Heterogeneity in cellular antigen retention structures

The mechanism of presentation of foreign antigens to helper T lymphocytes and the nature of the structures involved in this process are not totally understood. It is well documented that this event is carried out by antigen-presenting cells (APC) (e.g., macrophages, dendritic cells, and B lymphocytes) that internalize the antigen, process it, reexpress it on their membrane surface, and present it to the T cell in the context of major histocompatibility complex class II (Ia) molecules. Recent evidence supports the hypothesis that peptide antigens associate directly with Ia molecules on the APC surface membrane. However, the characteristics of other APC membrane structures potentially involved in antigen presentation are not entirely clear. Previous studies in our laboratories identified a guinea pig macrophage membrane-bound, non-Ia-containing antigenic complex (peak A) formed upon incubation of APC with the octapeptide antigen angiotensin (AII). This complex was capable of stimulating AII-immune guinea pig T cells and thus appeared to contain the immunologically relevant form of the antigen. For this reason it was important to establish whether such complex formation with peptides occurs with other cell types and with other peptide antigens. In the present study we found that other types of cells are also capable of forming such a membrane complex with antigen (peak A) and that this event is not unique to AII. Two other peptides, alpha-melanocyte-stimulating hormone and human fibrinopeptide B, both of which are antigenic in mice, were found to form peak A with a number of murine cell lines. As in our earlier studies with guinea pig macrophages, there was no evidence from these experiments for a role for major histocompatibility complex Ia antigens in the peptide binding observed. Differences in both the amount of peak A formation and the pattern of peptide antigen degradation were found from cell line to cell line for a given peptide, and from peptide to peptide for a given cell line, suggesting cellular heterogeneity in peptide processing and retention. In addition, cross-inhibition studies indicated that there was peptide specificity in the formation of peak A perhaps suggestive of molecular heterogeneity in the structure of peak A. These results indicate that there may be several types of cell surface molecules that specifically bind and retain peptide antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

T lymphocyte recognition of insolubilized peptide antigen

To study the role of antigen-presenting cells (APC) in T lymphocyte responses, the stimulation requirements of a murine T cell hybridoma specific for the peptide antigen human fibrinopeptide B (hFPB)/I-Ak was examined. The fine specificity of T cell recognition of this peptide was determined by using several hFPB homologs and analogs, which indicated that the intact 14-amino acid peptide must remain intact to preserve the antigenic determinant, and that the carboxyl terminal Arg14 was important for T cell responses. Of particular interest was the finding that APC-associated hFPB failed to stimulate the T cells, and that activation was only observed with soluble peptide or by brief hFPB treatment of the T cells and APC mixed together. In addition, hFPB covalently bound to agarose beads was able to cause T cell activation, provided that I-Ak+ APC were also present in the culture. A number of control experiments were performed that showed that hFPB was not released from the bead and that the antigenic peptide involved in T cell responses remained bound to the beads. These results indicate that the form of the hFPB peptide antigen recognized by this T cell can be provided separately from APC.     

Silencing of immunodominant epitopes by contiguous sequences in complex synthetic peptides.

We have previously shown that the T cell response to the synthetic peptide cI12-26:NP365-380 (covalently linked epitopes of lambda repressor (cI) and influenza A nucleoprotein (NP) polypeptides) requires amino acid sequences located in the junctional region between the cI12-26 and NP365-380 epitopes in the H-2d and H-2k haplotypes. In this study, we show that the dominant epitope of cI12-26:NP365-380 in H-2b mice is also located within the junctional region of the peptide, indicating that the same amino acid sequence is immunodominant in three different H-2 haplotypes. Based on results using fixed APC, there was no qualitative difference in epitope recognition due to antigen processing. In addition, antigen presentation by APC expressing mutant I-A molecules constructed by hemiexon shuffling of regions of the molecule containing primarily beta sheet or alpha helix showed that many different substitutions were permissive for at least one of the T hybridomas. More importantly, however, when the junctional sequences are covalently linked in composite synthetic peptides containing additional previously defined T cell epitopes, antigenicity of the immunodominant junctional region was silenced and a new epitope assumed immunodominance. Thus, immunodominance does not correlate with the primary amino acid sequence of the potential epitope. Instead, the immunodominant epitope is determined by complex interactions among the epitopes, which most likely depend on the structural conformation of the composite peptide.     

Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4⁺ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.     

Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein

Abstract Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli . The carboxyl terminal fusion (HBc_3–144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc_1–183) could not be purified or characterised immunologically, although it formed core like particles. HBc_3–144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7–17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc_3–144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.     

Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex.

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex. An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated. We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains. Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase. DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases.     

Functional and biochemical parameters of peptide antigen presentation

To understand the mechanism by which peptide antigens are processed and presented to T cells, we examined the T-cell response to the 13-amino-acid peptide alpha-melanocyte-stimulating hormone (alpha-MSH). To determine the fine specificity of T-cell recognition, T cells specific for alpha-MSH, and genetically restricted by I-Ab/d, were challenged with different alpha-MSH analogs and homologs. It was found that intact alpha-MSH, including the blocked amino and carboxy termini of the native molecule, was required for T-cell responsiveness. Antigen-presenting cells (APC) could be briefly pulsed with alpha-MSH and then present the alpha-MSH antigenic determinant to T cells, indicating that the relevant antigen was retained by the APC. APC stimulatory capacity was dramatically reduced by aldehyde treatment of the APC, or by pulsing the APC with alpha-MSH at low temperature. Efficient alpha-MSH pulsing was also impaired by treatment of the APC with the carboxylic ionophore, monensin, but not by the lysosomotropic agents chloroquine and methylamine. In addition, isolated APC plasma membranes added to the T cells in the presence of soluble alpha-MSH were not stimulatory. However, plasma membranes isolated from APC that had been previously pulsed with alpha-MSH retained stimulatory activity for T-cell responses. The only detectable alpha-MSH contained in these pulsed APC membranes was in an acid-stable complex of higher molecular weight than native peptide. The amount of alpha-MSH detected in the cellular membrane fraction isolated by density gradient sedimentation was also reduced by treatments that reduced the APC stimulatory capacity, such as pulsing at low temperature or in the presence of monensin. Taken together, these results suggest that processing of alpha-MSH is unlike that heretofore described for other peptide antigens and seems to involve APC handling to form the stimulatory moiety presented on the APC surface.     

Critical and distinct roles of amino- and carboxyl-terminal sequences in regulation of the biological activity of the Chp atypical Rho GTPase

Chp (Cdc42 homologous protein) shares significant sequence and functional identity with the human Cdc42 small GTPase, and like Cdc42, promotes formation of filopodia and activates the p21-activated kinase serine/threonine kinase. However, unlike Cdc42, Chp contains unique amino- and carboxyl-terminal extensions. Here we determined whether Chp, like Cdc42, can promote growth transformation and evaluated the role of the amino- and carboxyl-terminal sequences in Chp function. Surprisingly, we found that a GTPase-deficient mutant of Chp exhibited low transforming activity but that deletion of the amino terminus of Chp greatly enhanced its transforming activity. Thus, the amino terminus may serve as a negative regulator of Chp function. The carboxyl terminus of Cdc42 contains a CAAX (where C is cysteine, A is aliphatic amino acid, X is terminal amino acid) tetrapeptide sequence that signals for the posttranslational modification critical for Cdc42 membrane association and biological function. Although Chp lacks aCAAXmotif, we found that Chp showed carboxyl terminus-dependent localization to the plasma membrane and to endosomes. Furthermore, an intact carboxyl terminus was required for Chp transforming activity. However, treatment with inhibitors of protein palmitoylation, but not prenylation, caused Chp to mislocalize to the cytoplasm. Thus, Chp depends on palmitoylation, rather than isoprenylation, for membrane association and function. In summary, Chp is implicated in cell transformation, and the unique amino and carboxyl termini of Chp represent atypical mechanisms of regulation of Rho GTPase function.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.     

Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

Nonrandom catabolism of proteins in the formation of antigenic peptide fragments

To examine the role of protein catabolism in the formation of antigenic peptide fragments, human fibrinopeptide-immune guinea pig T cells were stimulated with the large native molecule, human fibrinogen. Two different systems were tested. In the first, we determined responses by human fibrinopeptide B (hFPB)-immune T cells, to which strain (St.) 2 guinea pigs are responders and St. 13 are nonresponders, and by human fibrinopeptide A (hFPA)-immune T cells to which St. 13 are responders and St. 2 are nonresponders. Of interest in this comparison is that both hFPA and hFPB are amino terminal peptides on the A and B chain of fibrinogen, respectively, and are readily cleaved by thrombin during fibrin formation and by other trypsin-like enzymes, leaving a carboxyl terminal Arg. Thus, if fibrinogen catabolism occurred, both antigenic peptides should be equally represented for availability in T cell responses. It was found that hFPB-immune St. 2 T cells responded to fibrinogen, but no response was observed with hPFA-immune St. 13 T cells cultured with fibrinogen. To rule out that there was a general catabolic defect in St. 13 antigen-presenting cells, fibrinogen was presented by (2 X 13)F1 macrophages to fibrinopeptide-immune parental T cells. Again it was found that F1 macrophages could present fibrinogen to hFPB-immune T cells but failed to present hFPA. In another comparison, responses with fibrinogen were also determined with des-ARg-hFPB, which lacks the carboxyl terminal Arg of hFPB, to which St. 13 are responders and St. 2 are nonresponders. The advantage of this comparison is that both antigenic determinants are contained within the same small peptide. St. 13 des-Arg-hFPB-immune T cells failed to respond in vitro by culture with human fibrinogen, suggesting that these antigenic determinants are not produced from larger peptides or proteins containing those determinants. To rule out the possibility that this was only an in vitro phenomenon, guinea pigs were immunized with the larger protein, the B chain of fibrinogen, and the immune T cells were examined for responses to fibrinopeptides derived from the B chain. Immune St. 2 T cells responded to hFPB but not to des-Arg-hFPB, whereas St. 13 T cells remained unresponsive with both peptides. These results indicate that proteolysis of larger proteins to form small antigenic peptides is not a random event and that not all potential antigenic determinants contained in a protein are produced during antigen processing.     

SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.

Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.     

Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling

BACKGROUND: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. RESULTS: Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. CONCLUSIONS: The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.     

Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.     

Signal peptide mutants ofEscherichia coli

Numerous secretory proteins of the Gram-negative bacteriaE. coli are synthesized as precursor proteins which require an amino terminal extension known as the signal peptide for translocation across the cytoplasmic membrane. Following translocation, the signal peptide is proteolytically cleaved from the precursor to produce the mature exported protein. Signal peptides do not exhibit sequence homology, but invariably share common structural features: (1) The basic amino acid residues positioned at the amino terminus of the signal peptide are probably involved in precursor protein binding to the cytoplasmic membrane surface. (2) A stretch of 10 to 15 nonpolar amino acid residues form a hydrophobic core in the signal peptide which can insert into the lipid bilayer. (3) Small residues capable of -turn formation are located at the cleavage site in the carboxyl terminus of the signal peptide. (4) Charge characteristics of the amino terminal region of the mature protein can also influence precursor protein export. A variety of mutations in each of the structurally distinct regions of the signal peptide have been constructedvia site-directed mutagenesis or isolated through genetic selection. These mutants have shed considerable light on the structure and function of the signal peptide and are reviewed here.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Eicosapentaenoic acid attenuates statin-induced ER stress and toxicity in myoblast

T cell activation requires both antigen specific and co-stimulatory signals that include the interaction of CD28 with its ligands CD80 and CD86. These signals are delivered by antigen presenting cells (APC) in the context of the immunological synapse (IS). Reorganization of the cytoskeleton is required for the formation and maintenance of the IS. Our results show that a highly conserved polylysine motif in CD86 cytoplasmic tail, herein referred to as the K4 motif, is responsible for the constitutive association of CD86 to the cytoskeleton in primary human APC as well as in a murine APC model. This motif is not involved in initial APC:T cell conjugate formation but mutation of the K4 motif affects CD86 reorientation at the IS. Importantly, APCs expressing CD86 with mutated K4 motif are severely compromised in their capacity to trigger complete T cell activation upon peptide presentation as measured by IL-2 secretion. Altogether, our results reveal the critical importance of the cytoskeleton-dependent CD86 polarization to the IS and more specifically the K4 motif for effective co-signaling.     

In vitro processing of insulin for recognition by murine T cells results in the generation of A chains with free CysSH.

Studies on the processing of insulin as an Ag for the presentation to MHC class II-restricted T cells revealed that the amino acid residues 1-14 of the insulin A chain are recognized by insulin-specific T cells. An A1-14 peptide containing three cys-residues that were protected by S-sulfonate groups still needed processing by APC for efficient presentation similar to native insulin. We suspected that reductive deblocking or opening of disulfide bonds that generates CysSH-residues may be an essential processing step for these Ag. Due to the instability of SH-groups it was not possible to test A chain peptides with free SH-groups in the usual way for processing-independent presentation by fixed APC. However, under acidic conditions (pH 5) during APC pulsing with the Ag we could demonstrate that the freshly reduced A1-14 fragment as well as reduced insulin are able to bind to Ia Ag and to stimulate appropriate T cells without further processing. Various substitutions of cys-residues by Ser within this peptide revealed that only CysA7 is critical for Ia binding and/or T cell recognition. In intact insulin, this residue links the A chain containing the T cell epitope to the B chain. Therefore, we propose that insulin processing is not dependent on proteolysis or on the generation of a conformational determinant but on the separation of A and B chains resulting in A chains whose cys-residues are converted into CysSH.     

Initiation of phage phi 29 DNA replication by the terminal protein modified at the carboxyl end

A mutant at the carboxyl end of the terminal protein, p3, of phage phi 29 DNA has been constructed by inserting an containing the stop translation codon TGA in the three possible reading frames, immediately downstream of a phage phi 29 DNA fragment coding for all but the last five amino acids of protein p3. The activity in the formation of the p3-dAMP initiation complex in vitro of this mutant as well as another one previously isolated, also mutated at the carboxyl end, have been tested. The results obtained suggest that an intact carboxyl end in the phage phi 29 terminal protein is essential for its normal primer function in DNA replication.     

MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.