8 T-cell reconstitution is the decisive parameter in the control of CMV after BMT. Clinical data have shown that both efficient reconstitution of CD8 T cells (41) and supplementation of antiviral CD8 T cells by preemptive cytoimmunotherapy with T-cell lines (42, 50) correlate with a reduced risk of human CMV disease, whereas combined in vivo-ex vivo T-cell depletion, intended as a prophylaxis against graft-versus-host (GvH) disease, accidentally resulted in an increased incidence of CMV infections in BMT patients (14). Aspects of these clinical problems can be approached experimentally in a murine model of BMT and concurrent infection with murine CMV (for an overview, see reference 35). Specifically, depletion of CD8 T cells, but not of CD4 T cells, performed in vivo during the phase of reconstitution after BMT abolished the development of protective antiviral immunity, with an inevitably lethal outcome (34, 47) resulting from multiple-organ pathology (34), including BM aplasia (29, 30). Likewise, an insufficient endogenous reconstitution was successfully supplemented by experimental adoptive cytoimmunotherapy with antiviral CD8 T cells. Again, CD4 T cells were not effective (36, 37, 39, 47). Altogether, clinical data on human CMV infection and experimental data from the murine model have so far been concordant and have identified CD8 T cells as the principal effectors controlling CMV infections after BMT.These findings imply that all conditions which lower the efficacy of CD8 T-cell reconstitution will increase the risk for progression of asymptomatic CMV infection to fatal CMV disease. Histoincompatibility between graft and recipient is a factor likely to negatively influence the restoration of antiviral immunity. Accordingly, even though cases of severe human CMV disease have been reported also after autologous BMT (27, 40), the incidence of CMV-related complications is generally higher after histoincompatible BMT (51). In clinical BMT, donor and recipient are usually matched in major histocompatibility complex (MHC) class II molecules, whereas differences in minor histocompatibility loci and in MHC class I loci are tolerated if unavoidable. Complications caused in the CMV-infected recipient by histoincompatibility may include (i) an impaired engraftment of transplanted cells in the recipient BM stroma, (ii) an immunological GvH response as well as a host-versus-graft (HvG) response directed against the foreign minor or major histocompatibility molecules, and (iii) a lack of antiviral T-cell control resulting from an inappropriate repertoire of viral antigenic peptides presented by infected tissue cells of the transplantation recipient.In a first attempt to dissect these possibilities, we have established a murine model of experimental BMT performed across a single MHC class I disparity, namely, the presence and absence of the L^d molecule in BALB/c mice (MHC class I molecules K^d, D^d, and L^d) and the L^d gene deletion mutant BALB/c-H-2^dm2 (44), respectively. Depending on the choice of donor and recipient for the BMT, immunogenetical GvH and HvG conditions can be studied separately (35). Work presented herein focuses on the HvG setting with BALB/c as the donor strain and the mutant as the recipient. Hence, after incomplete depletion of hematopoietic cells of the recipients, this model entails a risk for graft rejection caused by a recipient response directed against the donor MHC class I molecule L^d. In addition, presentation of viral peptides by L^d, including the immunodominant IE1 nonapeptide of murine CMV (18, 38), is confined to donor-derived hematopoietic cells and their progeny, whereas the parenchymal and stromal sites of cytocidal infection (34) lack L^d as the prevailing peptide presenter. The aim of the study was to investigate the influence of this particular MHC class I disparity on the control of murine CMV after BMT.     

CD4+ and CD8+ T Cells Make Discrete Contributions to Demyelination and Neurologic Disease in a Viral Model of Multiple Sclerosis

Following intracerebral infection with Theiler’s murine encephalomyelitis virus (TMEV), susceptible strains of mice (SJL and PLJ) develop virus persistence and demyelination similar to that found in human multiple sclerosis. Resistant strains of mice (C57BL/6) clear virus and do not develop demyelination. To resolve the controversy about the role of CD4⁺ and CD8⁺ T cells in the development of demyelination and neurologic deficits in diseases of the central nervous system, we analyzed TMEV infection in CD4- and CD8-deficient B6, PLJ, and SJL mice. Genetic deletion of either CD4 or CD8 from resistant B6 mice resulted in viral persistence and demyelination during the chronic stage of disease. Viral persistence and demyelination were detected in all strains of susceptible background. Although genetic deletion of CD8 had no effect on the extent of demyelination in susceptible strains, deletion of CD4 dramatically increased the degree of demyelination observed. Whereas strains with deletions of CD4 showed severe neurologic deficits, mice with deletions of CD8 showed minimal or no deficits despite demyelination. In all strains, deletion of CD4 but not CD8 resulted in a decreased delayed-type hypersensitivity response to viral antigen. We conclude that each T-cell subset makes a discrete and nonredundant contribution to protection from viral persistence and demyelination in resistant strains. In contrast, in susceptible strains, CD8⁺ T cells do not provide protection against chronic demyelinating disease. Furthermore, in persistent TMEV infection of the central nervous system, neurologic deficits appear to result either from the absence of a protective class II-restricted immune response or from the presence of a pathogenic class I-restricted response.Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system (CNS) in humans. MS lesions are characterized by foci of inflammation, myelin destruction, and formation of astrocytic scars known as plaques. The presence of CD4⁺ T cells, CD8⁺ T cells (11), and macrophages in lesions suggests that pathogenesis is immunologically mediated; however, the specific contribution of specific cell types remains unknown (12, 44, 45). Although the etiology of MS is unknown, virus infection is the only epidemiological factor consistently associated with clinical exacerbation (43), and beta interferon, a cytokine with multiple known antiviral properties (46), is the only therapeutic agent definitively shown to decrease exacerbation and limit disability in MS (46). Therefore, the study of viral models of demyelination is extremely relevant.Theiler’s murine encephalomyelitis virus (TMEV), a picornavirus, induces a pathological and clinical disease similar to MS (24). Intracerebral infection with the Daniel strain (DA) of TMEV induces transient, acute neuronal polioencephalitis followed by chronic white matter demyelination and neurologic deficits in mice with susceptible (H-2^f,p,q,r,s,v) major histocompatibility complex (MHC) haplotypes (15, 32). Mice with resistant (H-2^b,d,k) MHC haplotypes recover from the acute disease with no obvious long-term sequelae or demyelination. Although TMEV infection of severely immunodeficient SCID mice results in severe neuronal encephalitis and death within approximately 2 weeks, these mice do not develop demyelination in the spinal cord white matter (38). However, when the immune systems of SCID mice are reconstituted by the adoptive transfer of splenocytes from immunocompetent mice or splenocytes treated with antibodies to CD4 or CD8, infection with TMEV results in chronic demyelination (38). These data indicate that similar to human MS, myelin destruction in chronic TMEV infection is immunologically mediated and requires contributions from both CD4⁺ and CD8⁺ T cells.Various reports have implicated both MHC class I- and class II-restricted cells in the pathogenesis of TMEV infection. CD4⁺ T cells have been implicated by studies demonstrating that demyelination is decreased following treatment with antibodies to CD4 (47) or I-A (34), is increased by adoptive transfer of a CD4⁺ T-cell line specific for VP2 capsid protein (9), and, in some studies, correlates with the development of a CD4-mediated delayed-type hypersensitivity (DTH) response against virus antigen (5). Furthermore, β₂-microglobulin-deficient mice, which are deficient in MHC class I, CD8⁺ T cells, and natural killer cells, develop demyelinating disease (6, 16, 28). In contrast, a role for CD8⁺ T cells has been suggested by studies demonstrating that susceptibility to demyelination maps genetically to MHC class I (H-2D) (1, 35), differential expression of MHC class I in the CNS correlates with disease susceptibility (1), and depletion of CD8⁺ T cells diminishes demyelination (41). Myelin destruction and neurologic deficits develop in TMEV-infected Aβ⁰ mice which are deficient in functional MHC class II and CD4⁺ T cells (20). Of interest, both class I and class II-deficient mice share the resistant (H-2^b) haplotype. This suggests that although multiple studies have implicated CD4⁺ and CD8⁺ T cells in the pathogenesis of TMEV infection, each of these components of the immune response is independently required for maintenance of resistance to demyelination.In order to definitively establish the contribution of CD4⁺ and CD8⁺ T cells to demyelination and neurologic deficits, mice lacking surface expression of CD4 or CD8 were backcrossed onto genetically resistant C57BL/6 (H-2^b) and susceptible SJL (H-2^s) and PLJ (H-2^u) strains. In this report, we confirm that both CD4⁺ and CD8⁺ T cells are required for protection from viral persistence and demyelination in resistant strains of mice. We also demonstrate that genetic deletion of CD8 does not significantly affect the degree of demyelination or survival in susceptible strains; however, genetic deletion of CD4 greatly increases the degree of demyelination and worsens clinical disease. Of interest, genetic deletion of CD8 greatly reduces neurologic deficits in animals with demyelination.     

Endogenous Processing and Presentation of T-cell Epitopes from Chlamydia trachomatis with Relevance in HLA-B27-associated Reactive Arthritis

Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8⁺ T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8⁺ T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.Chlamydia trachomatis is an obligate intracellular parasite that infects the urogenital epithelium. It is a very common pathogen and one frequently inducing reactive arthritis (ReA)¹ (1). Multiple strategies, including down-regulation of major histocompatibility complex (MHC) class I and class II expression (2–4) and persistence, have been developed by the bacteria to evade the immune system. Yet both CD4⁺ and CD8⁺ T-cell responses are activated upon infection (5). In particular, HLA-B27-restricted CD8⁺ T-lymphocytes are found in patients with Chlamydia-induced ReA (6, 7). The role of these cells in the pathogenesis and evolution of ReA to chronic disease is probably mediated by IFN-γ. Secretion of this cytokine is critical for the protective immunity against Chlamydia (8) because it inhibits the bacterial growth (9). However, this is often insufficient to promote complete clearance of C. trachomatis, and actually IFN-γ-induced depletion of the tryptophan pool induces the differentiation of the metabolically active reticular bodies to persistent forms (10), which sustain chronic infection and ReA. The high prevalence of HLA-B27 among patients with Chlamydia-induced ReA (11), especially in its chronic form, suggests a pathogenetic mechanism based on interactive effects of the bacteria and HLA-B27 that seems unrelated to the capacity of C. trachomatis to infect or replicate into HLA-B27-positive cells (12). One such mechanism could be T-cell-mediated autoimmunity elicited by molecular/antigenic mimicry between chlamydial and self-derived HLA-B27 ligands. Antigenic mimicry between chlamydial and homologous α-myosin-derived peptides is crucial to inducing autoimmune myocarditis in mice (13). Breakdown of cytotoxic T-lymphocyte (CTL) tolerance to HLA-B27 was observed in transgenic rats upon exposure to C. trachomatis (14). Cross-reactivity between HLA-B27-restricted Chlamydia-specific CTL with self-derived HLA-B27 epitopes has not been reported. However, a biochemical basis for it was provided by the finding of an endogenously processed and presented peptide from the DNA primase of C. trachomatis with high homology to a self-derived HLA-B27 ligand (15, 16).Because of the likely involvement of HLA-B27 in the pathogenesis of chronically evolving ReA, the role of CD8⁺ T-cell responses in the protective immunity against C. trachomatis and the presence of HLA-B27-restricted T-cells in patients with Chlamydia-induced ReA, the identification of relevant chlamydial epitopes becomes crucial to establish the pathogenetic mechanism of this disease. Unfortunately a direct analysis of chlamydial HLA-B27 ligands expressed on infected cells is exceedingly difficult because of their extremely low amounts, which challenge even the most sensitive techniques of MS. In the case of Chlamydia, the situation is further complicated by the down-regulation of MHC class I expression shortly after infection (3, 4). To our knowledge, only one MHC class I ligand was recently identified, in the mouse system, from Chlamydia muridarum-infected cells using state-of-the-art MS techniques (17). Due in part to this difficulty, alternative approaches, such as expression cloning and synthetic peptide epitope mapping (18, 19) or MHC class I tetramer arrays (20), have been used to identify MHC class I-restricted chlamydial T-cell epitopes in mice. In a previous study (6) predictive algorithms were used to screen the whole genome of C. trachomatis for nonamer peptide sequences containing the HLA-B*2705 binding motif and a high probability of being generated by proteasomal cleavage. This led to identifying multiple sequences that, when used as synthetic peptides in vitro, stimulated CD8⁺ T-cells from patients with Chlamydia-induced ReA. Such cells could also be detected in the synovial fluid of these patients using HLA-B27 tetramers complexed to some of these peptides (7).Although these strategies identify chlamydial sequences that are recognized by CD8⁺ T-cells they do not prove that these peptides are the endogenously processed epitopes that activated the natural T-cell responses to the bacteria in vivo. Because of the intrinsic cross-reactivity of T-cells (21, 22), it is conceivable that synthetic peptides recognized in vitro may be different from the natural epitopes generated by endogenous processing of the chlamydial proteins that elicit the HLA-B27-restricted T-cell responses in ReA patients. To investigate this issue we focused on two predicted epitopes (6). Stable transfectants expressing the corresponding chlamydial proteins fused to green fluorescent protein (GFP) were generated in a B*2705-positive cell line. The endogenous processing and presentation of the predicted epitopes or other peptides from the same bacterial protein were analyzed by comparative immunoproteomics analysis of the B*2705-bound peptide repertoires from transfected and untransfected cells and sequencing of peptides differentially presented on the bacterial protein transfectant.     

The Effect of Proteasome Inhibition on the Generation of the Human Leukocyte Antigen (HLA) Peptidome

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.The repertoires and levels of peptides, presented by the major histocompatibility complex (MHC)¹ class I molecules at the cells'' surface, are modulated by multiple factors. These include the rates of synthesis and degradation of their source proteins, the transport efficacy of the peptides through the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), their subsequent processing and loading onto the MHC molecules within the ER, and the rates of transport of the MHC molecules with their peptide cargo to the cell surface. The off-rates of the presented peptides, the residence time of the MHC complexes at the cell surface, and their retrograde transport back into the cytoplasm, influence, as well, the presented peptidomes (reviewed in (1)). Even though significant portions of the MHC class I peptidomes are thought to be derived from newly synthesized proteins, including misfolded proteins, defective ribosome products (DRiPs), and short lived proteins (SLiPs), most of the MHC peptidome is assumed to originate from long-lived proteins, which completed their functional cellular roles or became defective (retirees), (reviewed in (2)).The main protease, supplying the MHC peptidome production pipeline, is thought to be the proteasome (3). It is also responsible for generation of the final carboxyl termini of the MHC peptides (4), (reviewed in (5)). The final trimming of the n-termini of the peptides, within the endoplasmic reticulum (ER), is thought to be performed by amino peptidases, such as ERAP1/ERAAP, which fit the peptides into their binding groove on the MHC molecules (6) (reviewed in (7)). Nonproteasomal proteolytic pathways were also suggested as possible contributors to the MHC peptidome, including proteolysis by the ER resident Signal peptide peptidase (8, 9), the cytoplasmic proteases Insulin degrading enzyme (10), Tripeptidyl peptidase (11–13), and a number of proteases within the endolysosome pathway (reviewed recently in (14–17)). In contrast to the mostly cytoplasmic and ER production of the MHC class I peptidome, the class II peptidome is produced in a special compartment, associated with the endolysosome pathway (18–20). This pathway is also thought to participate in the cross presentation of class I peptides, derived from proteins up-taken by professional antigen presenting cells (21), (reviewed in (15–17, 22)).The centrality of the proteasomes in the generation of the MHC peptidome was deduced mostly from the observed change in presentation levels of small numbers of selected peptides, following proteasome inhibition (3, 23). Even the location of some of the genes encoding the catalytic subunits of the immunoproteasome (LMP2 and LMP7) (24) within the MHC class II genomic locus, was suggested to support the involvement of the proteasome in the generation of the MHC class I peptidome (25). Similar conclusions were deduced from alterations in peptide presentation, following expression of the catalytic subunits of the immunoproteasome (26), (reviewed in (5)). Yet, although most of the reports indicated reductions in presentation of selected peptides by proteasome inhibition (3, 27–29), others have observed only limited, and sometimes even opposite effects (23, 30–32).The matter is further complicated by the indirect effects of proteasome inhibition used for such studies on the arrest of protein synthesis by the cells (33–35), on the transport rates of the MHC molecules to the cell surface, and on their retrograde transport back to the vesicular system (36) (reviewed in (37)). Proteasome inhibition likely causes shortage of free ubiquitin, reduced supply of free amino acids, and induces an ER unfolded protein response (UPR), which signals the cells to block most (but not all) cellular protein synthesis (reviewed in (38)). Because a significant portion of the MHC peptidome originates from degradation of DRiPs and SLiPs (reviewed in (2)), arrest of new protein synthesis should influence the presentation of their derived MHC peptides. Taken together, these arguments may suggest that merely following the changes in the presentation levels of the MHC molecules, or even of specific MHC peptides, after proteasome inhibition, does not provide the full picture for deducing the relative contribution of the proteasomal pathway to the production of the MHC peptidome (reviewed in (7)).MHC peptidome analysis can be performed relatively easily by modern capillary chromatography combined with mass spectrometry (reviewed in (39)). The peptides are recovered from immunoaffinity purified MHC molecules after detergent solubilization of the cells (40, 41), from soluble MHC molecules secreted to the cells'' growth medium (42, 43) or from patients'' plasma (44). The purified peptides pools are resolved by capillary chromatography and the individual peptides are identified and quantified by tandem mass spectrometry (40), (reviewed in (45–47)). In cultured cells, quantitative analysis can also be followed by metabolic incorporation of stable isotope labeled amino acids (SILAC) (48). Furthermore, the rates of de novo synthesis of both MHC peptides and their proteins of origin can be followed using the dynamic-SILAC proteomics approach (49) with its further adaptation to HLA peptidomics (50–52).This study attempts to define the relative contribution of the proteasomes to the production of HLA class I peptidome by simultaneously following the effects of proteasome inhibitors, epoxomicin and bortezomib (Velcade), on the rates of de novo synthesis of both the HLA class I peptidome and the cellular proteome of the same MCF-7 human breast cancer cultured cells. The proteasome inhibitors did not reduce the levels of HLA presentations, yet affected the rates of production of both the HLA peptidome and cellular proteome, mostly decreasing, but also increasing some of the synthesis rates of the HLA peptides and cellular proteins. Thus, we suggest that the degree of contribution of the proteasomal pathway to the production of the HLA-I peptidome should be re-evaluated in accordance with their effects on the entire HLA class-I peptidome, while taking into consideration the inhibitors'' effects on the synthesis (and degradation) rates of the source proteins of each of the studied HLA peptides.