Extrafollicular Antibody Responses Research Articles

B cell-intrinsic changes with age do not impact antibody-secreting cell formation but delay B cell participation in the germinal centre reaction.

Vaccines typically protect against (re)infections by generating pathogen‐neutralising antibodies. However, as we age, antibody‐secreting cell formation and vaccine‐induced antibody titres are reduced. Antibody‐secreting plasma cells differentiate from B cells either early post‐vaccination through the extrafollicular response or from the germinal centre (GC) reaction, which generates long‐lived antibody‐secreting cells. As the formation of both the extrafollicular antibody response and the GC requires the interaction of multiple cell types, the impaired antibody response in ageing could be caused by B cell intrinsic or extrinsic factors, or a combination of the two. Here, we show that B cells from older people do not have intrinsic defects in their proliferation and differentiation into antibody‐secreting cells in vitro compared to those from the younger donors. However, adoptive transfer of B cells from aged mice to young recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the GC during the early stages of GC formation. In contrast, by the peak of the GC response, GC B cells derived from the donor cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age‐related intrinsic B cell changes delay the GC response but are not responsible for the impaired antibody‐secreting response or smaller peak GC response in ageing. Collectively, this study shows that B cells from aged individuals are not intrinsically defective in responding to stimulation and becoming antibody‐secreting cells, implicating B cell‐extrinsic factors as the primary cause of age‐associated impairment in the humoral immunity.

IL-10 revisited in systemic lupus erythematosus.

IL-10 is a cytokine with pleiotropic functions, particularly known for its suppressive effects on various immune cells. Consequently, it can limit the pathogenesis of inflammatory diseases, such as multiple sclerosis (MS), inflammatory bowel disease, Crohn’s disease, and Epidermolysis bullosa acquisita, among others. Recent evidence however indicates that it plays dual roles in Systemic lupus Erythematosus (SLE) where it may inhibit pro-inflammatory effector functions but seems to be also a main driver of the extrafollicular antibody response, outside of germinal centers (GC). In line, IL-10 promotes direct differentiation of activated B cells into plasma cells rather than stimulating a GC response. IL-10 is produced by B cells, myeloid cells, and certain T cell subsets, including extrafollicular T helper cells, which are phenotypically distinct from follicular helper T cells that are relevant for GC formation. In SLE patients and murine lupus models extrafollicular T helper cells have been reported to support ongoing extrafollicular formation of autoreactive plasma cells, despite the presence of GCs. Here, we discuss the role of IL-10 as driver of B cell responses, its impact on B cell proliferation, class switch, and plasma cells.

EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, debilitating, multisystem illness of unknown etiology for which no cure and no diagnostic tests are available. Despite increasing evidence implicating EBV and human herpesvirus 6A (HHV-6A) as potential causative infectious agents in a subset of patients with ME/CFS, few mechanistic studies address a causal relationship. In this study we examined a large ME/CFS cohort and controls and demonstrated a significant increase in activin A and IL-21 serum levels, which correlated with seropositivity for antibodies against the EBV and HHV-6 protein deoxyuridine triphosphate nucleotidohydrolase (dUTPases) but no increase in CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation and for the generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A–dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH function in ME/CFS. In vitro studies revealed that virus dUTPases strongly induced activin A secretion while in vivo, EBV dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Together, our data indicate abnormal germinal center (GC) activity in participants with ME/CFS and highlight a mechanism by which EBV and HHV6 dUTPases may alter GC and extrafollicular antibody responses.

Marginal zone B cells mediate a CD4 T-cell–dependent extrafollicular antibody response following RBC transfusion in mice

AbstractRed blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell–deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell–mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

SummaryBackgroundThe spread of SARS-CoV-2 has caused a worldwide pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection and may enable the relaxation of social-distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection; therefore, it is desirable that any new vaccine candidates elicit a robust immune response in older adults.MethodsHere, we use in-depth immunophenotyping to characterize the innate and adaptive immune response induced upon intramuscular administration of the adenoviral vectored ChAdOx1 nCoV-19 (AZD-1222) COVID-19 vaccine candidate in mice.FindingsA single vaccination generates spike-specific Th1 cells, Th1-like Foxp3+ regulatory T cells, polyfunctional spike-specific CD8+ T cells. and granzyme-B-producing CD8 effectors. Spike-specific IgG and IgM are generated from both the early extrafollicular antibody response and the T follicular helper cell-supported germinal center reaction, which is associated with the production of virus-neutralizing antibodies. A single dose of this vaccine generated a similar type of immune response in aged mice but of a reduced magnitude than in younger mice. We report that a second dose enhances the immune response to this vaccine in aged mice.ConclusionsThis study shows that ChAdOx1 nCoV-19 induces both cellular and humoral immunity in adult and aged mice and suggests a prime-boost strategy is a rational approach to enhance immunogenicity in older persons.FundingThis study was supported by BBSRC, Lister institute of Preventative Medicine, EPSRC VaxHub, and Innovate UK.

T Cells Subsets in the Immunopathology and Treatment of Sjogren's Syndrome.

Sjogren’s syndrome (SS) is an autoimmune disease whose pathogenesis is characterized by an exacerbated T cell infiltration in exocrine glands, markedly associated to the inflammatory and detrimental features as well as the disease progression. Several helper T cell subsets sequentially converge at different stages of the ailment, becoming involved in specific pathologic roles. Initially, their activated phenotype endows them with high migratory properties and increased pro-inflammatory cytokine secretion in target tissues. Later, the accumulation of immunomodulatory T cells-derived factors, such as IL-17, IFN-γ, or IL-21, preserve the inflammatory environment. These effects favor strong B cell activation, instigating an extrafollicular antibody response in ectopic lymphoid structures mediated by T follicular helper cells (Tfh) and leading to disease progression. Additionally, the memory effector phenotype of CD8+ T cells present in SS patients suggests that the presence of auto-antigen restricted CD8+ T cells might trigger time-dependent and specific immune responses. Regarding the protective roles of traditional regulatory T cells (Treg), uncertain evidence shows decrease or invariable numbers of circulating and infiltrating cells. Nevertheless, an emerging Treg subset named follicular regulatory T cells (Tfr) seems to play a critical protective role owing to their deficiency that enhances SS development. In this review, the authors summarize the current knowledge of T cells subsets contribution to the SS immunopathology, focusing on the cellular and biomolecular properties allowing them to infiltrate and to harm target tissues, and that simultaneously make them key therapeutic targets for SS treatment.

The adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRβ clonotypes.

The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE-formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRβ clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines.

The continuing story of T-cell independent antibodies.

The purpose of this article is to review the role of extrafollicular and T-cell independent antibody responses in humoral immunity. We consider two interrelated questions: (a) do T-cell independent antibody responses dominated by IgM and/or IgA play unique functions in immunity and homeostasis; and (b) is it typical for these responses to result in lifelong protection? In addressing these questions, we consider the established advantages of T-cell driven responses including the unique role played by germinal center reactions in these responses, and contrast the processes and outcomes of germinal center-centric responses with germinal center- and T-cell independent antibodies. We suggest that T-independent and other extrafollicular responses contribute substantially to highly stable antibody repertoires in both the serum and the intestine, providing relatively constitutive humoral barriers with the collective dual function of protecting against invading pathogens and regulating the composition of non-pathogenic microbial communities.

Specific IgM and Regulation of Antibody Responses.

Specific IgM, administered together with the antigen it recognizes, enhances primary antibody responses, formation of germinal centers, and priming for secondary antibody responses. The response to all epitopes on the antigen to which IgM binds is usually enhanced. IgM preferentially enhances responses to large antigens such as erythrocytes, malaria parasites, and keyhole limpet hemocyanine. In order for an effect to be seen, antigens must be administered in suboptimal concentrations and in close temporal relationship to the IgM. Enhancement is dependent on the ability of IgM to activate complement, but the lytic pathway is not required. Enhancement does not take place in mice lacking complement receptors 1 and 2 (CR1/2) suggesting that the role of IgM is to generate C3 split products, i.e., the ligands for CR1/2. In mice, these receptors are expressed on follicular dendritic cells (FDCs) and B cells. Optimal IgM-mediated enhancement requires that both cell types express CR1/2, but intermediate enhancement is seen when only FDCs express the receptors and low enhancement when only B cells express them. These observations imply that IgM-mediated enhancement works through several, non-mutually exclusive, pathways. Marginal zone B cells can transport IgM-antigen-complement complexes, bound to CR1/2, from the marginal zone and deposit them onto FDCs. In addition, co-crosslinking of the BCR and the CR2/CD19/CD81 co-receptor complex may enhance signaling to specific B cells, a mechanism likely to be involved in induction of early extrafollicular antibody responses.

Germinal centers and autoimmune disease in humans and mice.

Antibodies are involved in the pathogenesis of many autoimmune diseases. Although the mechanisms underlying the antibody response to infection or vaccination are reasonably well understood, we still have a poor understanding of the nature of autoimmune antibody responses. The most well studied are the anti-nuclear antibody responses characteristic of systemic lupus erythematosus and studies over the past decade or so have demonstrated a critical role for signaling by TLR7 and/or TLR9 in B cells to promote these responses. These Toll-like receptors (TLRs) can promote T-cell-independent extrafollicular antibody responses with a heavy-chain class switch and a low degree of somatic mutation, but they can also strongly boost the germinal center response that gives rise to high-affinity antibodies and long-lived plasma cells. TLRs have been shown to enhance affinity maturation in germinal center responses to produce high-affinity neutralizing antibodies in several virus infection models of mice. Although more data are needed, it appears that anti-nuclear antibodies in mouse models of lupus and in lupus patients can be generated by either pathway, provided there are genetic susceptibility alleles that compromise B-cell tolerance at one or another stage. Limited data in other autoimmune diseases suggest that the germinal center response may be the predominant pathway leading to autoantibodies in those diseases. A better understanding of the mechanisms of autoantibody production may ultimately be helpful in the development of targeted therapeutics for lupus or other autoimmune diseases.

Loss of miR-182 affects B-cell extrafollicular antibody response.

MicroRNAs have been shown to play a role in B-cell differentiation and activation. Here, we found miR-182 to be highly induced in activated B cells. However, mice lacking miR-182 have normal B-cell and T-cell development. Interestingly, mutant mice exhibited a defective antibody response at early time-points in the immunization regimen when challenged with a T-cell-dependent antigen. Germinal centres were formed but the generation of extrafollicular plasma cells was defective in the spleens of immunized miR-182-deficient mice. Mutant mice were also not able to respond to a T-cell-independent type 2 antigen, which typically elicited an extrafollicular B-cell response. Taken together, the data indicated that miR-182 plays a critical role in driving extrafollicular B-cell antibody responses.

Follicular Helper T Cells

Although T cell help for B cells was described several decades ago, it was the identification of CXCR5 expression by B follicular helper T (Tfh) cells and the subsequent discovery of their dependence on BCL6 that led to the recognition of Tfh cells as an independent helper subset and accelerated the pace of discovery. More than 20 transcription factors, together with RNA-binding proteins and microRNAs, control the expression of chemotactic receptors and molecules important for the function and homeostasis of Tfh cells. Tfh cells prime B cells to initiate extrafollicular and germinal center antibody responses and are crucial for affinity maturation and maintenance of humoral memory. In addition to the roles that Tfh cells have in antimicrobial defense, in cancer, and as HIV reservoirs, regulation of these cells is critical to prevent autoimmunity. The realization that follicular T cells are heterogeneous, comprising helper and regulatory subsets, has raised questions regarding a possible division of labor in germinal center B cell selection and elimination.

Natural and vaccine-mediated immunity to Salmonella Typhimurium is impaired by the helminth Nippostrongylus brasiliensis.

BackgroundThe impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined.Methodology/Principal FindingsMice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection.ConclusionThese findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

A forward genetic screen reveals roles for Nfkbid , Zeb1 , and Ruvbl2 in humoral immunity

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble , cellophane , and Worker ascribed to mutations in Nfkbid , Zeb1 , and Ruvbl2 , respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid , is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.

Contribution of Toll‐like receptor signaling to germinal center antibody responses

Toll-like receptors (TLRs) have emerged as one of the most important families of innate immune receptors for initiating inflammation and also for promoting adaptive immune responses. Recent studies have examined the ability of TLRs to promote antibody responses, including T-cell-dependent antibody responses. Initial study suggested that TLR stimulation promotes primarily an extrafollicular antibody response, which rapidly produces moderate affinity antibodies made by short-lived plasma cells. Recent studies, however, have shown that TLRs can also enhance the germinal center response, which produces high affinity class-switched antibody made by long-lived plasma cells. TLR stimulation can increase the magnitude of the latter response and also enhance selection for high affinity IgG. This review summarizes recent advances in understanding the roles of TLRs in B cells and also in other cell types for enhancement of antibody responses, with an emphasis on T-cell-dependent and germinal center antibody responses.

B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells

T follicular helper cells (Tfh cells) localize to follicles where they provide growth and selection signals to mutated germinal center (GC) B cells, thus promoting their differentiation into high affinity long-lived plasma cells and memory B cells. T-dependent B cell differentiation also occurs extrafollicularly, giving rise to unmutated plasma cells that are important for early protection against microbial infections. Bcl-6 expression in T cells has been shown to be essential for the formation of Tfh cells and GC B cells, but little is known about its requirement in physiological extrafollicular antibody responses. We use several mouse models in which extrafollicular plasma cells can be unequivocally distinguished from those of GC origin, combined with antigen-specific T and B cells, to show that the absence of T cell-expressed Bcl-6 significantly reduces T-dependent extrafollicular antibody responses. Bcl-6(+) T cells appear at the T-B border soon after T cell priming and before GC formation, and these cells express low amounts of PD-1. Their appearance precedes that of Bcl-6(+) PD-1(hi) T cells, which are found within the GC. IL-21 acts early to promote both follicular and extrafollicular antibody responses. In conclusion, Bcl-6(+) T cells are necessary at B cell priming to form extrafollicular antibody responses, and these pre-GC Tfh cells can be distinguished phenotypically from GC Tfh cells.

Dendritic cell-B cell interactions in the marginal zone drive extrafollicular antibody responses. (61.4)

Abstract Targeting Ag directly to dendritic cell (DC) subsets through the use of monoclonal antibodies (mAb) is increasingly being employed as a means to generate CD4 and CD8 T cell responses. However, the ability of individual DC subsets to raise primary Ab responses following Ag targeting is not well characterized. Using the hapten 4-hydroxy-3-nitrophenyl (NP) coupled to mAbs that recognize DC-specific DEC205 or DCIR2, we found that MZ-associated DCIR2+ DCs, but not DEC205+ DCs, were able to induce robust T cell- and CD40-dependent anti-NP Ab responses following injection of soluble Ag. Anti-NP Ab responses were restricted to the IgG1 sub-class but failed to either persist over time or undergo affinity maturation, despite the addition of TLR or RLR agonists. Using adoptive transfers with NP-specific B cells, we show that the unique ability of DCIR2+ DCs to induce Ab responses was associated with rapid, MHC class II-independent activation of Ag-specific B cells that occurred specifically upon Ag delivery to DCIR2. Importantly, we found that NP-specific B cells accumulated in MZ-associated bridging channels in close association with CD11c+ cells as early as 24hr following Ag delivery to DCIR2+ DCs. Together, these results reveal a previously unrecognized role for DC-B cell interactions in splenic bridging channels, and provide a novel mechanism for the initiation of T-dependent extrafollicular Ab responses.

IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses

During T cell–dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell–intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell–autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1+ GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21.

Roquin Differentiates the Specialized Functions of Duplicated T Cell Costimulatory Receptor Genes Cd28 and Icos

During evolutionary adaptation in the immune system, host defense is traded off against autoreactivity. Signals through the costimulatory receptor CD28 enable T cells to respond specifically to pathogens, whereas those through the related costimulatory receptor, ICOS, which arose by gene duplication, are critical for affinity maturation and memory antibody responses. ICOS ligand, unlike the pathogen-inducible CD28 ligands, is widely and constitutively expressed in the immune system. Here, we show that crosstalk between these two pathways provides a mechanism for obviating the normal T cell dependence on CD28. Several CD28-mediated responses-generation of follicular helper T cells, germinal center formation, T helper 1 cell-dependent extrafollicular antibody responses to Salmonella and bacterial clearance, and regulatory T cell homeostasis-became independent of CD28 and dependent on ICOS when the E3 ubiquitin ligase Roquin was mutated. Mechanisms to functionally compartmentalize ICOS and CD28 signals are thus critical for two-signal control of normal immune reactions.

B cell clones that sustain long-term plasmablast growth in T-independent extrafollicular antibody responses

Some antigens induce Ab responses without T lymphocyte help. Among these, many polysaccharide-based antigens cause marginal zone B cells to proliferate and differentiate into plasma cells. B1 cells also respond to some of these antigens. In this article, we report that antigen-specific B1b cells, in response to the T-independent antigen (4-hydroxy-3-nitrophenyl)-acetyl (NP)-Ficoll, develop into clones that sustain Ab production for months with continued production of plasma cells and the accumulation of antigen-specific B cells in follicles. The persistence of this T-independent plasmablast response contrasts with the short-term plasmablast growth associated with T-dependent extrafollicular responses. The nature of the cells responding to NP-Ficoll was probed by using chimeras that have B1 cells but lack primary B lymphopoietic capacity and have very few B2 cells or T cells. The chimeras were constructed by transferring 10(5) IgM(+) IgD(-) peritoneal exudate cells into mice unable to produce their own T and B cells because of deficiency in recombinase-activating gene 1 (RAG-1). The chimeras mounted sustained IgM and IgG3 anti-NP Ab responses to NP-Ficoll. This finding was associated with continued NP-specific extrafollicular plasmablast growth and the accumulation of NP-specific B cells in follicles. B cells were not found in the marginal zones of chimeras, and they also lacked recirculating IgD(+) cells and CD3(+) cells. The absence of B2 and T cells confirms that hemopoietic cell chimerism leading to primary lymphopoiesis had not been established.