Evidence that intestinal IgA plasma cells in {micro},{varkappa} transgenic mice are derived from B-1 (Ly-1 B) cells

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived,Igh-C^a allotype) µ. heavy chain and light chain transgenes,specific for trinitrophenyl, on a C57BL background (Igh-C^b allotype).FACS analyses show that the majority of B cells in peripherallymphoid organs and bone marrow(BM) express transgenic IgM exclusively.A small proportion of the B cells, however, express endogenousIgM, usually concomitant with transgenic IgM. Three criteriaestablish that the endogenous IgM expressing B cells belongto the B-1 cell lineage. (I) Endogenous IgM expressing B cellsin B6-Sp6 mice have the same localization pattern as B-1 cellsfrom normal animals: they are enriched in the peritoneal cavity.(II) The endogenous IgM⁺ B cells have the phenotype of B-1 cells:the endogenous IgM⁺ peritoneal B cells express Mac-1 (CD11b)and low levels of IgD, and most also express CD5 (L-1). (III)B6-Sp6 BM poorly reconstitutes endogenous IgM⁺ B cells, justas adult BM from normal mice poorly reconstitutes B-1 cells.In contrast, B cells which only express the transgene are readilyreconstituted by B6-Sp6 BM. The few endogenous IgM⁺ cells inthe B6-Sp6 BM recipients are located in the peritoneal cavityand have the phenotype of B-1b cells (previously the Ly-1 Bsister population), which are known to be reconstituted by adultBM.Two-color immunofluorescence staining of tissue sectionsfrom the gut and from isolated gut lamina propria cells showsthe presence of many IgA containing cells, about one-third ofwhich simultaneously express cytoplasmic (transgenic) IgM. TheC-region of this IgA is produced by endogenous C a genes, becausethe transgene encodes only for C_µ. Furthermore, the majorityof gut IgA containing cells do not express the Idiotype of thetransgene, indicating that most of the gut IgA cells are encodedby endogenous V_H genes and thus the result of an isotype switchfrom endogenous IgM expressing B cells. Since the endogenousIgM⁺ cells are B-1 cells (both B-1a and B-1b), the data stronglyindicate that the intestinal IgA plasma cells also belong tothe B-1 cell lineage.     

Allotype suppression in the chicken. I. Generation of chronic suppression in heterozygous but not in homozygous chickens.

The injection of antibody directed against either the IgM-1 a or IgM-1 b allotype into heterozygous (M-l^a/M-l^b, G-l^g/G-lⁱ) B 14-line chickens produced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes in their serum, as quantitated by single radial immunodiffusion. Suppression of the IgM-1 and IgG-1 allotypes was associated with a compensatory increase in the alternative IgM-1 and IgG-1 serum allotype levels. This suppression was induced (a) by passive injection of anti-allotype antiserum into 13-day-old embryonal or neonatal recipients, or (b) by egg yolktransmitted antibody in chickens hatched from homozygous B 14 A (M-l^a, G-l^g) hens immunized against the IgM-l b of the homozygous B14C (M-l^b, G-lⁱ) rooster. Heterozygous chickens injected embryonally with anti-allotype antiserum were profoundly suppressed for at least 16 weeks after hatching, while neonatally injected chickens showed a gradual recovery of both IgM-1 and IgG-1 allotypes over the same period. Suppression of the IgM-1 b allotype could be induced in heterozygotes which had inherited the M-l^b allele either maternally or paternally. However, no suppression of either IgM-1 or IgG-1 levels could be detected in homozygous chickens injected with the relevant anti-allotype antiserum. Hence, allotype suppression only occurred in M-1 heterozygous chickens which had an alternative source of B cells available. The involvement of a B cell surveillance mechanism in allotype suppression is postulated and the possible role of suppressor cells is discussed.     

Peritoneal B-2 cells comprise a distinct B-2 cell population with B-1b-like characteristics

B-1 and B-2 cells are lymphocyte populations that differ in development, surface marker expression, tissue localization, and function. Though mainly found in the spleen, lymph nodes, and circulation of mice, small numbers of B-2 cells are found in the peritoneal cavity, a site predominantly populated by B-1 cells. Here, we characterized peritoneal B-2 cells, and determined their relationship to B-1 cells. We found that peritoneal B-2 cells appear to be intermediate between splenic B-2 and peritoneal B-1 cells in terms of surface marker expression of B220, CD80, and CD43, expression of several marker genes, and in vitro viability and IgM secretion. Adoptive transfer of peritoneal B-2 cells into severe combined immunodeficiency mice resulted in the acquisition of a phenotype reminiscent of B-1b cells, as shown by up-regulation of Mac-1 and CD43, and down-regulation of CD23. Moreover, adoptively transferred peritoneal B-2 cells recapitulated B-1 cell function by producing natural IgM in recipient mice. These data suggest that peritoneal B-2 cells express some characteristics of B-1b cells and that this similarity increases with additional time in the peritoneal cavity.     

Normal T splenocytes are able to induce immunoglobulin allotypic suppression in F1 hybrid mice

A chronic suppression of Igh-1b and Igh-3b (IgG2a and IgG2b of b haplotype) allotype expression has been induced by injecting T splenocytes from normal BALB/c or BC8 mice into newborn F1 hybrids of appropriate Igh congenic strains: BALB/c into (BALB/c Igha X CB20 Ighb)F1 and BC8 into (BC8 Igha X C57BL/6 Ighb)F1 or (C57BL/6 X BC8)F1. This suppression does not affect IgM (IgH-6b) or IgA (Igh-2b) expression. When the Ighb haplotype is paternally transmitted, the proportion of T splenocyte recipients showing allotypic suppression increases with time reaching 70% 40 weeks after birth. We also succeeded in inducing this pattern of suppression in 2 out of 13 cases when the Ighb was inherited from the mother. These normal T splenocytes are therefore clearly allotype specific. As Igh-6b production is not affected by the suppression, these T splenocytes are believed to influence B cells more or less committed to Igh-1b or Igh-3b production rather than more precocious Igh-6b (IgM of b haplotype) carrying precursors in the classical IgM-IgG filiation pathway.     

Allotype suppression of the responsiveness of adoptively transferred adult spleen cells induced by affinity prepared anti-allotype antibody.

In an adoptive transfer assay it has been shown that a form of allotype suppression can be induced in adult spleen cells. Use of SPF congenic mice, and either anti-allotype serum freed of IgM rheumatoid factor or affinity prepared antibody, has helped to eliminate the possibility of a role for non-specific factors in allotype suppression. Both memory-cell development and a conventional primary response are more susceptible to suppression than memory expression in a secondary response. Non-specific suppressive effects on isotypes other than that of the target allotype (CH) are present but are much smaller than for specific suppression.     

Expression of b 4 and b 5 chi light chain allotypes by B and pre-B cells in allotype-suppressed and neutralized b4b5 rabbits.

In previous studies, we described a primitive lymphoid cell found in fetal liver and in the bone marrow of older rabbits which contained cytoplasmic IgM but lacked surface IgM detectable by immunofluorescence. In heterozygous b⁴b⁵ rabbits, the pre-B cells in which we could detect these kappa chain allotypes appeared to exhibit allelic exclusion. In the present study, we investigated the effects of allotype suppression and its neutralization on the expression of the b4 and b5 allotypes by B and pre-B cells from the spleens and bone marrow of b⁴b⁵ rabbits. We found that in young allotype suppressed rabbits, pre-B cells of the suppressed allotype persist in bone marrow when B cells of the suppressed allotype are absent or severely depleted. The persistence of pre-B cells of the suppressed type supports the view that pre-B cells differ in their responsiveness to external influences such as anti-Ig compared to B lymphocytes. Injection of serum with b5 immunoglobulin into b⁴b⁵ animals suppressed 14-23 days previously for b5 was followed by the appearance of increased proportions of b 5 B cells in spleen within 24 h. Surviving pre-B cells are a likely source of these rapidly appearing B cells as well as of the B cells bearing surface immunoglobulin of the suppressed allotype which appear during the recovery phase of allotype suppression.     

Allotype suppression in the chicken III. Analysis of the recovery from suppression by neonatally injected or maternal antibodies

Injection of M-1 (Cμ), G-1 (CμgM) heterozygous chickens on the day of hatch with anti-IgM-1 antiserum induced allotype suppression from which chickens recovered over a period of approximately 4 months. The suppression of the serum IgM-1 levels was matched by a decrease in the number of splenic and peripheral blood B cells bearing the relevant IgM-1 allotype, and a compensatory increase in the number of cells bearing the alternative nonsuppressed IgM-1 allotype. However, the proportion of IgM-1-bearing bursal cells was only marginally altered. The recovery from suppression was due to B cell recruitment and could be abrogated by bursectomy. Allotype suppression induced in ovo or maintained by repeated injections of anti-IgM-1 anti-serum resulted in chronic suppression and depletion of the relevant peripheral as well as bursal IgM-1-bearing cells. Antibody titers of the relevant allotype in partially suppressed chickens generally correlated with serum allotype levels without clonal restriction in antibody response of the suppressed allotype.     

X-chromosome-linked immune-deficient mice have B-1b cells

The B lymphocyte subsets of X-chromosome-linked immune-deficient (XID) mice were examined by flow cytometric analyses of spleen and peritoneal cells. As shown in prior studies, young adult XID mice had reduced representation of the CD5+ (B-1a) subset in their peritoneal cavity. However, the CD11b+ (B-1b) B-cell subset was present and exhibited the IgM(hi) CD45(lo) CD23- phenotype characteristic of most B-1 cells. Although present at a lower frequency than that found in their normal counterparts, B-1b cells were evident in CBA/N and (XD2J)F1 male mice. With increasing age, B-1b cell number increased and in the oldest XID mice were present as B-cell chronic lymphocytic leukaemia. These results show that XID mice do have B-1 cells, particularly the B-1b subset.     

Monoclonal gammopathies in aging μ, x-transgenic mice: Involvement of the B-1 cell lineage

Monoclonal gammopathies (MG) are monoclonal proliferative disorders of B cells at the differentiation stage of Ig production. They can be detected in the serum, either as transient or as persistent homogenous immunoglobulin (H-Ig) components. The exact phenotype, localization, and cell lineage origin of the precursor cells of MG are unknown, but may be crucial for both the correct diagnosis and for timely efficient treatment of the malignant forms. We used for the first time transgenic (Tg) mice (Sp6; μ/x) to study the origin of MG. In the μ, x Tg mice a small proportion of B cells can still produce endogenous IgM. These cells are of B-1 cell origin. The MG in Tg mice showed a later onset and a lower frequency than those in littermate control mice, mainly due to a four times lower frequency of benign monoclonal gammopathy. The 10% of B-1 cells that were able to produce endogenous Ig led to the development of MG in a frequency that was half the number of MG found in normal littermates. None of the MG in Tg mice produced an Ig of the Tg origin and therefore it can be concluded that they originated from B-1 cells.     

Morphological characterization of mouse B-1 cells

At least three B cell subsets, B-1a, B-1b and B-2 are present circulating peripherally in the mouse. In these animals, B-1 cells constitute a minor fraction of B cells in spleen and are absent in lymph nodes although they represent the main B cell population in peritoneal and pleural cavities. Currently these cells are identified by a surface phenotypic repertoire; they express Mac-1, IgM(high), and B220(low). B-1a cells express CDS. The aim of this work emerged from the fact that the morphology of B-1 cells is not fully characterized. Here we identified B-1 cells using colloidal gold immunocytochemical assays and purified B-1 cells from supernatants of adherent peritoneal cell cultures by a magnetic bead technique. These techniques lead us to demonstrate that, in mice, either B-1a or B-1b cells have a unique morphology distinct from that of B-2 cells.     

Regulatory role of B-1 B cells in chronic colitis

According to the 'hygiene hypothesis', enhanced microbial exposure due to early childhood infections leads to a reduction of T(h)2-mediated allergic diseases and inflammatory bowel disease. To begin to elucidate the mechanisms underlying this hypothesis, we studied development of T(h)2-mediated colitis of the TCRalpha knockout (KO) mouse in both a specific pathogen-free (SPF) facility and a conventional (CV) facility. After more than five generations in each facility, TCRalpha KO mice kept in the CV facility developed dramatically less colitis than mice that were kept in the SPF facility. Surprisingly, the suppression of colitis in the CV facility correlated with a significant increase in natural IgM production by B-1 B cells. In contrast, B cell-deficient TCRalpha double-knockout (alphamu DKO) mice maintained in the CV facility continued to develop severe colitis, strongly suggesting that B-1 B cells contributed to the suppression of colitis. Indeed, the adoptive transfer of B-1 B cells isolated from the peritoneal cavity of TCRalpha KO mice (SPF) into alphamu DKO mice (CV) suppressed the development of colitis in the recipient mice. We conclude that B-1 cells play a regulatory role in T(h)2-mediated colitis under non-hygienic conditions, possibly by generating natural antibodies in response to microbial flora.     

Recipient age determines the success of intraperitoneal transplantation of peritoneal cavity B cells.

In vivo studies of lymphocyte biology have used intravenous (i.v.) injection as the primary mode of cell transfer, a protocol consistent with the anatomic distribution of most lymphocytes. However, for study of peritoneal cavity B cells, i.v. injection does not correlate with anatomical localization. This report describes the restoration of B-cell function in B lymphocyte-defective X-chromosome-linked immune-defective (XID) mice after intraperitoneal transfer of immunoglobulin heavy chain (Igh)-disparate peritoneal cavity (PerC) cells. In contrast to i.v. transfer, intraperitoneal (i.p.) transfer restored B-cell function in young, but not adult (> 8 weeks), XID mice. When host and donor Igh allotype matched, PerC B-cell engraftment was noted in older recipients; this reconstitution however, was also age-dependent. Migration from the peritoneum to systemic circulation was necessary for serum IgM production as shown by the presence of donor antibody-secreting cells in the host spleen. Host lymphocytes also influenced the success of i.p. transplantation as severe combined immune-deficient mice, regardless of age, exhibited donor serum IgM production. Recipient age, Igh allotype, and immune-deficiency were found to have an impact on the ability of i.p.-transferred PerC B cells to restore B-cell function in XID mice.     

Allelic exclusion of M1 (IgM) allotype on the surface of chicken B cells.

The membrane expression of M1 (IgM) and G1 (IgG) allotype markers on peripheral blood lymphocytes was examined by immunofluorescence. In homozygous chickens 15% lymphocytes stained with either anti-M1 or with polyspecific rabbit anti-Ig serum, suggesting that M1 is expressed on the surface of probably all B lymphocytes. In heterozygous M1a/M1b individuals antisera against either allelic antigens reacted with 50% of the total surface Ig positive cells as evidence for the allelic exclusion of surface M1 expression. Donor allotype synthesis was determined from serum allotype levels in congenic cyclophosphamide-treated recipients of lymphoid cells. Treatment of cells with anti-M1 serum prior to transfer inhibited both M1 and G1 allotype synthesis. The treatment of heterozygous (M1a/M1b, G1a/G1e) cells with anti-M1b serum inhibited specifically the synthesis of M1b and G1e allotypes controlled by linked genes from the same parental chromosome. Thus, B cells which had been the targets for anti-M1 antibody mediated suppression are also subject to allelic exclusion.     

Non-overlapping Fas- and BCL-2-regulated death pathways in IgG2a(b)-producing B cells

Using perforin (Pfp)- and/or Fas-dependent cytotoxic pathways, T splenocytes from Igh(a/a) mice are able in vivo to totally and chronically eliminate congenic Igh(b/b) B cells committed to IgG2a(b) production. This phenomenon leads to a characteristic absence of serum IgG2a(b) expression (IgG2a(b) allotype suppression) in, for instance, histocompatible Igh(a/b) or Igh(b/b) mice, having neonatally received such T cells. Because the study of the protective role of BCL-2 oncoprotein against Fas-mediated cell death has generated contradictory findings, we examined the possible impact of constitutive overexpression of transgenic human BCL-2 protein in Igh(b/b) B cells when the latter were exposed in vivo exclusively with the Fas-dependent, anti-IgG2a(b) T cell activity of Igh(a/a) Pfp(0/0) mice. We observed that, despite high intracellular expression of functional transgenic BCL-2 and no up-regulation of the principal BCL-2 inhibitors in whole Igh(b/b) B cells, total, chronic and specific IgG2a(b) suppression was exerted by Igh(a/a) Pfp(0/0) cytotoxic T cells. These data show that, in this model of negative regulation of Ig production, Fas- and BCL-2-regulated mechanisms belong to non-overlapping death pathways at the level of IgG2a(b)-producing B cells, targets of Igh(a/a) T cell-mediated cytotoxicity. Thus, in these mature B cells, the Fas signaling-directly operating via caspase 8-does not involve a mitochondria-dependent pathway regulated by BCL-2.     

Idiopathic paraproteinaemia V. Expression of Igh1 and Igh5 allotypes within the homogeneous immunoglobulins of ageing (C57BL/LiARij X CBA/BrARij)F1 mouse

The role of genetic factors linked to the immunoglobulin loci and the development of idiopathic paraproteinaemia (IP)--a benign B-cell proliferative disorder--was investigated in F1 hybrid mice of low (CBA/BrARij) and high (C57BL/LiARij) IP frequency strains. Igh1 and Igh5 allotypes were used as markers for the (parental type) origin of homogeneous immunoglobulins (H-Ig) which appeared in the sera of the F1 mice with ageing. The frequencies of H-Ig in the F1 mice were intermediate with those of the parental strains. The isotype distribution of the H-Ig was 27%, 24%, 12%, 12%, 11%, 10%, 3% and 1% for IgG2a, IgM, IgG1, IgG3, IgG2b, IgD, IgA and IgE, respectively. H-Ig of the IgG2 subclass carried the Igh1b (C57BL) allotype in 98% and the Igh1a (CBA) allotype in 2% cases. Of the IgD H-Ig, 70% carried the Igh5b and 30% the Igh5a determinant. The Igh1 allotype distribution in the bone marrow and spleen plasma cells showed a large variation in the Igh1a/Igh1b ratio among old individual mice and often also between bone marrow and spleen within a single animal with or without a H-Ig component. The categorization of the paraproteinaemias on the basis of their origin showed that 10% of the H-Ig were the result of a transient monoclonal B-cell proliferation; multiple myeloma or lymphoma was found to be responsible for about 1% of the paraproteinaemias; H-Ig fulfilling the criteria for IP were detected in about 42% of cases. The origin of the remaining old age paraproteinaemias could not be determined. These data indicate that the F1 mice develop monoclonal proliferative disorders in a manner more similar to the C57BL than to the CBA parental strain. The allotype associated genetic material from the parental C57BL strain was shown to be mainly responsible for the development of IP in ageing F1 mice.     

Igh-1b-specific CD4+CD8- T cell clones of the Th1 subset selectively suppress the Igh-1b allotype in vivo.

The demonstration of major histocompatibility complex (MHC)-restricted T helper (Th) cells specific for peptides from the variable (V) regions of syngeneic immunoglobulin (Ig) (idiopeptides) opens the possibility that Th cells regulate B cell functions via idiopeptide-based cognate T-B interactions. As a model for such interactions we investigated the influence of Ig allotype-specific T cells on the differentiation of H-2-syngeneic B cells expressing that particular Ig allotype. We established a BALB/c (H-2d, Iga) CD4+CD8- T cell line and clones of the Th1 subset (interleukin 2+, interleukin 4-, interferon-gamma+, tumor necrosis factor-alpha+) that recognized Igh-1 (IgG2a) of the b allotype (Igh-1b) together with I-Ad. These T cells specifically suppressed surface Igh-1b+ B cells in vitro and in vivo. In 12 out of 15 6-week-old (BALB/c X B10.D2)F1 mice neonatally injected with Igh-1b-specific T cells, the serum Igh-1b concentrations were less than 5% of the levels in the controls. Thus, allotype suppression can be accomplished solely by adoptive transfer of Igh-1b-specific CD4+ T cells. The in vivo suppression was specific for Igh-1b+ B cells as the recipients' levels of Igh-1a and Igh-4b (IgG1b) were unaffected. The V beta 14-specific anti-T cell receptor (TcR) monoclonal antibody 14-2 inhibited activation of hybridomas derived from two of the clones. Collectively the data indicate that suppression resulted from cognate interactions between allopeptide-specific TcR alpha/beta+ T cells and normal unmanipulated B lymphocytes presenting their endogenous Igh-1b in association with MHC class II molecules. The data support the possibility that normal B cells can be suppressed by idiopeptide-specific T cells in vivo.     

B-1a cells are imprinted by the microenvironment in spleen and peritoneum

B-1a cells are found mainly in the peritoneal cavity of mice but are also present in the spleen. Gene expression profiling defined many genes differentially expressed in B-1a cells from these two sites. To see whether this gene expression pattern was imprinted by the particular microenvironment, peritoneal or spleen cells from recombinant L2 mice mainly consisting of B-1a cells were adoptively transferred into Rag1-/- mice. Re-isolated peritoneal and splenic B-1a cells were analyzed for expression of three indicator genes--vcam-1, adamdec1 and spi-c. The expression of these genes was up-regulated in splenic and down-regulated in peritoneal cells. This particular pattern was observed for peritoneal or splenic donor cells transferred either intraperitoneally or intravenously. Similar results were obtained when levels of surface IgM or frequencies of Mac-1+ B-1 cells were compared after transfer. This suggests that the environment induces the particular genetic program of B-1a cells and argues against an independent ontogeny.     

Allotype suppression induced in the adoptive transfer system: the variables of the system and an apparent absence of a role for T cells.

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are; SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells are less susceptible heterozygous CBA spleen cells are less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system.     

Effect of cell:cell competition and BAFF expression on peripheral B cell tolerance and B-1 cell survival in transgenic mice expressing a low level of Igkappa-reactive macroself antigen

In mice carrying a synthetic Igkappa-reactive superantigen ("kappa macroself antigen"), low level expression induced split peripheral B cell tolerance in the sIgkappa+ compartment, with striking reductions in follicular and marginal zone (MZ) B cells and the retention of significant numbers of sIgkappa+ B-1a but not B-1b cells in the peritoneum. Here, we characterize the transgenic line pKkappa with this split tolerance phenotype and assess the effects of B cell competition and the survival cytokine BAFF (B cell activating factor belonging to the TNF family) on peripheral tolerance. In pKkappa mice the surviving peritoneal and splenic kappa+ B cells were largely lost in mice carrying one copy of the human Ckappa exon in place of the mouse version, a maneuver that generates additional antigen non-reactive competitor B cells in this model. Furthermore, overexpression of BAFF suppressed kappa-macroself antigen-induced deletion and promoted production of both IgM,kappa and IgA,kappa antibodies in mice with normal Igkappa alleles but not in mice carrying one copy of the human Ckappa allele. These findings suggest that BAFF overexpression has minimal effects on the survival of autoreactive B cells in a polyclonal immune system and that B cell:B cell competition plays a potent role in suppressing the survival of B-1 and splenic B cells with excessive autoreactivity.     

Age-dependent increase of peritoneal B-1b B cells in SCID mice

The impact of increasing age upon immunoglobulin production and B-lymphocyte generation in "leaky" severe combined immune-defective (SCID) mice was examined by enzyme-linked immunosorbent assay and flow cytometry. By 1 year of age, the mice had normal numbers of B cells in their peritoneal cavity, while their spleen had very few immunoglobulin M-positive (IgM+) cells. The majority of B cells expressed the CD11b marker characteristic of the B-1b subset. B-1a (CD5+) cells were present at a lower frequency and B-2 cells were absent. The frequency of mice producing detectable immunoglobulin increased with age, and isotype diversity within individual mice was variable. IgM production was most frequently observed followed by IgG3 and IgG2a, then IgG1, and finally IgA. The selective persistence of the B-1 B-cell subset in the peritoneal cavity of aging SCID mice is a natural model for the study of those genetic and environmental influences that determine lymphocyte longevity.