Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.     

An hour after immunization peritoneal B-1 cells are activated to migrate to lymphoid organs where within 1 day they produce IgM antibodies that initiate elicitation of contact sensitivity

Elicitation of contact sensitivity (CS), a classic example of T cell-mediated immunity, requires Ag-specific IgM Abs to trigger an initiation process. This early process leads to local recruitment of CS-effector T cells after secondary Ag challenge. These Abs are produced by the B-1 subset of B cells within 1 day after primary skin immunization. In this study we report the surprising observation that B-1 cells in the peritoneal cavity are activated as early as 1 h after naive mice are painted with a contact-sensitizing Ag on the skin of the trunk and feet to begin the initiation of CS. B-1 cells in the spleen and draining lymph nodes produce the initiating Abs by 1 day after immunization, when we found increased numbers of Ag-specific IgM Ab-producing cells in these tissues by ELISPOT assay. Importantly, we show that contact-activated peritoneal B-1 cells migrate to these lymphoid tissues and then differentiate into Ag-specific IgM Ab-producing cells, resulting in specific CS-initiating IgM Abs in the serum by 1 day. Furthermore, pertussis toxin, which is known to inhibit signaling via G protein-coupled chemokines, inhibited the migration of contact-activated peritoneal B-1 cells to the lymphoid tissues, probably due to BLR-1 (Burkitt lymphoma receptor-1). These findings indicate that within 1 h after contact skin immunization, B-1 cells in the peritoneal cavity are activated to migrate to the lymphoid tissues by chemokine-dependent mechanisms to produce serum Ag-specific IgM Abs within 1 day after immunization, leading to local recruitment of CS-effector T cells.     

The Bw cells, a novel B cell population conserved in the whole genus Mus

In common laboratory mouse strains, which are derived from the crossing between three subspecies, peritoneal B cells are enriched in B-1a cells characterized by the CD5(+)Mac-1(+)B220(low)IgM(high)IgD(low)CD43(+)CD9(+) phenotype. Intriguingly in other vertebrates, CD5(+)Mac-1(+) cells have never been found in a specific anatomic site. To ascertain the peculiarity of the CD5(+) peritoneal B cells in laboratory mice, we analyzed the peritoneal B cell subsets in 9 inbred and 39 outbred wild-derived mouse strains belonging to 13 different species/subspecies. We found that most of these strains do not have the CD5(+) B-1a cell population. However, all of these strains including classical laboratory mouse strains, have variable proportions of a novel B cell population: Bw, which is characterized by a unique phenotype (CD5(-)Mac-1(+)B220(high)IgM(high)IgD(high)CD43(-)CD9(-)) and is not restricted to the peritoneal cavity. Bw cells are also distinct from both B-1 and B-2 cells from a functional point of view both by proliferative responses, cytokine secretion and Ab synthesis. Moreover, transfer experiments show that bone marrow and fetal liver cells from wild mice can give rise to Bw cells in alymphoid mice. The conservation of this B cell population, but not of the CD5(+) B-1a, during evolution of the genus Mus, its readiness to respond to TLR ligands and to produce high concentration of autoantibodies suggest that Bw cells play a key role in innate immunity.     

Functional activity of natural antibody is altered in Cr2-deficient mice

The major source of natural IgM Abs are B-1 cells, which differ from conventional B cells in their anatomic location, cell surface phenotype, restricted usage of particular V(H) genes and limited use of N-region addition during V-D-J rearrangement. The origin of B-1 cells is unclear. However, they are capable of self-renewal and their development is sensitive to signaling via the B cell receptor, as genetic defects that impair the strength of the signal often result in limited development. These findings suggest that B-1 cells require either an intrinsic signal, or contact with Ag, for positive selection and expansion and/or maintenance in the periphery. In support of interaction with cognate Ag, deficiency in the complement receptors CD21/CD35 results in a 30-40% decrease in the CD5(+) B-1 population. To determine whether this reduction reflects a loss of certain specificities or simply a proportional decline in the repertoire, we examined peritoneal B cells isolated from Cr2(+) and Cr2(def) mice for recognition of a B-1 cell Ag, i.e., phosphatidylcholine, and assayed for injury in an IgM natural Ab-dependent model of reperfusion injury. We found a similar frequency of phosphatidylcholine-specific CD5(+) B-1 cells in the two strains of mice. By contrast, the Cr2(def) mice have reduced injury in the IgM-dependent model of reperfusion injury. Reconstitution of the deficient mice with pooled IgM or adoptive transfer of Cr2(+) peritoneal B cells restored injury. These results suggest that complement receptors CD21/CD35 are important in maintenance of the B-1 cell repertoire to some, but not all, specificities.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

IL-9-induced expansion of B-1b cells restores numbers but not function of B-1 lymphocytes in xid mice

Mice expressing the X-linked immunodeficiency (xid) mutation lack functional Bruton's tyrosine kinase and were shown to be specifically deficient in peritoneal B-1 lymphocytes. We have previously shown that IL-9, a cytokine produced by TH2 lymphocytes, promotes B-1 cell expansion in vivo. To determine whether IL-9 overexpression might compensate the xid mutation for B-1 lymphocyte development, we crossed xid mice with IL-9-transgenic mice. In this model, IL-9 restored normal numbers of mature peritoneal B-1 cells that all belonged to the CD5(-) B-1b subset. Despite this normal B-1 lymphocyte number, IL-9 failed to restore classical functions of B-1 cells, namely, the production of natural IgM Abs, the T15 Id Ab response to phosphorylcholine immunization, and the antipolysaccharide humoral response against Streptococcus pneumoniae. By using bromelain-treated RBC, we showed that the antigenic repertoire of these IL-9-induced B-1b lymphocytes was different from the repertoire of classical CD5(+) B-1a cells, indicating that the lack of B-1 function by B-1b cells is associated with distinct Ag specificities. Taken together, our data show that B-1b cell development can restore the peritoneal B-1 population in xid mice but that these B-1b cells are functionally distinct from CD5(+) B-1a lymphocytes.     

TLR9 in peritoneal B-1b cells is essential for production of protective self-reactive IgM to control Th17 cells and severe autoimmunity

The role of TLR9 in the development of the autoimmune disease systemic lupus erythematosus is controversial. In different mouse models of the disease, loss of TLR9 abolishes the generation of anti-nucleosome IgG autoantibodies but at the same time exacerbates lupus disease. However, the TLR9-dependent tolerance mechanism is unknown. In this study, we show that loss of TLR9 is associated with low peritoneal B-1b cell numbers and low levels of protective self-reactive IgM serum autoantibodies in lupus-prone FcγRIIB-deficient mice leading to the uncontrolled accumulation of proinflammatory CD4(+) cells and exacerbated autoimmunity. TLR7 signaling was not able to compensate for the loss of TLR9 signaling in peritoneal B-1b cells to induce IgM Abs. Transfer of TLR9-expressing peritoneal B-1b cells from FcγRIIB-deficient mice or of recombinant monoclonal self-reactive IgM Abs was sufficient to reduce the frequency of proinflammatory Th17 cells and lupus disease in FcγRIIB/TLR9 double-deficient mice. Taken together, these data provide evidence for a TLR9-dependent tolerance mechanism of peritoneal B-1b cells generating protective self-reactive IgM in lupus-prone mice to control Th17 cell development and severe autoimmunity.     

Association of B-1 B cells with follicular dendritic cells in spleen

Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.     

Mechanisms of peritoneal B-1a cells accumulation induced by murine lupus susceptibility locus Sle2

The abundance of B-1a cells found in the peritoneal cavity of mice is under genetic control. The lupus-prone mouse New Zealand Black and New Zealand White (NZB x NZW)F(1) and its derivative NZM2410 are among the strains with the highest numbers of peritoneal B1-a cells. We have previously identified an NZM2410 genetic locus, Sle2, which is associated with the production of large numbers of B-1a cells. In this paper, we examined the mechanisms responsible for this phenotype by comparing congenic C57BL/6 mice with or without Sle2. Fetal livers generated more B-1a cells in B6.Sle2 mice, providing them with a greater starting number of B-1a cells early in life. Sle2-expressing B1-a cells proliferated significantly more in vivo than their B6 counterparts, and reciprocal adoptive transfers showed that this phenotype is intrinsic to Sle2 peritoneal B cells. The rate of apoptosis detected was significantly lower in B6.Sle2 peritoneal cavity B-1a cells than in B6, with or without exogenous B cell receptor cross-linking. Increased proliferation and decreased apoptosis did not affect Sle2 peritoneal B-2 cells. In addition, a significant number of peritoneal cavity B-1a cells were recovered in lethally irradiated B6.Sle2 mice reconstituted with B6.Igh(a) bone marrow, showing radiation resistance in Sle2 B-1a cells or its precursors. Finally, B6.Sle2 adult bone marrow and spleen were a significant source of peritoneal B-1a cells when transferred into B6.Rag2(-/-) mice. This suggests that peritoneal B-1a cells are replenished throughout the animal life span in B6.Sle2 mice. These results show that Sle2 regulates the size of the B-1a cell compartment at multiple developmental checkpoints.     

Invariant NKT cells rapidly activated via immunization with diverse contact antigens collaborate in vitro with B-1 cells to initiate contact sensitivity

In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Valpha14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.     

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the J(H) region of chromosome 12. This T15H chain combines with a kappa22-33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id-. Analysis of L chains expressed in these T15-Id-, PC-specific B cells revealed that two L chains, kappa8-28 and kappa19-15, were expressed in this population. The V(kappa) region of these L chains was recombined to J(kappa)5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id- B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id- B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:kappa8-28L and T15H:kappa19-15L Abs revealed L chain differences from the kappa22-33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:kappa22-33L Ab.     

Cutting edge: absence of expression of RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent protein gene

It has been proposed that Ig gene rearrangement in the peritoneal cavity (Pc) B-1 cells might be involved in autoantibody generation. To study possible secondary B cell maturation, we prepared mice carrying a target integration of gfp gene into a rag1 locus (rag1/gfp mice). The GFP+ cells express rag1 mRNA and are undergoing Ig gene rearrangement. RAG1 expression was studied in Pc B-1 cells to detect cells during the stage of Ig gene rearrangement. In contrast to previous reports, Pc B-1 cells did not show RAG1 expression in adolescent or elderly mice. RAG1 expression was not induced in Pc B-1 cells in vivo after stimulation by oral or i.p. administration of LPS. Our results suggest that RAG1 expression in Pc B-1 cells is inhibited for a long period under normal condition and that this suppression is an essential state which maintains allelic exclusion of Ig genes.     

Intact active bone transplantation synergizes with anti-CD40 ligand therapy to induce B cell tolerance

Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-alpha1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-gamma and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.     

Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency

Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to N^ε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE^−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE^−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE^−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.     

Impaired clearance of apoptotic cells induces the activation of autoreactive anti-Sm marginal zone and B-1 B cells

Since apoptotic cell Ags are thought to be a source of self-Ag in systemic lupus erythematosus, we have examined the role of apoptotic cells in the regulation and activation of B cells specific for Sm, a ribonucleoprotein targeted in human and murine lupus. Using Ig-transgenic mice that have a high frequency of anti-Sm B cells, we find that apoptotic cell injection induces a transient splenic B cell response, while simultaneously causing extensive splenic and peritoneal anti-Sm B cell death. In contrast, mice deficient in the clearance of apoptotic cells develop a chronic anti-Sm response beginning at 1-2 mo of age. These mice have expanded marginal zone and B-1 B cell populations and anti-Sm B cells of both types are activated to form Ab-secreting cells. This activation appears to be Ag-specific, suggesting that activation is due to increased availability of apoptotic cell Ags. Since marginal zone and B-1 cells are positively selected, these data suggest a loss of ignorance rather than a loss of tolerance.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire

Intestinal ischemia-reperfusion (IR) injury is initiated when natural Abs recognize neoantigens that are revealed on ischemic cells. Cr2(-/-) mice, deficient in complement receptors (CR)1 and CR2, demonstrate defects in T-dependent B-2 B cell responses to foreign Ags and have also been suggested to manifest abnormalities of the B-1 subset of B lymphocytes. To determine whether these CRs might play a role in the generation of the natural Abs that initiate intestinal IR injury, we performed experiments in Cr2(-/-) and control Cr2(+/+) mice. We found that Cr2(-/-) mice did not demonstrate severe intestinal injury that was readily observed in control Cr2(+/+) mice following IR, despite having identical serum levels of IgM and IgG. Pretreatment of Cr2(-/-) mice before the ischemic phase with IgM and IgG purified from the serum of wild-type C57BL/6 mice reconstituted all key features of IR injury, demonstrating that the defect involves the failure to develop this subset of natural Abs. Pretreatment with IgM and IgG individually demonstrates that each contributes to unique features of IR injury. In sum, CR2/CR1 play an unanticipated but critical role in the development of a subset of the natural Ab repertoire that has particular importance in the pathogenesis of IR injury.     

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.