Rapid reconstitution of antibody responses following transplantation of purified allogeneic hematopoietic stem cells.

Allogeneic hematopoietic cell transplantation has broad clinical applications extending from the treatment of malignancies to induction of immunologic tolerance. However, adaptive cellular and humoral immunity frequently remain impaired posttransplantation. Here, recovery of T-dependent and T-independent Ab responses was evaluated in mice transplanted with purified hematopoietic stem cells (HSCs) devoid of the mature immune cells believed to hasten immune recovery. Mixed and full donor chimeras were created by conditioning recipients with sublethal or lethal irradiation, respectively, across different donor/host genetic disparities. By 6 wk posttransplantation, all animals demonstrated robust T-independent Ab responses, and all mixed chimeras and recipients of MHC-matched or haploidentical HSCs with a shared MHC haplotype had T-dependent Ab responses equivalent to those of untransplanted controls. Full chimeras that received fully MHC-disparate HSCs showed delayed T-dependent Ab responses that recovered by 12 wk. This delay occurred despite early reconstitution and proper migration to germinal centers of donor-derived T(follicular helper) (T(FH)) cells. Congenic transplants into T(FH)-deficient CD4(-/-) mice revealed restoration of T-dependent Ab responses by 6 wk, leading us to conclude that MHC disparity caused delay in humoral recovery. These findings, together with our previous studies, show that, contrary to the view that depletion of graft lymphocytes results in poor posttransplant immunity, elimination of immune-suppressing graft-versus-host reactions permits superior immune reconstitution. This study also provides insight into the regeneration of T(FH) cells and humoral immunity after allogeneic HSC transplantation.

Follicular B cell trafficking within the spleen actively restricts humoral immune responses.

Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.

Cell intrinsic TGF-beta 1 regulation of B cells.

TGF-beta family cytokines play multiple roles in immune responses. TGF-beta1-null mice suffer from multi-organ infiltration that leads to their premature death. T cells play a central role in the TGF-beta1 phenotype, as deficiency of TGF-beta1 only in T cells reproduces the lethal phenotype. Although it is known that TGF-beta1 controls B cells isotype switch and homeostasis, the source responsible for this control has not been characterized. Because of the major role that T cells play in regulating B cell responses, we addressed the T cell dependency of the TGF-beta1 control of B cells. The analysis of T cell-deficient, TGF-beta1 knockout mice and the production of chimeras in which B but not T cells lacked TGF-beta1 allowed us to show that B cells are controlled in part by cell autonomous production of TGF-beta1.

Axon growth and guidance genes identify T-dependent germinal centre B cells.

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.

Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection.

The analysis of molecular signatures of antigen-driven affinity selection of B cells is of immense use in studies on normal and abnormal B cell development. Most of the published literature compares the expected and observed frequencies of replacement (R) and silent (S) mutations in the complementarity-determining regions (CDRs) and the framework regions (FRs) of antibody genes to identify the signature of antigenic selection. The basic assumption of this statistical method is that antigenic selection creates a bias for R mutations in the CDRs and for S mutations in the FRs. However, it has been argued that the differences in intrinsic mutability among different regions of an antibody gene can generate a statistically significant bias even in the absence of any antigenic selection. We have modified the existing statistical method to include the effects of intrinsic mutability of different regions of an antibody gene. We used this method to analyse sequences of several B cell-derived monoclonals against T-dependent antigens, T-independent antigens, clones derived from lymphoma and amyloidogenic clones. Our sequence analysis indicates that even after correcting for the intrinsic mutability of antibody genes, statistical parameters fail to reflect the role of antigen-driven affinity selection in maturation of many clones. We suggest that, contrary to the basic assumption of such statistical methods, selection can act both for and against R mutations in the CDR as well as in the FR regions. In addition we have identified different methodological difficulties in the current uses of such statistical analysis of antibody genes.

STAT6 is required for IL-4-induced germline Ig gene transcription and switch recombination.

Transcription of the germline C gamma1 and C epsilon Ig genes is believed to be a necessary prerequisite for isotype switching to IgG1 and IgE, respectively. IL-4 stimulation and ligation of CD40 can each independently induce low level germline gamma1 and epsilon transcription in murine B cells. Together these signals act synergistically to promote high level germline transcription and are normally required for T-dependent isotype switching to IgG1 and IgE. The STAT6 transcription factor has been suggested to play a critical role in IL-4-induced activation of germline C gamma1 and C epsilon genes. To directly assess the role of STAT6 in IL-4R- and CD40-mediated germline transcription and switching, we have analyzed these events in splenic B cells from STAT6-deficient mice. Our results demonstrate that IL-4 does not induce detectable levels of germline gamma1 or epsilon transcripts in STAT6-deficient B cells. Germline transcript expression induced by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely ablated. Switch recombination to S gamma1, as measured by digestion-circularization PCR, is dramatically reduced in STAT6-deficient B cells stimulated with CD40 ligand plus IL-4. Similarly, germline gamma1 transcript expression and switch recombination to S gamma1 are also impaired in STAT6-deficient B cells stimulated with IL-4, IL-5, and anti-IgD Abs conjugated to dextran, a model for T-independent type II responses. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination in nontransformed B cells.

Regulation of idiotope expression. III. H-2 influences the magnitude and the idiotypy of a T-independent antibody response in mice of certain genetic backgrounds.

Antibody response to the phosphocholine (PC) epitope on Streptococcus pneumoniae R36a (Pn), a T-independent Ag type 2, was studied in H-2 congenic mouse strains. The PC-specific antibody plaque-forming cells (PFC) were enumerated in the spleen at various intervals after the primary Pn injection, and the proportion of PFC that produced antibody expressing the AB1-2 idiotope (Id) was determined by using the corresponding monoclonal anti-Id. AB1-2 is a cross-reactive Id, detectable on germline-encoded PC antibody of the T15 family, and on most, but not all, somatic variants of that antibody. The specific PFC responses in BALB/c (H-2d) and BALB.B (H-2b) strains were of comparable magnitude and most, if not all, PFC were ABl-1 Id-positive (AB1-2+). This was not the case in the responses of the B10D2 (H-2d) vs C57BL/10 (H-2b) strains and the D1.C (H-2d) vs D1.LP (H-2b) strains (on DBA/1 background). In each of these pairs, the H-2d mice were high responders, and the response was dominated by AB1-2 Id (greater than or equal to 80% AB1-2+ PFC at the peak, on day 5). The H-2b mice were low responders, and only a minor proportion of PFC (less than or equal to 30%) were AB1-2+; an increase of AB1-2+ was seen later in the response (d.10). The results of PFC assays were confirmed by measuring the PC-binding antibody and AB1-2 Id in the sera of D1.C and D1.LP mice immunized repeatedly with Pn. Moreover, D1.LP mice that had very low levels of AB1-2 Id had higher serum levels of antibody expressing two other T15 Id, B36-82, and B24-44. The B36-82 and B24-44 Id have been previously found on somatic variants of PC antibody expressed independently of the Ab1-2 Id. The concentrations of these two Id in D1.LP mice after repeated immunization approached those in D1.C. These results indicate that 1) the H-2 allelism may have a significant effect on TID antibody response in mice of a certain genetic background, but not in the BALB/c; and 2) the idiotypic repertoire of the response may be influenced by H-2 at the level of clonal variants of PC-reactive cells.

Immune memory expression to Tnp-Ficoll in CB.20 mice: evidence for a multigenic control.

Trinitrophenyl (Tnp)-Ficoll, a class 2 thymus-independent (TI) antigen, generates in most mouse strains Tnp-specific B-memory cells which can be detected in situ 1 week after priming by a heterologous stimulation with Tnp-lipopolysaccharide (LPS) but not by a homologous Tnp-Ficoll challenge. We have investigated the secondary responses raised in CB.20 congenic mice by a homologous challenge in situ occurring at various time intervals after priming. We report that a memory-type response is obtained, culminating when the challenge is performed at 4 weeks; this finding assesses definitely the ability of TI-2 antigens to produce immunological memory under standard conditions. However, the same immunization procedure elicits no memory-type response in the majority of other mouse strains, suggesting a possible genetic control of the expression of memory to class 2 TI antigens. The utilization of F1 hybrids between C57BL/6 and BALB/c and of appropriate congenic strains shows indeed that this memory expression is under multigenic control: Igh-V or closely linked genes are clearly involved but a complementation with other gene(s), located outside the H-2 complex, is required for a memory-type response to Tnp-Ficoll. We have also analyzed the secondary heterologous response to Tnp-LPS in CB.20 mice at different times after Tnp-Ficoll priming. The difference in the kinetic profile of the heterologous (TI-2----TI-1) versus homologous (TI-2----TI-2) secondary responses is discussed in terms of B-memory-cell ontogeny and humoral regulation.

In vitro analysis of allogeneic lymphocyte interaction. VIII. Characterization of helper components of allogeneic effect factor (AEF) that activate Lyb5+ and Lyb5- B cells to respond to thymus-dependent and thymus-independent antigens.

We analyzed the ability of an allogeneic effect factor (AEF), produced using alloactivated A.SW (H-2s) responder T cells and irradiated A/WySN (H-2a) T cell-depleted stimulator spleen cells, to enhance the responsiveness of Lyb5+ and Lyb5- B cells to thymus-dependent (TD) and thymus-independent type 1 (TI-1) and type 2 (TI-2) antigens. We found that normal adult male CBA/CaHN (Lyb5+ + Lyb5-) B cells are activated by unfractionated AEF in primary in vitro IgM antibody responses to the SRBC (TD), TNP-LPS (TI-1), and TNP-Ficoll (TI-2) antigens. Adult male xid CBA/N (Lyb5-) B cells are activated by unfractionated AEF to respond to TNP-LPS but to neither SRBC nor TNP-Ficoll. Gel filtration of AEF on ACA 54 resolves several helper components. One component, which is I-Ak restricted in its activity and may represent a secreted form of an Ia antigen T cell receptor, interacts with histocompatible and functionally competent CBA/CaHN or CBA/N antigen-presenting cells to activate a primary anti-SRBC response of CBA/CaHN B cells but not of CBA/N B cells. A second antigen-nonspecific and H-2-nonrestricted AEF component, which consists of interleukin 2 and possibly also interleukin 1, activates anti-SRBC and anti-TNP-Ficoll responses of both CBA/CaHN B cells and CBA/N B cells. Such CBA/N B cell responses were potentiated by this AEF component only after its biochemical fractionation and only in the presence of a limiting number of T cells. These data indicate that both Lyb5+ and Lyb5- B cells are responsive to TD, TI-1, and TI-2 antigens in the presence of ancillary antigen-nonspecific help. They are also consistent with the notion that T helper cell activation of Lyb5+ B cells is H-2 nonrestricted and that T helper cell activation of Lyb5- B cells is H-2 restricted.

Genetic analysis of the low responsiveness to dextran B512 in CBA/N and C57BL/10ScCr mice.

CBA/N and C57BL/10ScCr mice are low responders to the antigen dextran B512. This is due to the Xid gene in CBA/N mice and to unknown genes in C57BL/10ScCr mice, although this strain is unresponsive to lipopolysaccharide (LPS) due to a defective gene in the fourth chromosome. The female F1 hybrids (C57BL/10ScCr X CBA/N) and (CBA/N X C57BL/10ScCr) were low responders to dextran, although the Xid gene is not expressed in these hybrids, indicating lack of genetic complementation. In contrast, female F1 hybrids between the dextran high-responder strains CBA or C57BL/10 as one parental strain and the low-responder strains CBA/N or C57BL/10ScCr as the other parental strain, respectively, were responders to dextran. The C57BL/10ScCr mice did not appear to have an X-linked gene determining low responsiveness to dextran. The findings suggest that the only defect in CBA/N mice cannot be the Xid gene and the only defect in C57BL/10ScCr mice cannot be the gene determining unresponsiveness to LPS.

A new approach to immunological memory using thymus-independent antigens.

Contrary to what has been reported of thymus-independent antigens, we recently demonstrated that trinitrophenylated lipopolysaccharide (TNP-LPS), a class 1 thymus-independent antigen, elicited an anti-TNP anamnestic response in C57BL/6 mice. The question of whether or not class 2 thymus-independent antigens (DNP-Ficoll and DNP-dextran) could also induce immunological memory in this mouse strain was examined. Evidence induce immunological memory in this mouse strain was examined. Evidence is presented that priming with either of these class 2 thymus-independent antigens resulted in the induction of memory B lymphocytes. However, while the memory cells generated by these two antigens were able to be activated by TNP-LPS, they were not triggered by class 2 thymus-independent antigens. Genetic analysis of the capacity of different mouse strains to mount a secondary response to TNP-LPS revealed that major histo-compatibility-associated genes did not play an essential role, but that IgH-V or closely linked gene(s) controlled the immunological memory to TNP-LPS. These findings are discussed in terms of regulatory phenomena which govern the expression of memory response to thymus-independent antigens.

The selection and maintenance of the V region determinant repertoire is germ-line encoded and T cell-independent.

The number of lipopolysaccharide-sensitive precursor cells synthesizing immunoglobulin (Ig) which reacts with the monoclonal anti-M460 antibody F6(51) has been determined in the spleen and in the bone marrow of different strains of mice. These precursor frequencies fall into two quantitatively different groups. The first group includes mice with the same Igh haplotype as BALB/c animals (Igha). In this group, spleen cells contained between 1:10(4) to 1:5 X 10(4) B cell precursors secreting Ig which bound F6(51). The second level of precursor was obtained with animals with allotypic haplotypes other than Igha. These values were too low to allow accurate frequency determinations. The frequency of these cells in mice of the latter group, however, increased dramatically when these animals were hyperimmunized with the monoclonal anti-M460 antibody. Similar results were obtained when the frequencies were determined using the Ig- fraction of bone marrow cells. Surprisingly, the numbers of lipopolysaccharide-sensitive B cell precursors secreting F6(51)-binding Ig in spleen cells of nude mice was found to be similar to the one in splenocytes of normal mice, and even in this case, the frequencies reflected the genetic background of the animals tested. Taken together these data support the notion that the establishment and the maintenance of the M460 idiotypic repertoire is germ-line encoded and independent of regulatory T cells.

Analysis of the B cell subpopulations influenced by allogeneic effect factor. I. MHC restricted enhancement of b cell responses to thymic-independent antigens, types 1 and 2, in normal and CBA/N mice.

The ability of allogeneic effect factor (AEF) to enhance the in vitro antibody responses of murine T cell-depleted B cell populations to thymic-dependent (TD) antigens also occurs with thymic-independent (TI) antigens. AEF significantly enhanced the in vitro antibody responses of normal T cell-depleted spleen cell populations to both 2,4,6-trinitophenylated Brucella abortus (TNP-BA), a TI type 1 antigen, and 2,4,6-trinitrophenylated aminoethylcarbamylmethyl-Ficoll (TNP-Ficoll), a TI type 2 antigen. The enhancing activity of AEF on TI responses was analyzed further in immune-defective CBA/N mice that possess only Lyb-5- B cells, and thus fail to respond to TNP-Ficoll, but do respond to TNP-BA and sheep red blood cells (SRBC). AEF was able to reconstitute the in vitro antibody responses of CBA/NT cell-depleted B cell populations to SRBC and to augment the T cell-depleted response to TNP-BA. These data suggest that AEF can enhance the activity of both Lyb-5+ as well as Lyb-5- B cells in their responses to both type 1 and type 2 TI antigens, and to the TD antigen SRBC. This enhancing effect of AEF on the TD and TI responses was MHC restricted for both Lyb-5+ as well as Lyb-5- spleen cells.

Major histocompatibility complex-restricted self-recognition in responses to trinitrophenyl-Ficoll. A novel cell interaction pathway requiring self-recognition of accessory cell H-2 determinants by both T cells and B cells.

In vitro primary antibody responses to limiting concentrations of trinitrophenyl (TNP)-Ficoll were shown to be T cell dependent, requiring the cooperation of T helper (TH) cells, B cells, and accessory cells. Under these conditions, TH cells derived from long-term radiation bone marrow chimeras were major histocompatibility complex (MHC) restricted in their ability to cooperate with accessory cells expressing host-type MHC determinants. The requirement for MHC-restricted self-recognition by TNP-Ficoll-reactive B cells was assessed under these T-dependent conditions. In the presence of competent TH cells, chimeric B cells were found to be MHC restricted, cooperating only with accessory cells that expressed host-type MHC products. In contrast, the soluble products of certain monoclonal T cell lines were able to directly activate B cells in response to TNP-Ficoll, bypassing any requirement for MHC-restricted self-recognition. These findings demonstrate the existence of a novel cell interaction pathway in which B cells as well as TH cells are each required to recognize self-MHC determinants on accessory cells, but are not required to recognize each other. They further demonstrate that the requirement for self-recognition by B cells may be bypassed in certain T-dependent activation pathways.

Medial histocompatibility antigens.

Medial histocompatibility (H) antigens are weak H antigens, recognized by unrestricted T cells; they differ thus from both major and minor H antigens. An example, Qed-1, is described in detail, and other known medial H antigens of the mouse are reviewed. The structural and genetic relationships of major and medial H antigens and their role in T cell recognition are discussed.