Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein.

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.

IgG3 anti-Kell allotypic variation results in differential antigen binding and phagocytosis.

BACKGROUND: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT). STUDY DESIGN AND METHODS: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages. RESULTS: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients. CONCLUSION: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn.

IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding.

Because IgG1 allotypes might have different half-lives, their influence on infliximab (G1m17,1 allotype) pharmacokinetics was investigated in a group of spondyloarthritis patients. Infliximab was found to have a shorter half-life in patients homozygous for the G1m17,1 allotypes than in those carrying the G1m3 with no G1m1 (G1m3,-1) allotype. Because the neonatal FcR (FcRn) is involved in the pharmacokinetics of mAbs, the interaction of different IgG1 allotypes with FcRn was examined using cellular assays and surface plasmon resonance. G1m17,1 mAbs, such as infliximab and rituximab, were shown to bind more efficiently to FcRn and to be transcytosed better than the G1m3,-1 mAb cetuximab, which explains why infliximab is a better competitor for endogenous IgG1 in G1m3,-1 allotype-bearing patients. A set of four allotype variants of adalimumab (G1m17,1; G1m17,-1; G1m3,1; and G1m3,-1) was also tested for its binding to FcRn, revealing that the G1m3,1 variant, not present in commercial mAbs, binds more efficiently to FcRn and is transcytosed better than the other three variants, all of which are found in therapeutic mAbs.

Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

BACKGROUND: Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. STUDY DESIGN AND METHODS: A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. RESULTS: A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. CONCLUSION: These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported.

Identification of high-affinity anti-CD16A allotype-independent human antibody domains.

CD16A (FcγRIIIA) is an activating receptor mostly expressed on natural killer (NK) cells and monocytes/macrophages. It can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through low-affinity interaction with human immunoglobulin G (IgG) Fc. It can also mediate cell lysis if NK cells are guided by bispecific killer cells engagers (BiKEs). BiKEs showed some success in clinical trials of cancer and are promising candidate therapeutics. However, currently reported BiKEs are based on antibody fragments (scFvs) of relatively large size. The CD16A-specific antibodies are also typically from animal origin. Decreasing the BiKE size could result in enhanced penetration into solid tumor and normal tissues, and using fully human antibodies could decrease the likelihood of immunogenicity. Here we report the identification and characterization of two antibody domains, D6 and E11, isolated from a very large human VH antibody domain library displayed on phage. D6 and E11 bound CD16A with EC50 of 4nM and 8nM, respectively, but not other Fc gamma receptors (FcγRs) such as CD64 (FcγRI), CD32 (FcγRII) and CD16B (FcγRIIIB). They bound to both CD16A allotypes (158F,V) with equal affinity and competed with each other as well as with human IgG1 and the mouse anti-CD16A antibody 3G8. These and other results were used to build a molecular docking model predicting that D6 and E11 may bind to the CD16A membrane proximal D2 domain by interacting with its BC, C'E and EF loops. Importantly, cross-linked (bivalent) D6 and E11 induced secretion of IL-2 after binding to CD16A-expressing Jurkat T cells. The small size of these antibody domains combined with their high-affinity, specific, allotype-independent, activating interactions with CD16A could allow generation of novel highly effective BiKEs and other candidate protein therapeutics.

Association of complement factor B allotypes and serum biomarkers in rheumatoid arthritis patients and their relatives.

The aim of the study was to investigate the allotypic variability of complement factor B (BF) in patients and relatives with rheumatoid arthritis (RA) and its association with serological biomarkers and clinical features of the disease. BF allotypes were determined by high-voltage agarose gel electrophoresis in serum samples of 180 patients with RA, 198 relatives and 98 controls from Southern Brazil. Anticyclic citrullinated peptide (anti-CCP), antimutated citrullinated vimentin (anti-MCV) and IgA-rheumatoid factor (RF) were determined by ELISA and IgM-RF by latex agglutination in all samples. No significant differences were found in the allotypic variants of BF between patients with RA, relatives and controls, nor associations with gender and age of RA onset. BF*S07 allotype was significantly associated with extra-articular manifestations (EAMs; Secondary Sjögren Syndrome, pneumonitis, rheumatoid nodules) in patients with RA (P = 0.02; OR = 6.62). Patients with phenotype BF F had lower positivity for anti-MCV biomarker (P = 0.02; OR = 0.22) and those with allotype BF*S had higher prevalence of this autoantibody (P = 0.02; OR = 3.77). An increased frequency of RF-IgA was detected in relatives of patients with RA with BF FS07 phenotype (P = 0.02; OR = 7.78). Complement BF variability did not influence the development of RA in the studied patients, but BF variants may act as markers of disease prognosis, such as development of EAMs, corroborating with the role of the alternative pathway in the pathogenesis of RA.

Immunoglobulin genes influence the magnitude of humoral immunity to cytomegalovirus glycoprotein B.

Human cytomegalovirus (HCMV) is a risk factor for many human diseases, but among exposed individuals, not everyone is equally likely to develop HCMV-spurred diseases, implying the presence of host genetic factors that might modulate immunity to this virus. Here, we show that antibody responsiveness to HCMV glycoprotein B (gB) is significantly associated with particular immunoglobulin GM (γ marker) genotypes. Anti-HCMV gB antibody levels were highest in GM 17/17 homozygotes, intermediate in GM 3/17 heterozygotes, and lowest in GM 3/3 homozygotes (28.2, 19.0, and 8.1 µg/mL, respectively; P=.014). These findings provide mechanistic insights in the etiopathogenesis of HCMV-spurred diseases.

H435-containing immunoglobulin G3 allotypes are transported efficiently across the human placenta: implications for alloantibody-mediated diseases of the newborn.

BACKGROUND: The neonatal receptor (FcRn) extends the half-life of human immunoglobulin (Ig)G and transports it across the placenta, providing the newborn with humoral immunity. Of the four subclasses, IgG3 stands out with strong effector functions, short half-life (7 days vs. 21 days for other subclasses), and poor placental transport. We recently described how a single-amino-acid polymorphism at Position 435 in IgG3 is sufficient to explain the short half-life of R435-containing IgG3 and demonstrated that H435-IgG3 has a normal half-life of 21 days. Here, we investigated whether the R435 also explains the relatively poor placental transport of IgG3. STUDY DESIGN AND METHODS: Sera were collected from paired mothers and newborns at birth. The study included six mothers expressing R435-IgG diagnosed with fetal and neonatal alloimmune thrombocytopenia and treated with intravenous immune globulin (IVIG; containing H435-IgG3, also known as G3m16 or G3m(s,t) allotype), as well as 33 paired samples of both G3m16(-) and G3m16(+) mothers. Placental IgG transport was estimated by comparing cord and maternal concentrations of IgG subclass and G3m16 allotype. RESULTS: The placental transport of naturally occurring H435-IgG3 allotypes was significantly more efficient than that of other R435-IgG3 allotypes and was comparable to IgG1 transport. CONCLUSION: We demonstrate that the poor maternal-fetal transport of IgG3 is only true for most individuals of western populations where the G3m16 is not common. In G3m16(+) individuals, expressing H435-containing IgG3, IgG3 transport is similar to IgG1, which may give rise to enhanced complications in pregnancy-associated alloimmune disease in ethnic communities where this naturally occurring H435 containing IgG3 allotype is more frequent.

Characterization of a mutated IgA2 antibody of the m(1) allotype against the epidermal growth factor receptor for the recruitment of monocytes and macrophages.

IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.

B-1 cells in the bone marrow are a significant source of natural IgM.

Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies identified BM and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B-cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM-secreting cells in spleen and, for the first time, in the BM as IgM(+) IgD(lo/-) CD19(hi) CD43(+) CD5(+/-) B-1 cells. The newly identified population of BM B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and BM B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Overall, these data identify populations of non-terminally differentiated B-1 cells in spleen and BM as the most significant producers of natural IgM.

Regulatory B-cell compartment in transfused alloimmunized and non-alloimmunized patients with sickle cell disease.

Transfusion therapy is a life-sustaining treatment for patients with sickle cell disease (SCD), but can cause serious complications including alloimmunization. We previously reported diminished regulatory T cells (Tregs) and skewed Th2 responses in alloimmunized SCD patients. We hypothesized that the B cell regulatory (Breg) compartment, which controls Treg and Th differentiation, may also be compromised in allosensitized SCD patients. Phenotypically, we did not find differences in the frequency or numbers of CD24(hi) CD38(hi) and CD24(hi) CD27(+) B cell subsets, both previously identified as human Bregs, between alloimmunized and non-alloimmunized SCD patients on regular transfusions. However, at the functional level, CD19+ B cells from alloimmunized SCD patients expressed lower levels of IL-10 following stimulation as compared with non-alloimmunized patients (P < 0.05), and had reduced ability in inhibiting autologous CD14+ monocyte TNF-α expression (P < 0.05). These findings suggest that Bregs from alloimmunized and non-alloimmunized SCD patients differ in their ability to produce IL-10 and dampen monocyte activation, all consistent with an altered immunoregulatory state in alloimmunized SCD patients.

Intrinsic unresponsiveness of Mertk-/- B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression.

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk(-/-) mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk(-/-) mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk(-/-) mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk(-/-) mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk(-/-) mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II.

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016-2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.

T lymphocytes specific for immunoglobulin allotype. II. Cloned Igh-1b-specific cytotoxic T cells.

This study describes long-term-cultured lines and clones of cytotoxic T cells (Tc) with specificity for determinants of the Igh-1(b) immunoglobulin allotype. These Tc clones were initiated by repeated stimulation of immune spleen cells from BALB/c mice with an Igh-1(b)-producing myeloma, and then they were maintained in medium supplemented with mitogen-induced growth factors in the absence offurther antigenic stimulation . The lytic potency of these clones was 30-100-fold greater than the primary cultures from which they were derived, and specificity studies showed them to be lytic for Igh-1(b) targets and not for targets expressing Igh-1(a) or Igh-4(b), nor the lipopolysaccharide blasts . Finally, soluble preparations of Ig were tested for their ability to block lysis of labeled Igh-1(b)-expressing targets. The results showed that Igh-1(b) and not other immunoglobulin allotypes or isotypes could block lysis, and that the mechanism of lytic inhibition is due to Igh-1(b)-induced autolysis of the killer cells.

Production of predominantly polymeric IgA by human peripheral blood lymphocytes stimulated in vitro with mitogens.

Human peripheral blood lymphocytes (PBL) were cultured for various time periods (up to 8 d) in the presence of pokeweed mitogen (PWM), lipopolysaccharide, or Epstein-Barr virus. Cell-free supernates were fractionated on a standardized ultrogel AcA 22 column and the proportion of polymeric and monomeric IgA was determined by radioimmunoassay. The results demonstrate that PBL stimulated with these mitogens produce IgM and IgG with molecular characteristics identical to those found in serum, but that the IgA produced is predominantly of the polymeric type. To prove that this IgA represented disulfide bond-linked polymers rather than aggregated monomers, we have demonstrated that the high molecular weight IgA (a) maintains its polymeric form upon treatment with acidic buffers, (b) contains J chain, a glycoprotein associated only with polymeric immunoglobulins, and (c) dissociates to the monomeric form upon reduction of disulfide bonds. After 1 wk in culture, approximately 60% of the PWM-stimulated cells that contained IgA were positive for IgA2, whereas 40% were IgA1 positive as determined by immunofluorescence. Therefore, peripheral blood contains a population of lymphocytes with the potential to display, after appropriate stimulation and differentiation, characteristics similar to IgA cells found in external secretory tissues. The demonstration of the presence of such cells in the peripheral circulation suggests that these cells are precursors of IgA-producing plasma cells with the potential to populate mucosal tissues.

T lymphocytes specific for immunoglobulin allotype. I. Igh-1b-specific T cells demonstrated by suppression in vivo and cytotoxicity in vitro.

We show that determinants of IgG(2a) of C57BL/6 mice (Igh-1(b)) stimulate allotypespecific T cells in BALB/c mice. Such cells are detected in two different functional assays; chronic allotype suppression and T cell-mediated cytotoxicity. A population of suppressor T cells capable of inducing chronic Igh-1(b) suppression was demonstrated by rosetting procedures to possess Igh-1(b)-specific receptors, a result interpreted as indicating that suppressor T cells may act directly upon allotype-bearing B cells. From similar populations we were also able to demonstrate Igh-1(b)-specific cytotoxic T cells. Such cells were lytic for target myeloma cells expressing the Igh-1(b) allotype of IgG28, and were ineffective against a variant cell line failing to express Igh-1(b), and other target cell lines expressing different allotypes or isotypes. The similar specificity of suppressor T cells and cytotoxic T lymphocytes for Igh-1(b) allotype raises the possibility that the target in allotype suppression is a B cell, and that allotype-specific cytotoxic T cells may play some role in regulation of allotype expression in the suppressed state.

Regulation of natural antiallotype antibody responses by idiotype network-induced auto-antiidiotypic antibodies.

This study was designed to determine whether natural immune responses could elicit immunoregulatory auto-antiidiotypic antibodies. Female rabbits heterozygous at the a and b Ig loci were bred to homozygous males. Offspring of one such breeding were studied for natural production of antibodies specific for the noninherited allotypes and for the production of immunoregulatory auto-antiidiotypic antibodies. All offspring mounted natural antiallotype responses. The anti-a1 responses cycled as a function of time whereas the anti-b5 responses were invariant. Anti-a1 responses from two offspring were shown to change specificity for different a1 subsets as they cycled. Anti-a1 was purified from the first cycle and was used to assay for auto-antiidiotypic responses. Auto-antiidiotypic antibodies were detected and were found to cycle in an inverse way with the anti-a1 cycles. The idiotopes detected using the natural auto-antiidiotypic antisera were strongly cross-reactive. Subsequent deliberate immunization showed that antibodies specific for all a1 subsets could be elicited after auto-antiidiotypic regulation had functioned. The data support the interpretation that idiotype network interactions indeed function in naturally occurring immunologic situations and are not merely laboratory curiosities or artifacts.

Human monoclonal macroglobulins with specificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6-pyruvylated-D-galactose.

Two human IgM myeloma proteins, IgMWEA and IgMMAY, were found to react with agar and Klebsiella polysaccharides that contain pyruvylated D-galactose (DGal). Quantitative precipitin data and precipitin inhibition studies with methyl alpha- and beta-glycosides of 4,6-pyruvylated-D-galactose showed their combining sites to be different, although each was directed against the pyruvylated-D-Gal, one reacting most specifically with Klebsiella polysaccharides with terminal nonreducing beta-linked 2,4 pyruvylated-D-Gal, whereas the other reacted equally well with Klebsiella polysaccharides that contain 3,4 beta-linked and 4,6 alpha-linked terminal nonreducing pyruvylated-DGal. Inhibition studies showed that both sites are directed toward one of the two space isomers of 3,4- or 4,6-pyruvylated DGal, the form in which the methyl group of the pyruvate is equatorial, or endo, and its carboxyl group axial, or exo, to the plane of the acetal ring. Coprecipitation studies showed the combining site of IgMWEA to be located on an (Fab')2 fragment and not on the (Fc)5mu fragment. The monoclonal peak in the serum of IgMMAY was specifically precipitated by Klebsiella polysaccharide. Myeloma proteins with specificities of this type may occur with reasonable frequency in humans and may be a consequence of clonal expansion from inapparent infection, carrier states, or disease produced by various Klebsiella organisms.

Regulation of murine B cells through surface immunoglobulin. I. Monoclonal anti-delta antibody that induces allotype-specific proliferation.

We describe the identification of a monoclonal antibody that recognizes a determinant on the delta chain of mice of the Iga, allotype groups. The monoclonal Ig in soluble form induces allotype-specific proliferation by splenic B lymphocytes from normal animals of these haplotypes. Spleen cells from mice bearing the X-linked defect of CBA/N mice fail to respond, although they bear the determinant. Proliferation is independent of T lymphocytes. The data indicate a direct triggering function for sIgD.