Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide.

Filamentous bacteriophage are widely used as immunogenic carriers for "phage-displayed" recombinant peptides. Here we report that they are an effective immunogenic carrier for synthetic peptides. The f1.K phage was engineered to have an additional Lys residue near the N-terminus of the major coat protein, pVIII, so as to enhance access to chemical cross-linking agents. The dimeric synthetic peptide, B2.1, was conjugated to f1.K (f1.K/B2.1) in high copy number and compared as an immunogen to B2.1 conjugated to ovalbumin (OVA/B2.1) and to phage-displayed, recombinant B2.1 peptide. All immunogens were administered without adjuvant. The serum antibody titers were measured against: the peptide, the carrier, and, if appropriate, the cross-linker. All immunogens elicited anti-peptide antibody titers, with those elicited by OVA/B2.1 exceeding those by f1.K/B2.1; both titers were greater than that elicited by recombinant B2.1 phage. Comparison of the anti-peptide and anti-carrier antibody responses showed that f1.K/B2.1 elicited a more focused anti-peptide antibody response than OVA/B2.1. The anti-peptide antibody response against f1.K/B2.1 was optimized for the injection route, dose and adjuvant. Dose and adjuvant did not have a significant effect on anti-peptide antibody titers, but a change in injection route from intraperitoneal (IP) to subcutaneous (SC) enhanced anti-peptide antibody titers after seven immunizations. The optimized anti-peptide antibody response exceeded the anti-carrier one by 21-fold, compared to 0.07-fold elicited by OVA/B2.1. This indicates that phage as a carrier can focus the antibody response against the peptide. The results are discussed with respect to the advantages of phage as an alternative to traditional carrier proteins for synthetic peptides, carbohydrates and haptens, and to further improvements in phage as immunogenic carriers.

Antigen-specific T cell activation and proliferation during oral tolerance induction.

One of several routes of achieving immunologic tolerance is through functional inactivation of Ag-specific T cells. Oral administration of Ag can allow survival of the Ag-specific T cells that are functionally anergic. The aim of this study was to investigate whether functional inactivation of Ag-specific T cells is directed through an activation process and to further define the differentiative pathways and functional characteristics of anergic T cells. Mice were transplanted with OVA-specific TCR-transgenic T cells and either fed OVA or immunized s.c. with the OVA peptide 323-339 in CFA. OVA-specific T cells from OVA-fed mice were unresponsive to restimulation in vitro within 48-72 h after treatment. In vivo, however, T cell proliferation was detected by 5, 6-carboxy-succinimidyl-fluoresceine-ester intensity changes in OVA-specific T cells. The mesenteric lymph nodes (LNs) from OVA-fed mice more frequently contained OVA-specific dividing cells in vivo than those in the peripheral LNs, and the reciprocal was observed following s.c. immunization of the OVA peptide in CFA. The induction of anergy in OVA-fed mice was accompanied by rapid up-regulation of CD69 and CTLA-4, later down-regulation of CD45RB on OVA-specific T cells, and a marked decrease in T cell secretion of IL-2, IL-10, and IFN-gamma after OVA restimulation in vitro. Results from this study indicate that the inductive phase of oral tolerance is preceded by Ag-specific T cell activation in vivo, proliferation in the regional draining LNs, and differentiation into a memory-like state. These results indicate that Ag-directed differentiation occurs as a part of T cell tolerance through anergy.

The effect of class II major histocompatibility complex expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell type 1-mediated damage.

Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer. Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis. Using H. pylori as "bait" to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor. Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules. Moreover, binding of H. pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC. Since type 1 T helper cells are present during infection and produce interferon (IFN)-gamma, which increases class II MHC expression, gastric epithelial cell lines were exposed to H. pylori in the presence or absence of IFN-gamma. IFN-gamma increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells. In contrast to MHC II-negative cell lines, H. pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection. These data describe a novel receptor for H. pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.

Elevated expression of Bcl-2 and Bcl-x by intestinal intraepithelial lymphocytes: resistance to apoptosis by glucocorticoids and irradiation.

Administration of glucocorticoids or exposure to ionizing radiation in vivo results in a rapid cell death of thymocytes. We report that murine small intestinal intraepithelial lymphocytes (IEL) are resistant to both steroid- and radiation-induced deletion. This is due to resistance to apoptosis, as evidenced by the absence of detectable apoptotic IEL nuclei in situ after in vivo glucocorticoid treatment. IEL express normal levels of glucocorticoid receptors and these receptors bind [3H]dexamethasone to equivalent levels as other lymphocyte populations. Thus, their survival is due to post-receptor signaling mechanisms. Many IEL express high levels of Bcl-2 and that of these Bcl-2high IEL are largely TCR gamma delta +. Those IEL that do express high levels of Bcl-2 are CD8 alpha + beta - CD4-. In addition, IEL express Bcl-x, another protein shown to be involved in the protection of cells from apoptotic signals. IEL represent the first lymphocyte population in vivo shown to have high levels of expression of both molecules, that otherwise occur only in activated lymphocytes in vitro. These data suggest that the Bcl-2+Bcl-x+ IEL are activated cells and not an effete population of cells necessarily destined to die. Also, the high levels of Bcl-2 and Bcl-x in this in vivo activated population supports the in vitro correlate of protection from activation-induced cell death.

CD16-expressing CD8alpha alpha+ T lymphocytes in the intestinal epithelium: possible precursors of Fc gammaR-CD8alpha alpha+ T cells.

T lymphocytes normally express their Ag receptors in association with the CD3 proteins, which include CD3zeta. In CD3zeta eta(null) mice thymic and peripheral T lymphocytes do not express the TCR/CD3 complex on their surface due to retention in the endoplasmic reticulum of the remaining polypeptide chains. However, intestinal intraepithelial lymphocytes (iIEL) of CD3zeta eta(null) mice do express surface TCR, because the Fc epsilonRI gamma chain replaced the CD3zeta chain in the TCR/CD3 complex. Here we report that in a subset of CD8alpha alpha+ iIEL the presence of the Fc epsilonRI gamma chain could be accounted for by the surface expression of the Fc gammaRIII(CD16) complex. Because in wild-type (wt) mice only CD16+ iIEL coexpressed Fc epsilonRI gamma and CD3zeta, we concluded that the presence of Fc epsilonRI gamma was dictated by its required participation of CD16 complex. CD8alpha alpha+ iIEL bearing CD16 and B220 were also detected in the intestinal mucosa of RAG-2(null) mice from 12 days after birth onward. Two independent experimental settings were used in an attempt to demonstrate that CD16+ iIEL matured into CD16- T cells. First, in the RAG-2(null) mice, iIEL responded to in vivo administration of an anti-CD3epsilon mAb by progression to a more mature stage of development, characterized by a loss of CD16 and B220. Secondly, a conversion to CD16- iIEL occurred upon transfer of wt CD16+ iIEL into RAG-2(null) mice. We conclude from these experiments that in both RAG-2(null) and wt mice, a precursor/progeny relationship may exists between CD16+ B220+ CD8alpha alpha+ and CD16- B220- CD8alpha alpha+ iIEL.

Direct measurement of anergy of antigen-specific T cells following oral tolerance induction.

T cell tolerance induced by oral administration of Ag may be the result of either deletion or functional inactivation of Ag-specific T cells. OVA p(323-339)-specific TCR transgenic (Tg+) lymphocytes were transplanted into BALB/c recipients. Chimeric mice were fed OVA and subsequently challenged with the peptide in CFA. Tolerance was then assessed by measurement of lymph node (LN) cell proliferation in response to the peptide, and deletion was assessed by measuring the frequency Tg+ T cells by flow cytometry. Lymphocytes from chimeric mice fed OVA showed a dose-dependent decline in their proliferative response to the peptide in vitro, compared with immunized control mice that were not fed OVA. Calculation of proliferative potential per Tg+ cell demonstrates that nonresponsiveness due to feeding Ag results in the induction of anergy in the LN. In addition, analysis of intestinal intraepithelial lymphocytes following feeding of OVA did not show evidence of trafficking of LN T cells to the small intestine intraepithelial nor lamina propria compartments.

Parallels in T lymphocyte development between lpr and normal mice.

The unusual phenotype and function of lpr T cell subsets has considerably aided the determination of various stages in normal T lymphocyte development through the identification of parallel populations. This has included the description of memory T cells that express high levels of CD44. Lpr mature (CD4+ and CD8+) T cells are all CD44hi and produce large amounts of several cytokines, in contrast to normal murine peripheral T cells. However, the minor CD44hi subset of normal memory T cells produces similarly high levels of cytokines. The expression of intermediate density surface T cell antigen receptor (TCR)-alpha beta by lpr CD4-, CD8- (CD4-8-) cells guided the identification of a minor subset of Heat Stable Antigen--normal CD4-8- thymocytes that also expresses TCR-alpha beta. The unresponsiveness of lpr CD4-8- T cells correlates closely with their absence of CD2 expression. This is paralleled in normal mice by CD2- and CD2+ subsets of thymocytes and gut lymphocytes in which the proliferative capacity also segregates with CD2 expression. Finally, lpr CD4-8- T cells bear many similarities to anergic T cells, including high expression of p59fyn, lack of IL-2 production, and recovery of function following induction of cell cycling in the presence of IL-2. The developmental block in lpr CD4-8- T cells has therefore provided considerable insight into normal T cell development and function.

CD2-CD4-CD8- lymph node T lymphocytes in MRL lpr/lpr mice are derived from a CD2+CD4+CD8+ thymic precursor.

MRL lpr/lpr (lymphoproliferative, lpr) mice demonstrate an age-dependent lymphoproliferation and development of autoimmunity. Characteristic of the lymphoproliferation in these mice is the accumulation of large numbers of CD4-CD8-(CD4-8-),CD3+ T lymphocytes in their lymph nodes. The development of the CD4-8- cells, which also aberrantly express B220 and CD44 (Pgp-1) but are CD2-, has been shown to be thymus dependent. An unusual feature of lpr CD4-8-T lymphocytes is that although they appear unresponsive to stimulation, as defined by proliferation and IL-2 production, they have undergone thymic negative selection. As thymic deletion normally occurs at the CD4+CD8+ (CD4+8+) stage, this raises the dilemma that lpr CD4-8- T lymphocytes have either previously been CD4+8+, or they are able to undergo thymic selection as CD4-8- cells. We have addressed this question by examining the methylation status of the CD8 gene in MRL lpr CD4-8- lymph node cells. Demethylation of the CD8 gene has been shown to be an indicator of previous CD8 expression. We find that the CD8 gene in lpr CD4-8- lymph node cells, as well as in the abnormal B220+ CD4-8- lpr thymocytes, is demethylated, suggesting that these cells have previously expressed CD8. In addition, we find that the lpr CD4+8+ thymocyte population contains an increased percentage of atypical B220+, CD44+ cells that are virtually all CD2+. Taken together, these data are consistent with the lpr CD2-CD4-8- population of LNC having arisen from a CD2+ CD4+8+ thymic stage of differentiation.

CD2 expression on murine intestinal intraepithelial lymphocytes is bimodal and defines proliferative capacity.

The CD2 molecule is normally expressed on nearly all murine lymphocytes, and is co-stimulatory in T cell activation via the antigen receptor (TCR). A naturally occurring T lymphocyte population that is bimodal for CD2 expression was found in the intestinal intraepithelial lymphocytes (IEL). TCR alpha beta + IEL contain CD2- and CD2+ cells of approximately equal proportion, while TCR gamma delta + IEL are predominantly CD2-. The proliferative response of IEL to stimulation with an anti-CD3 mAb or with PMA plus ionomycin co-segregated with CD2 expression; the CD2+ subset proliferated vigorously under these conditions while the CD2- subset was much less responsive. The responding CD2+ IEL contained both TCR alpha beta + and TCR gamma delta + cells. However, activation of the CD2- IEL with anti-CD3 mAb resulted in only the expansion of TCR gamma delta + IEL, while activation with PMA plus ionomycin did not promote expansion of either the TCR alpha beta + or the TCR gamma delta + IEL. These findings parallel observations in the autoimmune lpr mouse, where massive numbers of peripheral TCR alpha beta + CD4-CD8- T cells that lack CD2 expression are also hyporesponsive to mitogenic stimulation. The apparent anergy of CD2- TCR alpha beta + IEL, as well as CD2- T cells from lpr mice, demonstrates that the absence of CD2 on TCR alpha beta + T lymphocytes co-segregates with nonresponsiveness.

Accelerated programmed cell death of MRL-lpr/lpr T lymphocytes.

MRL-lpr/lpr (lpr) mice develop a polyclonal accumulation of abnormal peripheral T lymphocytes, which bear surface alpha beta TCR, CD3, and the B220 isoform of CD45, but lack CD4, CD8, and CD2. These T cells have a constitutively phosphorylated CD3 zeta chain and manifest a defect in signal transduction that results in a lack of IL-2 production and proliferation. We investigated whether this signaling abnormality might contribute to their accumulation via a defect in T cell elimination in the periphery. T cell deletion occurs through a process of programmed cell death with DNA degradation, or apoptosis. Viable lymphocytes from lpr mice were found to undergo rapid programmed cell death in culture within 4 h without additional activation, which was not observed in lymphocytes from normal MRL-+/+ or C57BL/6-+/+ mice. Both nonmature B220+ and mature B220- T lymphocytes from lpr mice display this accelerated programmed cell death, indicating that this is a defect affecting all peripheral T lymphocytes in lpr mice. In vitro apoptosis of lpr T cells could be inhibited with PMA, a stimulator of protein kinase C. Thus, the massive accumulation of T lymphocytes in the lymphoid tissue of lpr mice is not due to a defect in their ability to undergo programmed cell death in vitro. The activation state of lpr T cells may contribute to their rapid degradation of DNA in vitro.

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.

Genetics of Coxsackie B3 (CVB3) myocarditis.

The pathogenic T cell responsible for myocarditis following CVB3 infection differs between BALB/c and DBA/2 mice. Since these two strains are identical at the major histocompatibility (MHC) loci (both H-2d), our aim was to investigate the genetic basis for this difference in T cell immunity, as well as the tissue origin responsible. As previously described, DBA/2 mice are protected from developing myocarditis by CD4+ T cell depletion; BALB/c CUM mice could be protected by CD8+ depletion. TxBM DBA/2 mice were irradiated and reconstituted with thymus and bone marrow cells of either DBA/2 or BALB/c CUM origin. These mice were then tested for susceptibility to CVB3 myocarditis following specific T cell depletion. These studies demonstrated that the pathogenic mechanism is inherent in the bone marrow of the animal and does not reflect thymic selection during T cell ontogeny. Further studies involving C.D2 mice, which are BALB/c AnPt mice congenic with DBA/2, were performed to analyse the susceptibility pattern following specific T cell depletion and virus infection. Analysis of BALB/c AnPt and congenics revealed a pattern consistent with the DBA/2 strain, and not the BALB/c CUM strain. This indicates that the differences in immune pathogenicity lie within subfamilies of BALB/c mice, making information on strain origin important when comparing experimental results in this system.

Selection of an attenuated Coxsackievirus B3 variant, using a monoclonal antibody reactive to myocyte antigen.

Previously, we described a heart-reactive monoclonal antibody (MAb), 10A1, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectively inhibits infection of HeLa cells and myocytes with the myocarditic virus variant (CVB3W). A plaque-purified variant (H3) of CVB3W was isolated from the heart of an infected animal, and a second virus (H3-10A1) was obtained by growing H3 in HeLa cells in the presence of MAb 10A1. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb 10A1, but the antibody-selected H3-10A1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 10(6) PFU of CVB3W, H3, or H3-10A1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10A1, suggesting that the escape mutant is less pathogenic. Additionally, hearts from animals infected with H3-10A1 demonstrated only half the amount of myocarditis observed in either CVB3W- or H3-infected mice. Cardiac virus titers were also reduced approximately 200-fold in H3-10A1-infected animals compared with those in mice given the pathogenic variants. In vitro studies indicate that H3-10A1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.

Coxsackievirus-B3-induced myocarditis: virus receptor antibodies modulate myocarditis.

Two variants of coxsackievirus group B, type 3 (CVB3) differ in ability to induce myocarditis in Balb/cCUM mice. Infection with the highly pathogenic variant (CVB3M) stimulates autoimmunity to normal cardiocyte antigens, and tissue injury results primarily from an autoreactive cytolytic T lymphocyte (ACTL). Animals infected with the less pathogenic CVB3o variant do not develop ACTL, although CVB3o replicates to high titers in the heart and polyclonal neutralizing antisera fail to distinguish between the two variant virions. The present study uses two IgM mAb derived by fusing spleen cells from CVB3M-infected mice with NS-1 cells. These mAb investigate important differences between the virus variants that may explain why only selected infections trigger autoimmunity. mAb 8A6 is a virus-neutralizing antibody that prevents infection of HeLa cells and cultured cardiocytes by attaching to the virus. mAb 10A1 also interferes with infection but presumably reacts to the virus receptor on the susceptible cells and shows little or no binding to the virions. While 8A6 is equally effective in neutralizing both CVB3o and CVB3M, suggesting that antigenic epitopes on both variants are either identical or highly cross-reactive, 10A1 distinguishes between the variants, suggesting that the pathogenic and less pathogenic viruses use distinct cell surface receptors. Competitive binding studies using radiolabeled CVB3M and either of the unlabeled variants confirm this hypothesis. Both mAb effectively prevent CVB3M-induced cardiac damage in vivo. mAb 10A1 also inhibits autoreactive ACTL lysis of cardiocytes, indicating that the autoimmune effectors may recognize the virus receptor, and that the receptor utilized by a virus may prove important in triggering auto-sensitization.

Early commitment to neoplasia in murine B- and T-cell lymphomas arising late in life.

Lymphomas arise spontaneously in C57BL/10.H-2aH-4b (2a4b) mice greater than 1 year old. The purpose of this study was to determine when cells become committed to neoplasia and whether genes linked to H-2a or H-4b are a factor in predisposition to genesis of lymphoma. Following transplantation of a pool of spleen cells in large groups of mice, lymphoma incidence increased 1.8- to 2.1-fold and mean latent periods decreased, compared with those in controls. Lymphomas arising in mice that received the same pool of spleen cells often displayed identical rearrangements of genes for immunoglobulin and T-cell receptor. This finding indicates that the lymphomas were derived from expanding clones of cells present in the donor mice prior to transfer. The age of the donor mice determined the latency of lymphomas arising after spleen cell transfer. When donors were 3-4 months old at the time of spleen cell transfer, lymphomas were detected in the recipients 1 year later. When mice 1.5-2 years old were used as donors, tumors were observed 3 months after transfer. Almost all of these tumors were clonally related B-cell lymphomas. We conclude that the lymphomas seen late in life arise from premalignant clones of cells that have become committed to neoplasia in relatively young animals. These clones normally follow an indolent course and are manifested clinically only in old age. Neither H-2a nor H-4b alone appeared to alter susceptibility to the genesis of lymphoma in similar experiments with H-2a and H-4b congenic mice.