The mucin-like protein CD43 is excluded from the immune synapse, and regulates T-cell proliferation as well as T-cell migration. While the CD43 cytoplasmic domain is necessary for regulation of T-cell activation and proliferation, the mechanism via which CD43 regulates trafficking is not well defined. To investigate whether CD43 phosphorylation regulates its function in T cells, we used tandem mass spectrometry and identified Ser76 in murine CD43 as a previously unidentified site of basal phosphorylation. Interestingly, mutation of this single serine to alanine greatly diminishes T-cell trafficking to the lymph node, while CD43 exclusion and CD43-mediated regulation of T-cell proliferation remain intact. Furthermore, the CD43 extracellular domain was also required for T-cell trafficking, providing a hitherto unknown function for the extracellular domain, and suggesting that the extracellu- IntroductionCell-surface mucins, characterized by large, highly glycosylated extracellular domains, are important regulators of tumor progression as well as lymphocyte trafficking. 1 Overexpression and altered glycosylation of mucins on cancer cells, compared with normal cells, makes them ideal candidates for tumor-based vaccines. 2,3 CD43 is one such member of the mucin family, 4 and is widely expressed on the surface of hematopoietic cells. 5 Reduced expression and aberrant glycosylation of CD43 is found on the T cells of patients suffering from Wiskott-Aldrich syndrome, 6-8 as well as HIV infection. 9,10 In contrast, CD43 is overexpressed on lymphomas and leukemias, and while not normally expressed on nonhematopoietic cells, it is present on colon adenomas and carcinomas. 11,12 In spite of its abundance on T cells, the precise function of CD43 remains elusive. T cells from CD43 Ϫ/Ϫ mice exhibit increased proliferation and adhesion in vitro, 13,14 and increased immune responses in vivo. 15 Strikingly, CD43 is excluded from the immune synapse of activated T cells, 16 and often localizes to a protein complex distal to the antigen-presenting cell (APC) contact site, referred to as the distal pole complex (DPC). 17,18 Several groups have reported that failure to exclude CD43 results in inhibition of IL-2 production. 19,20 Based on these data, CD43 has been characterized as a negative regulator of T-cell function.The prevailing hypothesis to explain the mechanism of CD43-mediated negative regulation was that the CD43 extracellular domain acted as a barrier to cell-to-cell interactions. 13,[21][22][23] However, we and others have shown that the cytoplasmic domain is not only necessary, but also sufficient for movement of CD43 away from the immunologic synapse, as well as CD43-mediated attenuation of T-cell proliferation and homotypic adhesion. 14,17,20 These data underscore a critical function for the CD43 cytoplasmic tail in regulating T-cell proliferation and adhesion. Despite significant progress in elucidating the function of the CD43 cytoplasmic tail, there is no well-defined function for its extracellular domain. In the abs...

SummaryThe cytosolic tail of the major histocompatibility complex class II-associated invariant chain (Ii) molecule is thought to contain the endosomal localization signal that directs and/or retains newly synthesized class II within the endosomal antigen processing compartment . To determine the role ofthis signal in class II transport and antigen presentation we have generated class II-positive L cell transfectants that coexpress wild type or truncated forms of Ii. Deletion of the endosomal localization signal from Ii results in rapid transport of class II-Ii complexes to the cell surface . Once at the cell surface, the complex is efficiently internalized, Ii is degraded, and class II free of Ii is recycled back to the plasma membrane. Interestingly, the truncated form of Ii is still able to increase the efficiency of antigen presentation to T cells. These data suggest that the ability of Ii to enhance antigen presentation is not limited to Golgi apparatus-endosomal sorting and raise the possibility that endocytosed class II can form immunogenic complexes with newly processed antigen . steady state endosomal localization of Ii and results in the transport of Ii to the cell surface.These results demonstrate that Ii can have profound effects on class II folding and transport and predict that association with Ii could increase the opportunity for class II to associate with peptides generated in endosomal compartments. Ii has been shown to increase the efficiency of antigen presentation, but for only a subset of antigens (15,16) . Although the characteristics of protein antigens that determine Ii dependence are not known, the ability of class II to present some antigens in the absence of Ii suggests that class II can gain access to endosomal compartments independently of Ii. Class II could enter endosomes either by direct transport from the transGolgi network (Chervonsky, A., and A. J. Sant, personal communication) or after internalization of class II from the plasma membrane (17)(18)(19). In most cases the ability of Ii to enhance antigen presentation is restricted to the alternatively spliced Ii gene product, p41 (16). The p41 form of Ii differs from p31 by the inclusion of an additional exon that encodes a 64-amino acid segment near the COOH terminus of Ii (20). The p41 form of Ii is able to impart the same folding (16) and peptide blocking functions as does p31 (Anderson, M. S., and J. Miller, manuscript in preparation) . Because p31 and p41 only differ in the lumenal domain, both proteins contain the same endosomal localization signal located in the rytosolic tail. Nevertheless, efficient antigen presentation often requires association of class II with p41 (16) .As a first step in dissecting the mechanism by which p41 1959 J. Exp. Med. m The Rockefeller University Press "

Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes.

The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II–restricted T helper cells. Invariant chain-deficient (Ii −/−) mice have impaired ability to present major histocompatibility complex class II–restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii −/− mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-γ, Ii −/− mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii −/− mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-γ–or IL-4–secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

In Ag-presenting cells, MHC class II molecules bind antigenic peptides in endocytic compartments and transport them to the cell surface for presentation to CD4+ T cells. Newly synthesized class II alpha beta heterodimers associate with a third polypeptide, invariant chain (Ii), in the endoplasmic reticulum. This association may prevent class II molecules from binding peptides until they are transported to endocytic compartments where Ii is proteolyzed. Signals in the Ii cytosolic tail are believed to be responsible for the targeting of class II-Ii complexes to endocytic compartments, but it is unclear whether this targeting event occurs at the trans face of the Golgi or at the plasma membrane. In this report, we address whether the endosomal localization signal in the Ii cytosolic tail can be functionally substituted with a tyrosine-based signal for rapid internalization from the cell surface. A chimeric protein was generated in which the Ii cytosolic tail was replaced in its entirety with the cytosolic tail from transferrin receptor. In cells expressing this chimeric form of Ii, newly synthesized class II-Ii complexes travel rapidly to the cell surface and are internalized efficiently, but Ii proteolysis is delayed and class II Ag presentation is inhibited. These results suggest that targeting class II-Ii complexes to early/recycling endosomes from the cell surface does not in itself lead to Ii proteolysis; subsequent delivery to later endosomes may be required. The data suggest that signals for this targeting event may lie in residues 18 to 29 of the Ii cytosolic tail.

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.

Antigen presentation by MHC class II (class II) is facilitated by the accessory molecules, invariant chain (Ii) and H2-M. Ii associates with class II during biosynthesis and promotes transport of class II to Ag-loading compartments. One function of H2-M is the removal of Ii fragments from MHC class II. We have previously demonstrated that Ii-deficient mice, unlike class II-deficient mice, are resistant to L. major infection. In the present study, we found that H2-M-deficient (H2-M0) mice were susceptible to progressive infection with L. major. The dispensability of Ii for control of L. major allowed genetic analysis of whether H2-M functions by association with or independently of Ii. In contrast to Ii-deficient (Ii0) mice, Ii0H2-M0 mice were as susceptible to L. major as H2-M0 mice. Thus, H2-M has an essential, Ii-independent function during presentation of microbial pathogens.

During biosynthesis, MHC class II molecules are diverted to endocytic compartments in which they bind antigenic peptides to be displayed on the surfaces of APC. For many Ags, the efficiency of class II presentation is enhanced by the intracellular association of class II with invariant chain (li), consistent with a role for newly synthesized class II molecules in Ag presentation. For a subset of Ags, however, efficient presentation does not require li. These Ags may also be bound by class II molecules en route to the cell surface. Alternatively, li-independent Ag presentation may utilize a pool of preexisting class II molecules that may gain access to endosomes following internalization from the cell surface. To examine the role of newly synthesized class II in the presentation of the li-independent Ag, RNase, we placed class II biosynthesis under the translational control of an iron response element. Chelation of iron from the media resulted in efficient diminution of class II synthesis and a marked decrease in the efficiency of RNase presentation. When compared with other cells expressing varying amounts of class II, we found that the ability to present RNase correlates with the level of class II biosynthesis and not with the level of class II surface expression. Because these cells internalize class II at a significant rate, we conclude that even in the absence of li, class II molecules can reach endocytic compartments containing antigenic peptides and they do so on their biosynthetic pathway.