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CD4 Raft Association and Signaling Regulate Molecular Clustering at the Immunological Synapse Site¹

Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510

	Abstract

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T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKC raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKC clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of CD4 T cells occurs upon recognition of a cognate MHC class II (MHC II)³-peptide complex, resulting in a complex series of signaling events which ultimately leads to T cell proliferation and differentiation. It has become increasingly clear that the macromolecular organization of molecules at the T cell surface plays an important role in determining functional outcomes. Specifically, both lateral association of the TCR, CD4, and signaling molecules with sphingolipid-rich membrane microdomains (rafts), as well as the higher order molecular patterning observed in the immunological synapse, have been shown to be correlated with T cell activation (1, 2, 3, 4). Several recent studies have detailed the molecular components both of lipid raft domains and of the immunological synapse and have elegantly described the dynamic nature of molecules participating in these processes during the course of T cell activation (5, 6, 7, 8, 9). However, regulatory mechanisms governing recruitment of molecules to rafts and synapse organization are less well understood.

Recently, we have shown that CD4 plays an important role in governing TCR/raft association in primary T cells. Specifically, we showed that CD4 is required for efficient recruitment of the TCR to lipid microdomains (10). In addition, CD4 has been shown to be required for MHC clustering and efficient lck recruitment to the immunological synapse (6). However, the way in which CD4 governs mature immunological synapse formation defined by specific localization of the TCR and protein kinase C (PKC) is unclear. Furthermore, the respective roles of the different functional domains of CD4 in regulating synapse formation remains poorly understood. Regions of particular interest include the CD4 ectodomain which is involved in MHC II interactions, the CD4 cytoplasmic tail which is involved in CD4 mediated signaling events, and the palmitoylation sequences in the juxtatransmembrane region of CD4 which act as raft targeting motifs (11).

To gain insight into the role that CD4 plays in synapse formation, we used specific mutants of CD4 to study their ability to regulate synapse formation. Specifically, we questioned whether CD4 association with lipid rafts is critical for TCR/PKC clustering at the synapse site. We found that CD4 is not only required for TCR association with lipid microdomains, but is also required for TCR/PKC clustering at the synapse site. Furthermore, the cytoplasmic tail and palmitoylation sequences of CD4 appear to play distinct roles in mediating raft localization and clustering at the immunological synapse. We demonstrate that raft localization of CD4 in T cells is dependent on palmitoylation sequences in the juxtatransmembrane region of CD4 but not on the cytoplasmic tail. Interestingly, however, both the cytoplasmic tail of CD4 and palmitoylation sequences play important roles in mediating TCR/PKC clustering at the synapse site. These data reveal a novel component of CD4 coreceptor function in governing the formation of higher ordered molecular patterns seen during T cell activation. We suggest that the raft association of CD4 is necessary but not sufficient for organized synapse formation and that there is an additional requirement for CD4 signaling through its cytoplasmic tail to result in TCR/PKC clustering at the T/APC contact zone. These studies indicate that there may be unique roles for a raft-specific subset of CD4 molecules which are particularly poised to regulate cellular processes in novel ways.

	Materials and Methods

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Antibodies

The following mAbs were used in this study: anti-CD4 (GK1.5), anti-CD8 (TIB 210), anti-Thy-1 (Y19), anti-MHC II (212.A1 or TIB 92 or 14.4.4), anti-CD32/16 (24G2), and anti-B220 (TIB 164). All Abs were purified from culture supernatants on protein G columns and dialyzed against PBS before use. Purchased mAbs are as follows: purified anti-TCR (H57), anti-TCR (H28), FITC-anti-V3, and anti-CD4 (RM 4-5) were purchased from (BD PharMingen, San Diego, CA); anti- tubulin (Sigma-Aldrich, St. Louis, MO). Rhodamine-conjugated cholera toxin was purchased from List Biological Laboratories (Campbell, CA). Biotinylated anti-human CD4 was purchased from BD PharMingen. The following fluorescent secondary Abs were purchased from Molecular Probes (Eugene, OR): Alexa-488 anti-FITC, Alexa 594 anti-goat, and streptavidin 647.

Mice

B10.BR and B10.A(5R) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The AND TCR-transgenic mice have been previously described (12). The CD4 ^–/– and CD4 cyt mice were generously provided by D. Littman (New York University, New York, NY) and then crossed with the AND TCR-transgenic mice (13). All mice used in these studies were 6–10 wk old.

Peptides

The peptide used in these studies is derived from moth cytochrome c (MCC; peptide 88–103) and has an arginine substituted for lysine at position 99 (K99R = VFAGLKKANERADLIAYLRQATK) and acts as a weak agonist peptide. A peptide derived from BSA, pBSA (peptide 141–154) = GKYLYEIARRHPYF, was used in some studies as a peptide control that binds to I-E but fails to stimulate AND TCR CD4 T cells. All peptides were synthesized by the W.M. Keck Foundation Biotechnology Resource Laboratory (New Haven, CT) and were purified by HPLC before use.

Preparation of APC and CD4⁺ T cells

T cell-depleted APC were prepared by Ab-mediated complement lysis of B10.A(5R) splenocytes as previously described (14). CD4⁺CD8^– T cells from lymph nodes and spleens of transgenic mice were isolated using immunomagnetic negative selection as previously described (14).

Effector T cell activation

Th1 cells were generated by in vitro cytokine skewing as previously described (13). Th1 cells were restimulated by coculture with T-depleted splenocytes from B10.BR mice pulsed with 50 µM of the indicated peptide as previously described (10).

Purification of raft fractions

Rafts were prepared by sucrose gradient fractionation as previously described (8). Four hundred-microliter fractions were collected from the top of the gradient. Fractions were pooled and subsequently analyzed directly or following immunoprecipitation. Pooled raft (fractions 1, 2, 3, 4) and nonraft (fractions10, 11, 12) fractions were internally normalized for protein content.

Immunoprecipitation and Western blotting

Immunoprecipitations were done using 1% Brij 96/97 (Sigma)-Aldrich lysis buffer (20 mM Tris-Cl (pH7.5), 150 mM NaCl, and 5 mM EDTA) supplemented with protease and phosphatase inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 1 mM Na₃VO₄, and 50 mM NaF) as previously described (10).

Immunocytochemistry and microscopy

T cells were mixed with Ag-pulsed APC as described above for 10 min at 37°C. T cell-APC conjugates were then placed on Alcian blue-coated coverslips in serum-free medium and incubated for 30 min at 37°C to permit adherence. Cells were fixed, permeabilized, stained, and analyzed by microscopy as previously described (15).

Cholera toxin colocalization

T cells were treated with rhodamine-labeled cholera toxin B subunit (List Biochemicals) on ice for 30 min, followed by treatment with goat anti-cholera toxin B subunit (Calbiochem, La Jolla, CA) on ice for 30 min. Cells were then placed at 37°C for 10 min to allow cross-linking, adhered to coverslips, fixed, stained, and analyzed by immunofluorescent microscopy as above for CD4.

Generation of CD4 mutants

Wild-type (WT) and palmitoylation mutant human CD4 cDNA were generously provided by J. Rose (Yale University, New Haven, CT). These cDNAs were then subcloned into the pMSCV retroviral vector. Three constructs were created: empty vector, WT human CD4, and palmitoylation mutant CD4, which has both palmitoylated cysteine residues mutated to serines (C394S plus C397S) and have been previously described (16).

Retroviral infection

Retroviral plasmids containing WT or palmitoylation mutant CD4 were transfected into Phoenix packaging cells. Twenty-four hours following transfection, cells were transferred to 32°C to allow viral integration, and supernatants were collected 24 and 48 h later. Viral supernatants were stored at –70°C until use. Retroviral supernatants were then used to infect CD4 T cells after 24 h in culture. Retroviral supernatant was preincubated with 5 µl/ml Lipofectamine 2000 (Invitrogen, San Diego, CA) for 30 min on ice and then added to 24-h T cell cultures. Virus was centrifuged onto CD4 knockout (KO) T cells at 2200 rpm for 90 min. Cells were then incubated at 37°C for 48 h, rested for 48 h, and sorted for green fluorescence protein (GFP)-positive cells using the FACSVAntage^SE cell sorter (BD Biosciences, Mountain View, CA). Cells were analyzed for CD4 expression by flow cytometry. Sorted GFP⁺ cells were then restimulated with K99R-pulsed syngeneic APC and analyzed by fluorescence microscopy as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD4 is required for clustering TCR/PKC at the synapse site

We have previously demonstrated in primary T cells that CD4 plays an important regulatory role in governing recruitment of the TCR to rafts following peptide stimulation. By analysis of lipid raft composition in activated Th1 and Th2 cells, we showed that the recruitment of the TCR to lipid rafts in Th1 cells was dependent on the presence of CD4 and correlated with the existence of raft-associated CD4 before activation in Th1 cells (10). Using Th1 effector cells as a model system in which to further study the role of the CD4 coreceptor in governing organization of T cell signaling molecules, we tested whether CD4 plays a role in TCR/PKC clustering at the synapse site. To do this, Th1 cells were prepared from AND TCR-transgenic mice either expressing or lacking CD4. It has been shown previously that selection of MHC II-restricted T cells bearing the AND TCR and the differentiation of Th1 cells can occur in the absence of functional CD4 (13)^, (17). Th1 cells from WT and CD4-deficient (CD4 KO) mice were tested for the ability to cluster the TCR and PKC at the T cell/APC interface following stimulation with K99R, a weak agonist peptide that is known to elicit a CD4 dependent response in these T cells (13). As seen in Fig. 1, CD4⁺ cells from WT mice showed effective clustering of both the TCR and PKC at the T/APC contact zone, whereas CD4-deficient cells showed a defect in clustering of both the TCR and PKC (Fig. 1, A and B). Microtubule organizing center (MTOC) reorientation, an event that is dependent on TCR signaling, remained intact in both WT and CD4-deficient T cells, indicating that competent conjugates had in fact formed. The average number of conjugates per high-power field was similar in WT and CD4-deficient cells. We observed an average of 28 and 30 conjugates per field, respectively. of at least 5 fields counted per experimental condition. Stable conjugates did not form in the presence of an irrelevant peptide which binds I-E^k but does not stimulate the AND TCR (data not shown). Similar results were seen when WT CD4 T cells were treated with blocking Abs to CD4 before conjugation in that the MTOC reoriented but PKC failed to cluster, indicating that the observed effects are not due to a selection defect in CD4-deficient T cells (Fig. 1D). Control immunostaining for the TCR and B220 demonstrates that these conjugates are in fact B/T conjugates. Staining for the cytoskeletal adaptor molecule talin serves as a second control for productivity of conjugation. We have observed that talin clustering occurs even in the absence of TCR and PKC clustering and serves as an excellent functional marker of effective conjugation. In this experiment, talin clustering can be observed even in the absence of TCR clustering in CD4-deficient T cells, indicating that these conjugates are functional (Fig. 1C). Therefore, CD4 is required for efficient clustering of the TCR and PKC at the synapse site.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. CD4 is required for TCR/PKC clustering at the synapse site in Th1 effector cells. A, T cells from AND TCR-transgenic mice either expressing (WT) or lacking (KO) CD4 were stimulated for 10 min with syngeneic APC pulsed with K99R and adhered to coverslips, fixed, permeabilized, and stained for the V3 TCR in green and PKC in red. B, Quantitation of PKC clustering at the T/APC contact zone. Data represent the percentage of conjugate pairs observed that had clustered PKC or reoriented MTOC at the T/APC contact zone. At least 100 conjugates were examined for each condition. Data shown represent the mean value derived from three independent experiments. C, T cells from WT and CD4 KO mice were stimulated and stained as in A. Staining is for the V3 TCR in green, talin in red, and B220 in blue. D, Quantitation of PKC clustering at the T/APC contact zone in WT Th1 cells that were either untreated or treated with anti-CD4 Ab (GK1.5) before incubation with peptide-bearing APC. Cells were then stimulated, fixed, and stained as described above. Quantitation was performed as described in B. E, WT or CD4 KO Th1 cells were stimulated with syngeneic K99R-pulsed APC for 10, 30, or 60 min and were then adhered to coverslips, fixed, and stained as described above. Quantitation was performed as described in B.

TCR/PKC clustering defects in CD4-deficient Th1 cells are not due to delayed kinetics

Recent studies have indicated that in the absence of CD4 there is delayed accumulation of phosphorylated lck in the immunological synapse in CD4-deficient T cells compared with WT T cells (18). To determine whether there is a similar delay in PKC clustering in the absence of CD4, CD4-deficient Th1 cells were analyzed for PKC clustering for varying periods of time after peptide stimulation. Interestingly, the defect in PKC clustering persisted over this time course, indicating that a delay in kinetics is not a likely explanation for these findings (Fig. 1E).

Palmitoylation sequences of CD4 are required for TCR/PKC clustering at the synapse site

Because CD4 regulates TCR recruitment to lipid rafts, we questioned whether the localization of CD4 itself to lipid rafts is critical for clustering of the TCR and rafts at the T/APC interface. To investigate the differential role of raft-associated vs nonraft-associated CD4 molecules in governing clustering at the T/APC contact site, a CD4 construct with mutant palmitoylation sequences, C396/399S, was studied. These mutants have been previously described and exhibit normal trafficking to the cell surface as well as lck binding (16). Human CD4 molecules have been shown to be functional in mouse T cells in that they are able to rescue T cell development and function in CD4-deficient mice (19, 20). These constructs were then used to infect CD4-deficient T cells, and sorted GFP-expressing cells were analyzed. Cell surface levels of CD4 were similar in cells transduced with both WT and mutant CD4 (data not shown). CD4 raft association was assessed by cross-linking GM1 ganglioside, a raft-associated sphingolipid, with cholera toxin B subunit and subsequently determining whether CD4 colocalized with the aggregated raft patches. We first demonstrated that CD4 palmitoylation sequences were in fact required for CD4 raft association (Fig. 2A), confirming in primary cell data what has recently been reported in Jurkat cells (11).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. CD4 palmitoylation sequences are required for raft localization and clustering at the synapse site. A, AND TCR+CD8– T cells were isolated from CD4 KO mice and stimulated with syngeneic APC, pMCC peptide, and IL-2 for 24 h and then infected with retroviral supernatants expressing WT CD4, C396/399S CD4, or GFP alone. Forty-eight hours later, GFP+ sorted cells were treated with rhodamine-cholera toxin on ice for 30 min, followed by anticholera toxin on ice for 30 min. Cross-linking was conducted for 10 min at 37°C. Cells were then adhered to coverslips, fixed, and stained for CD4 as described above. B, T cells from CD4 KO mice were infected with either WT or palmitoylation mutant retroviral constructs as described above. Sorted GFP+ cells were then incubated for 10 min with syngenic APC that had been pulsed with K99R peptide, fixed, and stained for TCR and PKC by immunofluorescence as described above. C, Quantitation was performed as described in Fig. 1. D, T cells from AND TCR CD4 KO mice were retrovirally transduced, stimulated, and stained as in B. Staining is for the V3 TCR in green, talin in red, and B220 in blue.

The cytoplasmic tail of CD4 is required for clustering and TCR raft association following peptide stimulation

To test whether clustering required additional signals from the cytoplasmic tail of CD4, PKC and TCR clustering in T cells from WT, CD4-deficient, and CD4 cyt mice were determined. CD4 cyt is a CD4 molecule that lacks the cytoplasmic tail of the CD4 molecule needed to bind lck but possesses intact palmitoylation sequences (21). Cholera toxin cocapping studies demonstrate that WT CD4 and CD4 cyt both colocalize with cholera toxin, indicating that the cytoplasmic tail is not required for CD4 raft association (Fig. 3A). The ability of CD4 cyt molecules to colocalize with rafts was confirmed by sucrose gradient studies which, interestingly, indicate enhanced raft localization of CD4 cyt molecules compared with WT CD4 molecules (Fig. 3E). However, in contrast to T cells expressing WT CD4, TCR and PKC clustering were not observed in cells deficient in CD4 or expressing cyt CD4 (Fig. 3, B and C). Control immunostaining for TCR and B220 demonstrates that the conjugates are in fact T cell/B cell pairs, and staining for talin confirms the productivity of the conjugates (Fig. 3D). The average number of conjugates per high-powered field in these experiments was similar with CD4 WT, KO, and cyt T cells at 30, 33, and 34 respectively. These values represent the average number of conjugates per high-powered field of at least five fields counted per experimental condition. These data indicate that although CD4 cyt molecules are able to colocalize with rafts, this association is not sufficient to mediate clustering of the TCR and PKC and that clustering requires an additional signal provided by the cytoplasmic tail of CD4.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. The cytoplasmic tail of CD4 is not required for CD4 raft association, but is required for TCR/PKC clustering at the site of T/APC contact. A, T cells from CD4 WT, CD4 KO, and CD4 tailess (cyt) mice were treated with cholera toxin and anticholera toxin and stained as described in Fig. 2. B, Th1 cells were generated from WT, CD4 KO, or CD4 cyt mice. Cells were stimulated and stained as described above. C, Quantitation was performed as described in Fig. 1. Data shown represent the mean value derived from three independent experiments. D, T cells from WT and CD4 cyt mice were stimulated and stained as in B. Staining is for the V3 TCR in green, talin in red, and B220 in blue. E, Raft and nonraft fractions were prepared from CD4 T cells from CD4 WT and cyt mice by sucrose gradient centrifugation. Pooled raft fractions were then either immunoprecipitated for CD4 or run as whole cell lysates on SDS-PAGE and analyzed by SDS-PAGE for the presence of CD4 and lck.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. CD4 cytoplasmic tail is required for TCR raft association following peptide stimulation. A, Raft and nonraft fractions were prepared from WT, CD4 KO, or CD4 cyt Th1 cells by sucrose gradient centrifugation. Pooled raft and nonraft fractions were then immunoprecipitated for TCR, resolved by SDS-PAGE, and analyzed by Western blotting for TCR. B, Quantitation was performed by scanning densitometry and are presented as raft fraction/nonraft fraction x 1000.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

These data enhance our understanding of the ways in which CD4 regulates the formation of the macromolecular TCR complex. We have previously demonstrated a role for CD4 in regulating the association of CD45 and the TCR. The present study implicates CD4 in mediating additional levels of TCR macromolecular complex formation through the recruitment of molecules to lipid rafts and clustering of TCR/PKC at the synapse site. CD4 has been postulated to be a "kinetic proofreading" molecule which would bind to MHC II and provide a stable interaction between a T cell and an APC during which time the TCR could scan for antigenic peptide (22). The participation of CD4 in lipid raft organization and immunological synapse formation are consistent with such a role: CD4 binding would allow productive T/APC conjugate pairs to form, and it is only in this context that higher order structures such as immunological synapses would form in the presence of cognate ligand.

The studies discussed above used the MCC-derived partial agonist peptide K99R. We chose to use the K99R peptide as Ag in these studies because it has been demonstrated previously to induce a CD4 T cell response that is CD4 coreceptor dependent (13). We have shown that the high-affinity agonist peptide MCC does not require CD4 signaling for proliferation, calcium mobilization, or IFN- production (13). Furthermore, we have shown, not unexpectedly, that there is less of a requirement for CD4 in governing molecular clustering at the immunological synapse site induced by MCC stimulation. Whereas PKC was clustered in 80% of WT cells stimulated with either MCC or K99R, CD4 KO cells had PKC clustered in only 6% of CD4 T cells stimulated with K99R-stimulated cells compared with 58% of CD4 T cells stimulated with MCC.

This finding is consistent with our previous data in Th2 cells which also indicated that raft recruitment and clustering at the site of T/APC contact was more important for a cell’s ability to respond to low-affinity antigenic stimulus. This notion is supported in part by a recent study demonstrating that CD4 was required for productive recognition of a small number of cognate ligands, but as the number of antigenic peptide-MHC II complexes increased, the requirement for CD4 decreased (23) Although Ag dose and affinity are clearly not identical, the notion that there is some threshold level of signal beyond which the requirement for CD4 decreases remains valid.

This may be explained if the requirement for T cell activation requires triggering of a certain number of TCRs over a given time period. In this scenario, high-affinity ligands would provide sufficient signal even without the higher ordered structures of lipid rafts and raft/TCR clustering. Low-affinity ligands, however, would not result in a sufficiently sustained signal unless the TCR and relevant signaling molecules are clustered in such a way that the signal supercedes the threshold for activation.

Studies using palmitoylation mutants of CD4 demonstrate that palmitoylation of CD4 is in fact required for CD4 association with lipid rafts and also for lipid raft aggregation upon CD3 cross-linking in Jurkat cells (11). Palmitoylation has also been shown to be critical for linker for activation of T cell localization to lipid rafts and signaling function (8). These data support the experiments described here and we have extended these observations to investigate the effect of CD4 palmitoylation on PKC and TCR clustering at the site of antigenic contact in primary T cells. Interestingly, there is some evidence to suggest that protein palmitoylation is a reversible, regulatable event (24). Under these circumstances, it is possible that the palmitoylation status of CD4 varies depending on the differentiation or activation state of a T cell and that the state of lipid modification may in fact be able to dictate some of these parameters. Ongoing studies of T cell differentiation using the palmitoylation mutants described here, as well as studies investigating the palmitoylation status of CD4 in differentiated Th1 and Th2 cells, will begin to allow us to answer these questions.

These studies also indicate that although palmitoylation of CD4 is necessary for immunological synapse formation to proceed, it is not sufficient. The tailess CD4 molecule used in these studies has both palmitoylation sequences intact, yet is still unable to rescue immunological synapse formation in Th1 cells. This is likely due to a signaling event downstream of CD4 which is required to allow clustering of the TCR and PKC. A likely candidate to mediate these events is CD4-associated lck. In support of such a scenario, the activation of vav and WASP, which are known to lead to actin polymerization, have been shown to be lck dependent (25, 26). The Src homology 3 domain of lck has been shown to be required for T/B cell adhesion/conjugate formation, indicating that proteins that interact with this domain are required for the "inside-out" signaling pathway which leads to accumulation of actin, talin, and LFA-1 at the T/APC contact zone (27). Lck has also been shown to play a role in tyrosine phosphorylation of other cytoskeletal proteins such as ERM proteins (28), which are involved in shuttling and organizing molecules distal to the immunological synapse (29).

In summary, these data indicate that CD4 is an important regulatory molecule that governs both recruitment of the TCR to rafts and TCR/PKC clustering at the immunological synapse site. These studies provide evidence that the CD4 coreceptor plays an increasingly complex role in T cell activation. Furthermore, we suggest that coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in microdomains of the plasma membrane.

	Acknowledgments

 
We thank Patricia Ranney for expert animal husbandry, Linda Isakson for assistance in preparing this manuscript, and David Leitenberg for his critical reading of this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants R37 AI26791-16 and RO1 CA 38350-23. F.B. is supported by the Medical Scientist Training Program.

² Address correspondence and reprint requests to Dr. Kim Bottomly, Section of Immunobiology, Yale University School of Medicine, 330 Cedar Street, Room 546, New Haven, CT 06510. E-mail address: Kim.Bottomly{at}yale.edu

³ Abbreviations used in this paper: MHC II, MHC class II; PKC, protein kinase C; MCC, moth cytochrome c; KO, knockout; GFP, green fluorescence protein; WT, wild type; MTOC, microtubule organizing center.

Received for publication October 1, 2003. Accepted for publication March 1, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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