Anti-class II monoclonal antibody-targeted Vibrio cholerae TcpA pilin: modulation of serologic response, epitope specificity, and isotype.

Toxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.

Immune response genes modulate serologic responses to Vibrio cholerae TcpA pilin peptides.

Cholera is an enteric disease caused by Vibrio cholerae. Toxin-coregulated pilus (TCP), a type 4 pilus expressed by V. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at the H-2 locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.

Evaluation of cholera vaccines formulated with toxin-coregulated pilin peptide plus polymer adjuvant in mice.

Cholera is an acute diarrheal disease that is caused by the gram-negative bacterium Vibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute to V. cholerae pathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine by V. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD(50)) challenge and 50% protection against a 10-LD(50) challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.

Molecular dynamics of point mutated I-A(k) molecules expressed on lymphocytes.

We have recently reported the lateral and rotational diffusion parameters for I-A(k) molecules expressing various cytoplasmic truncations (Int. Immunol. 12 (2000) 1319). We now describe the membrane dynamics of I-A(k) with various mutations in the presumed contact region between alphabeta-heterodimers in an (alphabeta)2 dimer of dimers structure. Such mutations are known to strongly affect the antigen presentation ability of these molecules (Int. Immunol. 10 (1998) 1237-1249) but cause relatively small changes in the molecular dynamics of I-A(k). Lateral diffusion coefficients of I-A(k) wild-type molecules and mutants obtained via fringe fluorescence photobleaching recovery (FPR) ranged from 1.1 to 2.3x10(-10)cm2/s at room temperature while fractional mobilities averaged 75+/-6%. For all cell types examined, treatment with either hen egg lysozyme 46-61 peptide or db-cAMP reduced the I-A(k) mobile fraction by about 10% relative to untreated cells, suggesting that these treatments may increase lateral confinement of class II in lipid rafts or cytoskeletal interactions of the molecules. Wild-type I-A(k) and mutants capable of normal or partial antigen presentation exhibited, as a group, slightly longer rotational correlation times (RCT) at 4 degrees C than did mutants inactive in antigen presentation, 14+/-4 versus 10+/-1 micros, respectively. Moreover, peptide, cAMP and anti-CD40 mAb treatment all increased rotational correlation times for fully- and partially-functional I-A(k) but not for non-functional molecules. For example, 16 h peptide treatment yielded average RCTs of 28+/-12 and 10+/-1 micros for the groups of functional and non-functional molecules, respectively. Such modulation of the dynamics of functional class II molecules is consistent with these treatments' stabilization of class II or induction of new gene expression. Measurements of fluorescence resonant energy transfer between I-A(k), though complicated by cellular autofluorescence, averaged 6+/-7% over 15 cells or treatments, a result consistent with the presence of a small fraction of I-A(k) as a dimer of dimers species. In summary, our results suggest subtle changes in the molecular motions of class II molecules correlate with a significant impact on class II function. Molecules active in antigen presentation exhibit more restricted motion in the membrane, and thus presumably more extensive intermolecular interactions, than non-functional molecules. Further, treatments, such as db-cAMP and anti-CD40, which rescue antigen presentation by partially defective mutants, appear to increase such interactions, several types of which have already been reported for class II. A more detailed understanding of these phenomena will require both more sensitive biophysical tools and a more refined model of the role of class II intermolecular interactions in antigen presentation.

Class II-targeted antigen is superior to CD40-targeted antigen at stimulating humoral responses in vivo.

We examined the efficacy of using monoclonal antibodies to target antigen (avidin) to different surface molecules expressed on antigen presenting cells (APC). In particular, we targeted CD40 to test whether the "adjuvant" properties of CD40 signaling combined with targeted antigen would result in enhanced serologic responses. We targeted avidin to class II as a positive control and to CD11c as a negative control. These surface proteins represent an ensemble of surface molecules that signal upon ligation and that are expressed on professional APC, in particular dendritic cells (DC). We observed that targeting class II molecules on APC was superior to targeting CD40, or CD11c. However, CD40 and CD11c could function as targets for antigen bound monoclonal antibodies under certain conditions. Interestingly, inclusion of anti-CD40 mAb with the targeting anti-class II-targeted antigens negatively affects humoral response, suggesting that CD40 signaling under certain conditions may suppress processing and/or presentation of targeted antigen.

Fc alpha receptor cross-linking causes translocation of phosphatidylinositol-dependent protein kinase 1 and protein kinase B alpha to MHC class II peptide-loading-like compartments.

A20 IIA1.6 B cells cotransfected with FcalphaR and wild-type gamma-chain (wt-ITAM (immunoreceptor tyrosine-based activation motif)) or FcalphaR and gamma-chain, in which the wt-ITAM was substituted with the FcgammaRIIA ITAM (IIA-ITAM), were used to investigate cell signaling events influencing presentation of FcalphaR-targeted exogenous Ag in the context of MHC class II. wt-ITAM cells presented FcalphaR-targeted OVA more efficiently than IIA-ITAM transfectants to OVA-specific T cell hybridomas. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibition abrogated Ag presentation, suggesting that FcalphaR may trigger a PI 3-kinase-dependent signal transduction pathway, and thus phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B alpha (PKBalpha) activation. Cross-linking FcalphaR on wt-ITAM or IIA-ITAM cells triggered equivalent PI 3-kinase-dependent activation of PKBalpha. Furthermore, FcalphaR cross-linking triggered recruitment of PDK1 and serine-phosphorylated PKBalpha to capped cell surface FcalphaR irrespective of the gamma-chain ITAM. Although FcalphaR endocytosis was accompanied by translocation of PDK1 and phospho-PKBalpha to FcalphaR-containing vesicles in both transfectants, this was decreased in IIA-ITAM cells, and a significant proportion of PDK1 and PKBalpha remained at the plasma membrane. In wt-ITAM cells, PDK1 and serine-phosphorylated PKBalpha translocated to lysosomal-associated membrane glycoprotein 1- and cathepsin B-containing vesicles, consistent with MHC class II peptide-loading compartments (MIIC) described by other groups. Our data indicate that translocation of signal transduction mediators to MIIC-like compartments accompanies efficient presentation of receptor-targeted Ag, and suggest a mechanism connecting signaling to the Ag-processing pathway.

Dendritic cell longevity and T cell persistence is controlled by CD154-CD40 interactions.

Inflammatory mediators facilitate the maturation of dendritic cells (DC), enabling them to induce the activation, proliferation and differentiation of cognate T cells. The role of CD40 on DC and CD154 on T cells has been studied by the co-adoptive transfer of antigen-pulsed DC and TCR-transgenic (Tg) T cells in vivo. It is shown that in the absence of CD40-CD154 interactions, initial Tg T cell expansion occurs in vivo, but over time, T cell expansion cannot be sustained. The basis for the demise of the T cell population is likely due to the disappearance of the antigen-pulsed DC in the draining lymph nodes when CD154-CD40 interactions are interrupted. These findings show that both T cell and DC persistence in vivo is dependent on CD40-CD154 interactions. In addition to the physical persistence of the DC, CD40 triggering of DC also greatly increases the period for which they can productively present antigen to Tg T cells. Hence DC persistence and antigen-presenting cell capacity are both dependent on CD40 signaling. While TNF-alpha can mature DC as measured by a variety of criteria, the unique capacity of CD40 signaling to sustain T cell responses and induce DC maturation is underscored by the inability of TNF-alpha to rescue the immune deficiency of CD40(-/-) DC. Hence, the profound impact of CD154 deficiency on cell-mediated immunity may be due to its ability to limit the duration of antigen presentation in vivo and cause the premature demise of antigen-specific T cells.

Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor gamma-chain signaling.

The mechanism of enhanced presentation of ovalbumin (OVA) internalized as immunoglobulin A (IgA)-OVA via the IgA Fc receptor (FcalphaR) was analyzed by focusing on the role of the FcalphaR-associated gamma chain. Comparison of B-cell transfectants expressing FcalphaR plus wild-type (WT) gamma chain or gamma chain in which the immunoreceptor tyrosine-based activation motif (ITAM) was altered by tyrosine mutation or substitution with the ITAM of FcgammaRIIA showed that signaling-competent ITAM was not required for endocytosis of IgA-OVA. However, antigen presentation was impaired by ITAM changes. Signaling-competent gamma-chain ITAM appeared necessary for transport of ligated FcalphaR to a lamp-1(+) late endocytic compartment for remodeling and/or activation of that compartment and also for efficient degradation of IgA complexes. Moreover, FcalphaR ligation also activated efficient processing of nonreceptor-targeted antigen. The results suggest that gamma-chain signaling activates the antigen processing compartment.

Rotational and lateral dynamics of I-A(k) molecules expressing cytoplasmic truncations.

Rotational and lateral diffusion of I-A(k) molecules with various alpha and beta chain cytoplasmic truncations known to affect class II function were measured to assess the role of cytoplasmic domains in regulating I-A(k) molecular motions. Deletion of all 12 alpha chain C-terminal residues and all 18 corresponding beta chain residues (alpha-12/beta-18) is known to abrogate translocation of protein kinase C to the nucleus upon class II cross-linking. Similarly, truncation of the entire cytoplasmic alpha chain domain and the 10 C-terminal residues of the beta chain impairs presentation of antigenic peptides to T cells. The rotational correlation time of the wild-type molecule, 11.9 +/- 2.6 micros as measured by time-resolved phosphorescence anisotropy, decreased to 7. 2 +/- 3.7 micros in the fully truncated alpha-12/beta-18 protein. Other truncated class II molecules exhibited only small changes in molecular rotation rates relative to the wild-type. The rate of lateral diffusion of the fully truncated molecule, measured with two independent methods, 2.3 x 10(-10) cm(2)/s, was comparable with that of the wild-type molecule. Thus, it appears that the alpha and beta chain cytoplasmic domains regulate the molecular motions of unperturbed I-A(k) molecules only modestly, despite the known involvement of these regions in class II signaling. Various explanations for this behavior are discussed, e.g. the possibility that class II membrane complexes are sufficiently large that association and dissociation of specific signaling proteins during antigen presentation do not significantly perturb the apparent molecular motions of the complex.

The ins and outs of getting in: structures and signals that enhance BCR or Fc receptor-mediated antigen presentation.

Antigen-presenting cells internalize antigen by fluid-phase pinocytosis or by endocytosis via surface receptors such as the B cell receptor (BCR) and Fc receptors for IgG, IgA and IgE (FcR). While both modes of internalization lead to antigen presentation it is recognized that receptor-mediated endocytosis greatly enhances the efficiency of processing and antigen presentation. Receptors facilitate the entry of antigen into the endocytic pathway by interaction of their internalization motifs with the endocytic machinery. These motifs include tyrosine-based, dileucine and casein kinase-like motifs. However these structures appear insufficient to support processing of cryptic epitopes, leading to a limited immune response. Cryptic epitope processing appears dependent on receptor signaling which is mediated by immunoreceptor tyrosine activation motifs (ITAMs). The signaling cascade which follows receptor crosslinking promotes reorganization and acidification of the late endocytic compartment or MIIC. Signaling events downstream of Syk, in particular calcium flux and protein kinase C activation, are necessary for MIIC induction. PI(3) kinase is also involved at multiple steps in antigen presentation, including production of PIP3 and transport of cathepsins. PIP3 is crucial both as a binding substrate for proteins implicated in vesicle transport and for the recruitment of signaling molecules to the plasma membrane. Among PIP3 activated molecules, protein kinase B (PKB) has been linked to endocytic function. We observe association of activated PKB with the MIIC after signaling through antigen presentation-competent receptors, but not mutant, presentation-defective receptors.

Mutations in specific I-A(k) alpha(2) and beta(2) domain residues affect surface expression.

A previous investigation demonstrated that several mutations in class II dimer-of-dimers contact residues interfere with antigen presentation by transfectants but not with plasma membrane expression of the mutant class II. In the present study we examined other class II mutations in this region that did inhibit plasma membrane expression of mutant class II molecules. Molecules containing both mutations H alpha 181D in the alpha(2) domain and E beta 170K in the beta(2) domain exhibited low plasma membrane expression, but molecules with only one of these mutations were expressed normally. The mutant class II molecules were transported to organelles that were accessible to a fluid-phase protein, hen egg lysozyme (HEL). Culture of transfectants with lysozyme enhanced the amount of class II compact dimer (alpha beta plus peptide; CD), and this was especially marked for the class II mutant H alpha 181D/E beta 170K and for other molecules possessing both mutations. Formation of class II CD was not paralleled by an increase in class II surface expression. Thus the joint mutation of H alpha 181 and E beta 170 has two effects. In the absence o high concentrations of exogenous peptide, it prevents efficient CD formation, possibly by affecting invariant chain (Ii) proteolysis and/or the stability of the class II after Ii/CLIP is removed. At high peptide concentrations supplied by exogenous HEL, the mutations allow CD formation, but not expression of class II on the plasma membrane. Molecular modeling of the possible interaction of class II and Ii suggests that the mutant amino acids H alpha 181D and E beta 170K, besides affecting the overall stability of class II, might also interact with Ii via two loops in class II's alpha(2) and beta(2) domains respectively.

Dynamics of molecules involved in antigen presentation: effects of fixation.

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-Ad on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-Ad at 4 degrees C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 micros unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from approximately 2x10(-10) cm2 s(-1), while the fractional recovery remained unchanged at about 40-50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (>500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.

Gamma-chain dependent recruitment of tyrosine kinases to membrane rafts by the human IgA receptor Fc alpha R.

We show that the human IgA receptor, Fc alpha R, redistributes to plasma membrane rafts after cross-linking and that tyrosine kinases are relocated to these sites following Fc alpha R capping. We demonstrate by confocal microscopy that Fc alpha R caps in membrane rafts by a gamma-chain-independent mechanism but that gamma-chain expression is necessary for Lyn redistribution. Immunoblotting of rafts isolated by sucrose density gradient centrifugation demonstrated recruitment of gamma-chain and phosphorylated tyrosine kinases Lyn and Bruton's tyrosine kinase to membrane rafts after Fc alpha R cross-linking. Time-dependent differences in Lyn phosphorylation and Bruton's tyrosine kinase distribution were observed between cells expressing Fc alpha R plus gamma-chain and cells expressing Fc alpha R only. This study defines early Fc alpha R-triggered membrane dynamics that take place before Fc alpha R internalization.

Dichotomy between naïve and memory CD4(+) T cell responses to Fas engagement.

Engagement of Fas (APO-1, CD95), a member of the tumor necrosis factor receptor superfamily, can induce apoptotic cell death. However, Fas engagement also can costimulate lymphocyte proliferation. The physiologic regulation of these two outcomes is poorly understood. Here, we have used two systems, the first in vitro and the second in vivo, to demonstrate that naïve and memory CD4(+) T cells display dichotomous responses to Fas ligation. Naïve CD4(+) T cells (CD44(lo), CD45RB+, CD62L+) die as a consequence of Fas ligation in the presence of anti-CD3 antibody, whereas memory T cells (CD44(hi), CD45RB-, CD62L-), freshly isolated from the same starting population and subjected to the same stimulation conditions, are costimulated to proliferate by Fas ligation. In vitro, we demonstrate that CD28-mediated signals or T helper 1 and T helper 2 differentiation cytokines alter the response of naïve T cells, but not of memory T cells, to Fas ligation. In vivo experiments in hen egg lysozyme (HEL) T cell receptor transgenic mice show that CD4(+) T cells from HEL-naïve mice are killed by Fas ligation, but CD4(+) T cells from long-term HEL-exposed mice are costimulated by Fas ligation. Thus, the physiological outcome of Fas ligation in CD4(+) T cells is determined primarily by the antigenic history of the T cell.

Salt bridge residues between I-Ak dimer of dimers alpha-chains modulate antigen presentation.

Class II dimers of dimers are predicted to have functional significance in antigen presentation. The putative contact amino acids of the I-Ak class II dimer of dimers have been identified by molecular modeling based on the DR1 crystal structure (Nydam et al., Int. Immunol. 10, 1237,1998). We have previously reported the role in antigen presentation of dimer of dimers contact amino acids located in the C-terminal domains of the alpha- and beta-chains of class II. Our calculations show that residues Ealpha89 and Ralpha145 in the alpha2-domain form an inter alpha-chain salt bridge between pairs of alphabeta-heterodimers. Other residues, Qalpha92 and Nalpha115, may be involved in close association in that part of the alpha-chain. We investigated the role of these amino acids on class II expression and antigen presentation. Class II composed of an Ealpha89K substituted alpha-chain paired with a wt beta-chain exhibited inhibited antigen presentation and expression of alpha-chain serologic epitopes. In contrast, mutation of Ralpha145E had less affect on antigen presentation and did not affect I-Ak serologic epitopes. Interchanging charges of the salt bridge residues by expressing both Ralpha145E and Ealpha89K on the same chain obviated the large negative effect of the Ealpha89K mutation on antigen presentation but not on the serologic epitopes. Our results are similar for those reported for mutation of DR3's inter-chain salt bridge with the exception that double mutants did not moderate the DR3 defect. Interestingly, the amino acids differences between I-A and DR change the location of the inter-chain salt bridges. In DR1 these residues are located at positions Ealpha88 and Kalpha111; in I-Ak these residues are located at position Ealpha89 and Ralpha145. Inter alpha-chain salt bridges are thus maintained in various class II molecules by amino acids located in different parts of the alpha2-domain. This conservation of structure suggests that considerable functional importance may attach to the ionic interactions.

Mutations in MHC class II dimer of dimers contact residues: effects on antigen presentation.

The recent solutions of the MHC class II crystal structure reveal dimerization of the alphabeta heterodimers. These dimer of dimers structures may also exist either on resting cells or after engagement by TCR, and may be involved in B cell signaling and up-regulation of co-stimulatory molecules such as B7 which facilitate T cell activation. By combining crystallographic data on HLA-DR1 with the sequence of murine I-Ak and refining the resulting structure through energy minimization calculations, we have predicted the contact amino acids expected to stabilize the I-Ak dimer of dimers structure. As in HLA-DR1, three salt bridges in I-Ak (D alpha62-Hbeta112, H alpha181-E beta163, E alpha183-Hbeta113) appear to provide the main interaction. Guided by this structural data, we prepared 45 B cell transfectants representing 20 different class II mutation phenotypes in the contact region containing these salt bridges. We examined their abilities to activate three T cell hybrids. Antigen-specific h4Ly50.5 cells were not greatly affected by changes in the dimer of dimer contact residues. In contrast, autoreactive C8.A3 T cells were very sensitive to changes in this region but presentation of class II of many mutation phenotypes could be rescued by treatments that up-regulate B7-1. The alloreactive hybridoma 2H40.2.5 was less sensitive to changes in the contact residues. A simple model was developed that summarizes the effects of the mutations for the T cells tested. Mutations at D alpha162, E alpha183, H alpha181 and Rbeta106 had the largest negative impact, while D alpha166, E alpha185, Hbeta112, Hbeta113 and E beta163 were less disruptive. Results are consistent with mutations interfering with class II interaction with another molecule which might or might not be another class II heterodimer. However, the larger negative impact of alpha chain mutations in salt bridge pairs suggests that these sites also help maintain some essential conformation of the alpha chain apart from any possible impact on dimer of dimers stability.

Interferometric fringe fluorescence photobleaching recovery interrogates entire cell surfaces.

Fluorescence photobleaching recovery (FPR) measurements of cell surface protein lateral diffusion typically employ an interrogated spot of 0.5 microm 1/e2 radius. The effective spot area represents only 1/500 of the total surface of an 8-microm cell. An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100 molecules. This limits the precision and reproducibility of FPR measurements. We describe a method for interferometric fringe pattern FPR that permits simultaneous interrogation of the entire cell's surface. Fringe patterns are generated interferometrically within the optical path of an FPR system. Methods for interpreting fluorescence recovery kinetics on cells and for determining the protein mobile fraction are presented. With fringe FPR, the murine major histocompatibility complex class II antigen I-Ak expressed on M12.C3.F6 cells has 100-fold improved fluorescence signals relative to spot FPR, with corresponding improvements in signal-to-noise ratios of recovery traces. Diffusion coefficients (+/- standard deviation) of (2.1 +/- 0.4) x 10(-10) and (1.8 +/- 1.0) x 10(-10) cm2 s-1 with corresponding mobile fractions of I-Ak of 66.1 +/- 7.8% and 63.4 +/- 18.0% were obtained by fringe and spot methods, respectively. The improved reproducibility of fringe over spot results is less than signal improvements predict. There may thus be substantial variation from cell to cell in protein dynamics, and this method may permit the assessment of such variation.

Increased inhibition of proliferation of human B cell lymphomas following ligation of CD40, and either CD19, CD20, CD95 or surface immunoglobulin.

Non-Hodgkin's (NHL) B cell lymphomas are growth-inhibited by ligation of their CD40 molecules. This inhibition is not absolute in that approximately 50% of the cells are not inhibited. We conducted studies to see if other signals that have been reported to inhibit B cell lymphoma growth could be used in combination with anti-CD40 signaling to completely inhibit growth. Ligation of surface immunoglobulin (Ig), CD19, CD20, CD37 or CD95 with soluble antibody did not affect growth of the panel of NHL cells examined. Ligation of CD20, CD19 or CD95 was inhibitory for some NHL cell lines if the primary antibody was crosslinked with a secondary antibody. Combining anti-CD40 with anti-CD19, anti-CD20, or anti-Ig resulted in increased inhibition past that produced by anti-CD40 alone. The additive effect of anti-CD40 and other antibodies to selected surface markers was not observed in all NHL cell lines. Crosslinking of CD95 was also growth inhibitory for the majority of the NHL, and when combined with anti-CD40 under conditions that afforded crosslinking of the two receptors, increased inhibition was seen in three of the NHL cell lines. We found that cAMP or sodium butyrate (NaB) were also effective at inhibiting growth of the NHL cells; this was a profound inhibition (approaching 100%) compared to the 50% inhibition seen with anti-CD40 treatment. The potential for anti-CD40 and either cAMP or NaB to be additive was tested and not found to be the case. The ability to inhibit proliferation of the NHL was very dynamic with some antibody combinations being either inhibitory for multiple cells, not having an effect at all, or in some cases being stimulatory. This suggests that the NHL may represent unique stages of B cells that might serve as a model system which could be developed to precisely categorize patient NHL.

Lateral dynamics of major histocompatibility complex class II molecules bound with agonist peptide or altered peptide ligands.

We examined the lateral diffusion of I-Ad on A20 cells following the binding of ovalbumin-derived peptides. The peptides were OVA323-339 and OVA325-335 and a related peptide OVA325-335s substituted H331Q. Only OVA323-339 and OVA325-335 were effectively presented by A20 cells to DO-11.10/S4.4 T cells as assessed by IL-2 production. Fluorescence photobleaching recovery (FPR) measurements showed anti-I-Ad to have a lateral diffusion coefficient on untreated A20 cells of 1.8 +/- 1.0 x 10(-10) cm2 s-1 at 25 degrees C with fluorescence recovery after photobleaching greater than 50%. After 24 h incubation of A20 cells with OVA323-339 or OVA325-335, a subpopulation of A20 cells appeared that were approximately half the size of untreated A20 cells. Culture of A20 with OVA325-355s did not stimulate DO-11.10 cells or induce a size change in A20 cells. Class II molecules were laterally immobile on these small cells with fluorescence recoveries after photobleaching of less than 20%. The relative number of small cells in the A20 cell population was correlated with the immunogenicity of the peptides. These results suggest that immobilization of surface I-Ad may be an important event in antigen presentation.

Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Ak molecules. Ak beta TM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Ak conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Ak beta chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Ak beta chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to assess a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the Ak alpha Cy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.