Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.     

Early development of Ig-secreting cells in young of germ-free BALB/c mice fed a chemically defined ultrafiltered diet

The influence of antigenic stimulation on the early development of the "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora, the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP), 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM- and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.     

The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.     

Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.     

Immunodominance is altered in T cell receptor (beta-chain) transgenic mice without the generation of a hole in the repertoire.

Despite the tremendous plasticity of the TCR repertoire, T cells recognize a limited number of antigenic sites (frequently a single site, or immunodominant epitope) on a complex protein Ag. Current models suggest that the immunodominant epitope of a complex protein is the processed peptide that binds to the MHC molecule with the highest affinity. Conversely, the inability of the T cell population to recognize a specific epitope, termed a "hole" in the repertoire, can prevent the immunodominance of a peptide despite efficient processing and MHC binding of the peptide. The role of specific TCR alpha- or beta-chains in determining MHC restriction and recognizing specific epitopes is complex and incompletely understood. To evaluate the contribution of each TCR chain to the functional diversity of the T cell repertoire, we investigated in vivo the T cell response to phage lambda-repressor protein in transgenic mice expressing a single rearranged beta-chain gene (C57L beta mice) in association with the complete germline alpha-chain repertoire. Our results demonstrate that expression of the TCR beta-chain transgene alters the immunodominant epitope recognized by T cells. However, after immunization with the appropriate peptide the transgenic mice can also respond to the nonimmunodominant epitope; thus, the expression of the TCR beta-chain transgene does not create a hole in the repertoire. These data indicate that the primary site, or immunodominant epitope, of an Ag recognized by T cells can be altered by the preimmune TCR repertoire independent of antigen processing and MHC affinity.     

Cutting edge: gamma delta T cells provide help to B cells with altered clonotypes and are capable of inducing Ig gene hypermutation

It has not been resolved whether gammadelta T cells can collaborate with germinal center B cells and support Ig hypermutation during an Ab response to a truly defined T-dependent Ag. In this study, we show that in the absence of alphabeta T cells, immunization with the well-defined T-dependent Ag, (4-hydroxy-3-nitrophenyl) acetyl (NP) conjugate, was able to induce Ig hypermutation. However, the clonotypes of B cells responding to NP were dramatically altered in TCR beta(-/-) mice. Unlike B cells in wild-type mice that use canonical VDJ rearrangements, most NP-responding B cells in mutant mice use analog genes of the J558 gene family. In addition, the majority of anti-NP Abs produced in mutant mice use kappaL chain instead of lambda1L chain, which dominates in mice of Igh(b) background. Thus, the B cell population that collaborates with gammadelta T cells is distinct from B cells interacting with conventional alphabeta Th cells.     

Differences in the repertoire expressed by peritoneal and splenic Ly-1 (CD5)+ B cells.

Purified populations of B cells expressing the Ly-1 and/or Mac-1 surface Ag were isolated from normal unmanipulated mice by cell sorting. The number of lymphocytes in each population secreting antibodies reactive with DNA, bromelain-treated mouse RBC, phosphorylcholine and TNP-keyhole limpet hemocyanin was quantitated by ELISA spot assay. The proportion of B cells secreting Ig in vivo and the repertoire of antibodies they produced varied as a function of B cell phenotype and location. Among peritoneal lymphocytes, those that were Ly-1+ or Ly-1- Mac-1+ secreted Ig 10 times more frequently that Mac-1- Ly-1- B cells from the same location. In addition, the former populations expressed repertoires that were significantly skewed toward the production of antibodies reactive with bromelain-treated mouse RBC (p less than 0.001). In contrast, splenic B cells expressing the Ly-1 surface Ag did not differ significantly from splenic Ly-1- B cells in their expressed repertoire or frequency of Ig production. B cells isolated from the spleen and peritoneum tended to differ in antibody specificity from bone marrow and lymph node-derived lymphocytes. For example, B cells from the spleen secreted anti-DNA antibodies two to four times more frequently than B cells from other organs. These results demonstrate that phenotype and microenvironment influence the repertoire of antibodies expressed by B cells in vivo.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Antigen-specific T cell repertoire modification of CD4+CD25+ regulatory T cells

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).     

Neonatal immunity develops in a transgenic TCR transfer model and reveals a requirement for elevated cell input to achieve organ-specific responses

In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host's spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN-gamma and the splenic T cells, although unable to proliferate or produce IFN-gamma, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN-gamma production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN-gamma-dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.     

Analysis of CD4+ T cells that provide contact-dependent bystander help to B cells.

Although cognate, MHC-restricted interaction of Th cells with Ag-presenting B cells provides effective help to a resting B cell, substantial B cell responses have also been seen with preactivated T cell clones that cannot recognize Ag on the B cell but apparently interact in a noncognate fashion (the bystander response). Here, we have investigated the ability of distinct Th cell subsets and T cells activated by different stimuli to support such bystander B cell responses. We have also determined which cytokines are involved. We generated distinct CD4+ T cell subsets specific for both alloantigen (using normal mice) and cytochrome c (using TCR transgenic mice). To compare cognate and bystander help, we analyzed the response of allogeneic (cognate) vs syngeneic (bystander) resting B cells in the former case, and the response of syngeneic B cells in the presence vs absence of Ag, in the latter case. Both approaches gave similar results. T cells stimulated with Ag for 24 h (naive and memory cells) or generated from naive cells over 4 days in the presence of exogenous IL-2 ("Th1-like" effectors) induced B cells to secrete minimal amounts of bystander Ig (20 to 700 ng/ml), less than 6% of the Ig induced under cognate conditions. In contrast, effectors generated in IL-4 or IL-6 ("Th2-like" and "Th0-like") induced significantly more bystander Ig (4 to 9 micrograms/ml), which was 18 to 30% of the amount produced during a cognate response. Restimulation of Th cell populations with anti-CD3, instead of Ag/APC, enhanced their ability to induce bystander Ig to levels 40 to 100% of those produced through cognate interaction. The addition of anti-cytokine Ab to bystander responses indicated that the cytokines utilized were similar to those mediating response after cognate interaction. Addition of exogenous cytokines did not specifically enhance the extent of the bystander response as a function of the cognate response. These results suggest that most Th cells can efficiently activate only those B cells that present relevant Ag on class II MHC, but that highly activated/differentiated Th effectors also have the ability to induce significant bystander B cell responses through noncognate interactions. We also conclude that the mode of Th cell activation and the cytokines encountered during Th differentiation play a major role in the capacity of helper cells to initiate a bystander response.     

Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with th2 phenotype acquisition in vivo.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vbeta repertoire, different from the TCR Vbeta profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IA(d)-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.     

Similarities in B cell repertoire development between autoimmune and aging normal mice.

B cell repertoire changes that characterize systemic autoimmune disease may be linked to an acceleration of normal immune aging. To examine this issue, the repertoires expressed by lupus-prone and geriatric normal mice were compared. An ELISA-spot assay was used to identify and quantitate individual lymphocytes secreting antibodies reactive with a panel of five autoantigens and three conventional Ag. Over half of autoimmune NZB and MRL/lpr mice developed repertoires biased toward the production of specific autoantibodies by 8 mo of age. The B cell repertoires expressed by normal BALB/c mice were stable over this period but developed a similar bias toward the production of autoantibodies by 18 to 22 mo of age. As both normal and autoimmune mice grew older, they expressed repertoires that increasingly diverged from those of other members of the same strain--a process whose onset and rate of development was accelerated in lupus-prone animals. By analyzing B cells from individual MRL/lpr mice at multiple time points, we found that 1) autoreactivity developed over a specific time period, 2) individual animals developed increased responsiveness against different autoantigens, and 3) this increased responsiveness persisted for life. These results suggest that the repertoires of adult autoimmune mice are generated and maintained by a process of continuous (auto)antigenic stimulation similar to that associated with normal immune aging.     

Vκ polymorphisms in NOD mice are spread throughout the entire immunoglobulin kappa locus and are shared by other autoimmune strains

The diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes available to form the lymphocyte repertoire has the capacity to produce a broad array of both protective and harmful specificities. In type 1 diabetes (T1D), the presence of antibodies to insulin and other islet antigens predicts disease development in both mice and humans, and demonstrate that immune tolerance is lost early in the disease process. Anti-insulin T cells isolated from T1D-prone non-obese diabetic (NOD) mice use polymorphic TCRα chains, suggesting that the available T cell repertoire is altered in these autoimmune mice. To probe whether insulin-binding B cells also possess polymorphic V genes, Ig light chains were isolated and sequenced from NOD mice that harbor an Ig heavy chain transgene. Three insulin-binding Vκ genes were identified, all of which were polymorphic to the closest germline sequence matches present in the GenBank database. Additional analysis of over 300 light chain sequences from multiple sources, including germline DNA, shows that polymorphisms are spread throughout the entire NOD Igκ locus, as these polymorphic sequences represent 43 distinct Vκ genes which belong to 14 Vκ families. Database searches reveal that a majority of polymorphic Vκ genes identified in NOD are identical to Vκ genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igκ haplotype may be present. Predicted amino acid changes preferentially occur in CDR, and thus could alter antigen recognition by the germline B cell repertoire of autoimmune versus non-autoimmune mouse strains.     

Systemic autoimmune disease caused by autoreactive B cells that receive chronic help from Ig V region-specific T cells

B cells present BCR V region-derived Id-peptides on their MHC class II molecules to Id-specific CD4+ T cells. Prolonged Id-driven T-B collaboration could cause autoimmune disease, but this possibility is difficult to test in normal individuals. We have investigated whether mice doubly transgenic for an Id+ Ig L chain and an Id-specific TCR develop autoimmune disease. Surprisingly, T cell tolerance was not complete in these mice because a low frequency of weakly Id-reactive CD4+ T cells accumulated with age. These escapee Id-specific T cells provided chronic help for Id+ B cells, resulting in a lethal systemic autoimmune disease including germinal center reactions, hypergammaglobulinemia, IgG autoantibodies, glomerulonephritis, arthritis, skin affection, and inflammatory bowel disease. Inflamed tissues contained foci of Id-driven T-B collaboration, with deposition of IgG and complement. The disease could be transferred with B and T cells. The results demonstrate a novel mechanism for development of autoimmune disease in which self-reactive Id+ B cells receive prolonged help from Id-specific T cells, thus bypassing the need for help from T cells recognizing conventional Ag.     

Impaired CD4 T cell activation due to reliance upon B cell-mediated costimulation in nonobese diabetic (NOD) mice

Diabetes in nonobese diabetic (NOD) mice results from the activation of I-A(g7)-restricted, islet-reactive T cells. This study delineates several characteristics of NOD CD4 T cell activation, which, independent of I-A(g7), are likely to promote a dysregulated state of peripheral T cell tolerance. NOD CD4 T cell activation was found to be resistant to antigenic stimulation via the TCR complex, using the progression of cell division as a measure. The extent of NOD CD4 T cell division was highly sensitive to changes in Ag ligand density. Moreover, even upon maximal TCR complex-mediated stimulation, NOD CD4 T cell division prematurely terminated. Maximally stimulated NOD CD4 T cells failed to achieve the threshold number of division cycles required for optimal susceptibility to activation-induced death, a critical mechanism for the regulation of peripheral T cell tolerance. Importantly, these aberrant activation characteristics were not T cell-intrinsic but resulted from reliance on B cell costimulatory function in NOD mice. Costimulation delivered by nonautoimmune strain APCs normalized NOD CD4 T cell division and the extent of activation-induced death. Thus, by disrupting the progression of CD4 T cell division, polarization of APC costimulatory function to the B cell compartment could allow the persistence and activation of diabetogenic cells in NOD mice.     

B cell-dependent TCR diversification

T cell diversity was once thought to depend on the interaction of T cell precursors with thymic epithelial cells. Recent evidence suggests, however, that diversity might arise through the interaction of developing T cells with other cells, the identity of which is not known. In this study we show that T cell diversity is driven by B cells and Ig. The TCR V beta diversity of thymocytes in mice that lack B cells and Ig is reduced to 6 x 10(2) from wild-type values of 1.1 x 10(8); in mice with oligoclonal B cells, the TCR V beta diversity of thymocytes is 0.01% that in wild-type mice. Adoptive transfer of diverse B cells or administration of polyclonal Ig increases thymocyte diversity in mice that lack B cells 8- and 7-fold, respectively, whereas adoptive transfer of monoclonal B cells or monoclonal Ig does not. These findings reveal a heretofore unrecognized and vital function of B cells and Ig for generation of T cell diversity and suggest a potential approach to immune reconstitution.     

Effect of environmental antigens on the Ig diversification and the selection of productive V-J joints in the bursa

In chickens, a single set of unique functional segments of both Ig H and L chain genes is rearranged during early embryogenesis to generate a pool of B cell progenitors that will be diversified in the bursa by gene conversion, forming the preimmune repertoire. After hatching, bursal cells are exposed to environmental Ags in the bursal lumen. We prepared B cells from each single bursal follicle and used PCR-directed Ig L chain gene analysis to study the differentiation of B cells and the effect of antigenic stimulation from the bursal lumen on the neonatal chicken B cell repertoire formation. Selective amplification of B cell clones with a productive V-J joint was observed during the late embryonic stage, possibly by the interaction with ligands expressed on the bursal stroma and further accelerated in the neonatal chicken. Administration of the artificial Ags into the bursal lumen before the isolation of bursa by bursal duct ligation in the embryo caused a significant increase in lymphocytes with a productive V-J joint in the neonatal chicken bursa compared with the isolated bursa. Intra- and interclonal diversity of a complementarity-determining region measured by an evolutionary distance increased during bursal development. Clonal diversification did not require stimulation by artificial Ags from the bursal lumen. Thus, the preimmune repertoire in the bursa is generated by gene conversion during Ag-independent B cell proliferation, and antigenic stimulation from the bursal epithelium to bursal B cells plays roles in the selection of clones with a productive V-J joint.