Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine

Both SDF-1 and CXCR4 disruption are lethal to mice at the embryonic stage and cause abnormalities in B lymphopoiesis, myelopoiesis, cardiogenesis, vasculogenesis, and cerebellar development. To investigate the role of SDF-1 and CXCR4 in hematopoiesis during the adult stage, mice reconstituted with bone marrow-derived hematopoietic progenitor cells transduced with either the SDF-1 or a genetically modified SDF-1-intrakine gene using a retroviral expression vector were analyzed. Flow cytometric (FCM) analysis showed a dramatic reduction of CXCR4 expression on the cells of intrakine-transduced mice, whereas CCR7 and CCR1 expression was unchanged or marginally decreased on splenocytes. Migration of splenocytes and bone marrow cells to SDF-1 was markedly suppressed in intrakine-transduced mice. FCM analysis of bone marrow cells of intrakine-transduced mice exhibited decreased numbers of pro-B (B220(+) CD43(+)), pre-B (B220(+) CD43(-)), and immature B (B220(+) IgM(+)) cells and a decreased number of granulocytes/myeloid (Gr1(+) CD11b(+)) cells. Impaired B lymphopoiesis and myelopoiesis in intrakine-transduced mice were confirmed by an in vitro colony-forming assay of bone marrow cells. In contrast, B lymphopoiesis and myelopoiesis were enhanced in SDF-1-transduced mice. Interestingly, T-cell maturation in the thymus was impaired both in intrakine- and SDF-1-transduced mice, suggesting that SDF-1 and CXCR4 play an important role in T lymphopoiesis as well as in B lymphopoiesis and myelopoiesis in adults. These results demonstrate an essential role of CXCR4 and its ligand SDF-1 in adult hematopoiesis, and they indicate the intrakine method as a powerful tool for functional analysis of chemokines/chemokine receptors in vivo and as a potential therapeutic approach for acquired immunodeficiency syndrome.     

Tolerance to apoptotic cells is regulated by indoleamine 2,3-dioxygenase

Tolerance to self-antigens present in apoptotic cells is critical to maintain immune-homeostasis and prevent systemic autoimmunity. However, mechanisms that sustain self-tolerance are poorly understood. Here we show that systemic administration of apoptotic cells to mice induced splenic expression of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO expression was confined to the splenic marginal zone and was abrogated by depletion of CD169(+) cells. Pharmacologic inhibition of IDO skewed the immune response to apoptotic cells, resulting in increased proinflammatory cytokine production and increased effector T-cell responses toward apoptotic cell-associated antigens. Presymptomatic lupus-prone MRL(lpr/lpr) mice exhibited abnormal elevated IDO expression in the marginal zone and red pulp and inhibition of IDO markedly accelerated disease progression. Moreover, chronic exposure of IDO-deficient mice to apoptotic cells induced a lupus-like disease with serum autoreactivity to double-stranded DNA associated with renal pathology and increased mortality. Thus, IDO limits innate and adaptive immunity to apoptotic self-antigens and IDO-mediated regulation inhibits inflammatory pathology caused by systemic autoimmune disease.     

B cell homeostasis and plasma cell homing controlled by Krüppel-like factor 2

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress from lymphoid organs by regulating sphingosin-1 phosphate receptor 1 (S1Pr1). Here we show that this is not the case for B cells. Instead, KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of plasma cells to the bone marrow, presumably by controlling the expression of β(7)-integrin. In mice with a B cell-specific deletion of KLF2, S1Pr1 expression on B cells was only slightly affected. Accordingly, all splenic B cell subsets including B1 cells were present, but their numbers were increased with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches harboring fewer B cells were found, and fewer B1 cells in the peritoneal cavity as well as recirculating B cells in the bone marrow were detected. Upon thymus-dependent immunization, IgG titers were diminished, and antigen-specific plasma cells were absent in the bone marrow, although numbers of antigen-specific splenic plasmablasts were normal. KLF2 plays also a role in determining the identity of follicular B cells, as KLF2-deficient follicular B cells showed calcium responses similar to those of MZ B cells and failed to down-regulate MZ B cell signature genes, such as CD21 and CXCR7.     

Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL)

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 oncogene (TCL1) in mouse B cells causes a CD5(+) leukemia similar to aggressive human CLL. To examine the mechanisms by which Tcl1 protein exerts its oncogenic activity in B cells, we performed proteomics experiments to identify its interacting partners. We found that Tcl1 physically interacts with de novo DNA methylthansferases Dnmt3A and Dnmt3B. We further investigated the effects of Tcl1 up-regulation on the enzymatic activity of Dnmt3A and found that Tcl1 overexpression drastically inhibits Dnmt3A function. In addition, B cells from TCL1 transgenic mice showed a significant decrease in DNA methylation compared with WT controls. Similarly, CLL samples with high Tcl1 expression showed a decrease in DNA methylation compared with CLL samples with low Tcl1 expression. Given the previous reports of inactivating mutations of DNMT3A in acute myelogenous leukemia and myelodysplastic syndrome, our results suggest that inhibition of de novo DNA methylation may be a common oncogenic mechanism in leukemogenesis.     

Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive, immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) "memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis, complementarity-determining region 3 (CDR3) spectratyping during a T-independent response, and gene-expression profiling of the different blood and splenic B-cell subsets, we show here that blood IgM(+)IgD(+)CD27(+) cells correspond to circulating splenic marginal zone B cells. Furthermore, analysis of this peripheral subset in healthy children younger than 2 years shows that these B cells develop and mutate their immunoglobulin receptor during ontogeny, prior to their differentiation into T-independent antigen-responsive cells. It is therefore proposed that these IgM(+)IgD(+)CD27(+) B cells provide the splenic marginal zone with a diversified and protective preimmune repertoire in charge of the responses against encapsulated bacteria.     

A splenic marginal zone-like peripheral blood CD27+B220- B cell population is preferentially depleted in HIV type 1-infected individuals

Peripheral blood CD27(+) B cells are reduced in HIV-1-infected individuals. In healthy individuals, the human peripheral blood CD27(+) B cell pool consists of two subsets defined by the expression, or lack thereof, of the CD45 isoform B220. We investigated the presence of circulating B220(+) and B220(-) memory B cells in HIV(+) individuals and found that the reduction in CD27(+) memory B cells occurs primarily among CD27(+)B220(-) B cells. Studies conducted using healthy controls indicate that CD27(+)B220(-) B cells have a splenic marginal zone like the immunophenotype IgM(hi)IgD(lo)CD21(+)CD23(-), express TLR9, and proliferate and secrete IgG and IgM in response to B cell-specific ODN. CD27(+)B220(+) B cells have the immunophenotype IgM(lo)IgD(hi)CD21(+)CD23(+), express activation-induced cytidine deaminase, and proliferate in response to SAC but do not secrete immunoglobulin. The AICD expression, along with CD86 expression, by CD27(+)B220(+) suggests these cells are of germinal center origin. The preferential depletion of CD27(+)B220(-) B cells mirrors alterations in spleen morphology and resident B cell populations due to HIV infection reported by other investigators and may play an important role in the defective B cell immunity against T-independent pathogens such as pneumococcus observed in HIV-1-infected individuals.     

Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL

Epidemiologic data show that the immune system may control or promote the emergence and growth of neoplastic lymphomatous clones. Conversely, systemic lymphomas, especially myeloma and chronic lymphocytic leukemia (CLL), are associated with clinical immunodeficiency. This prospective controlled study demonstrates substantially reduced circulating T helper cells, predominantly naive CD4(+) cells, in patients with nonleukemic follicular lymphoma and extranodal marginal zone lymphoma, but not in monoclonal gammopathy and early CLL. These changes were correlated with a preactivated phenotype, hyperreactivity in vitro, pre-senescence, and a T helper 2 shift of peripheral T helper cells. No prominent alterations existed in the regulatory T-cell compartment. Gene expression profiling of in vitro-stimulated CD4(+) cells revealed an independent second alteration of T helper cell physiology, which was most pronounced in early CLL but also detectable in follicular lymphoma/extranodal marginal zone lymphoma. This pattern consisted of down-regulation of T-cell receptor signaling cascades and globally reduced cytokine secretion. Both types of T-cell dysfunction may contribute to significant immunodeficiency in nonleukemic indolent B-cell lymphomas as demonstrated by unresponsiveness to hepatitis B vaccination. The precise definition of systemic T-cell dysfunction serves as the basis to study its prognostic impact, its relationship to the established influence of the lymphoma microenvironment, and its therapeutic manipulation.     

Hoxb3 deficiency impairs B lymphopoiesis in mouse bone marrow

OBJECTIVE: Hox genes are involved in hematopoietic lineage commitment and differentiation. In this study, we investigated the roles of Hoxb3 in hematopoiesis by examining the phenotypes of a Hoxb3 knockout mutant mouse line. RESULTS: Despite previous reports describing the apparently normal phenotype of these mutant mice, we found that by 6 months of age, Hoxb3(-/-) mice began to exhibit significantly impaired B lymphopoiesis in the bone marrow (BM). The cellularity was reduced by 30% in mutant BM compared to age- and sex-matched heterozygous and wild-type controls. The population size of B220(+)CD43(+) progenitor B cells showed a twofold reduction while that of B220(+)CD43(-)IgM(-) precursor B cells was decreased fivefold. Sorting-purified Hoxb3(-/-) progenitor B cells displayed significantly reduced proliferative response to IL-7 in culture, consistent with our findings of reduced IL-7 receptor expression in Hoxb3(-/-) progenitor B cells. However, the peripheral B cell pool in the spleen of Hoxb3(-/-) mice was maintained with a similar size as in wild-type littermates. CONCLUSION: Analysis of T-cell development in the thymus and B1 cell compartment in the peritoneal cavity showed no significant changes. Thus, our findings suggest that the Hoxb3 gene plays an essential role in regulating B lymphopoiesis in the BM of adult mice.     

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.     

IFN‐γ mediates early B‐cell loss in experimental African trypanosomosis

African trypanosomes infect humans and animals throughout the African continent. These parasites maintain chronic infections by various immune evasion strategies. While antigenic variation of their surface coat is the most studied strategy linked to evading the host humoral response, African trypanosomes also induce impaired B‐cell lymphopoiesis, the destruction of the splenic B‐cell compartment and abrogation of protective memory responses. Here we investigate the mechanism of follicular B‐cell destruction. We show that during infection follicular B cells undergo apoptosis, correlating to enhanced Fas death receptor surface expression. Investigation of various type 1 cytokine knockout mice indicates a crucial role of IFN‐γ in the early onset of FoB cell destruction. Indeed, both IFN‐γ^?/? and IFN‐γR^?/? mice are protected from trypanosomosis‐associated FoB cell depletion, exhibiting an inhibition of B‐cell apoptosis as well as a reduced activation of FoB cells during the first week post‐infection. The data presented herein offer new insights into B‐cell dysfunctioning during experimental African trypanosome infections.     

Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice

We herein tested the effect of B-cell depletion on tolerance induction to factor VIII (FVIII) in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential depletion effects were used. Thus, IgG1 anti-CD20 selectively depleted follicular B cells and spared marginal zone B cells, whereas IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice, a single dose of either IgG1 or IgG2a anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice by subsequent daily, high-dose FVIII intravenous injection as a model for immune tolerance induction. However, the IgG1, but not the IgG2a, anti-CD20 pretreatment led to a significant increase of regulatory T cells in the spleen. Importantly, 3 months after the partial B-cell depletion with IgG1 anti-CD20, the FVIII-specific hyporesponsive state remained. We suggest a tolerogenic role of the remaining marginal zone B cells as a potential mechanism for anti-CD20 therapy.     

Stromal cell-derived factor 1/CXCR4 signaling is critical for early human T-cell development.

The present study investigated the potential role of stromal cell-derived factor 1 (SDF-1) in human intrathymic T-cell differentiation. Results show that SDF-1 is produced by human thymic epithelial cells from the subcapsular and medullary areas, and its receptor, CXCR4, is up-regulated on CD34(+) precursor cells committed to the T-cell lineage. Chimeric human-mouse fetal thymus organ culture (FTOC) seeded with purified CD34(+) thymic progenitors and treated with neutralizing antibodies against SDF-1 or CXCR4 showed a significant reduction of the number of human thymocytes and an arrested thymocyte differentiation in the transition between CD34(+) precursor cells and CD4(+) immature thymocytes. SDF-1-treated FTOC showed an increase of human thymocyte numbers, mainly affecting the most immature subpopulations. Moreover, these results suggest that CXCR4/SDF-1 signaling is not critical for the CD34(+) cell precursor recruitment to the thymus. On the other hand, SDF-1 significantly increased the viability of CD34(+) T-cell precursors modulating the expression of BCL-2 and BAX genes, and stimulated the proliferation of CD34(+) thymic precursor cells, particularly in synergy with interleukin 7 (IL-7), but not with other cytokines, such as stem cell factor or flt3-ligand. Accordingly, only IL-7 was able to up-regulate CXCR4 expression on CD34(+) thymic progenitors. In addition, deprivation of SDF-1 partially inhibited human thymocyte expansion induced by IL-7 in human-mouse FTOC. This study indicates that SDF-1/CXCR4 signaling is required for the survival, expansion, and subsequent differentiation of human early thymocytes and identifies a new mechanism by which IL-7 mediates its effects on human thymopoiesis.     

Crucial role of FLT3 ligand in immune reconstitution after bone marrow transplantation and high-dose chemotherapy

Almost 5 decades after the first clinical transplantations, delayed immune reconstitution remains a considerable hurdle in bone marrow transplantation, and the mechanisms regulating immune reconstitution after transplantation remain to be established. Whereas adult fms-like tyrosine kinase 3 ligand-deficient (FL(-/-)) mice have reduced numbers of early B- and T-cell progenitors, they sustain close to normal levels of mature B and T cells. Herein, we demonstrate that adult bone marrow cells fail to reconstitute B-cell progenitors and conventional B cells in lethally irradiated FL(-/-) recipients, which also display delayed kinetics of T-cell reconstitution. Similarly, FL is essential for B-cell regeneration after chemotherapy-induced myeloablation. In contrast, fetal progenitors reconstitute B lymphopoiesis in FL(-/-) mice, albeit at reduced levels. A critical role of FL in adult B lymphopoiesis is further substantiated by an age-progressive decline in peripheral conventional B cells in FL(-/-) mice, whereas fetally and early postnatally derived B1 and marginal zone B cells are sustained in a FL-independent manner. Thus, FL plays a crucial role in sustaining conventional B lymphopoiesis in adult mice and, as a consequence, our findings implicate a critical role of FL in promoting immune reconstitution after myeloablation and bone marrow transplantation.     

CBFB-MYH11 hinders early T-cell development and induces massive cell death in the thymus

Recent studies suggest that the chromosome 16 inversion, associated with acute myeloid leukemia M4Eo, takes place in hematopoietic stem cells. If this is the case, it is of interest to know the effects of the resulting fusion gene, CBFB-MYH11, on other lineages. Here we studied T-cell development in mice expressing Cbfb-MYH11 and compared them with mice compound-heterozygous for a Cbfb null and a hypomorphic GFP knock-in allele (Cbfb(-/GFP)), which had severe Cbfb deficiency. We found a differentiation block at the DN1 stage of thymocyte development in Cbfb-MYH11 knock-in chimeras. In a conditional knock-in model in which Cbfb-MYH11 expression was activated by Lck-Cre, there was a 10-fold reduction in thymocyte numbers in adult thymus, resulting mainly from impaired survival of CD4+CD8+ thymocytes. Although Cbfb-MYH11 derepressed CD4 expression efficiently in reporter assays, such derepression was less pronounced in vivo. On the other hand, CD4 expression was derepressed and thymocyte development was blocked at DN1 and DN2 stages in E17.5 Cbfb(-/GFP) thymus, with a 20-fold reduction of total thymocyte numbers. Our data suggest that Cbfb-MYH11 suppressed Cbfb in several stages of T-cell development and provide a mechanism for CBFB-MYH11 association with myeloid but not lymphoid leukemia.     

Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells

VAF347 is a low-molecular-weight compound, which activates the aryl hydrocarbon receptor (AhR). Herein, we report that oral administration of a water-soluble derivative of VAF347 (VAG539) promotes long-term graft acceptance and active tolerance in Balb/c mice that receive a transplant of MHC-mismatched pancreatic islet allografts. In vivo VAG539 treatment results in increased frequency of splenic CD4(+) T cells expressing CD25 and Foxp3, markers associated with regulatory T (Tr) cells, and in vitro VAF347 treatment of splenic CD4(+) T cells improved CD4(+)CD25(+)Foxp3(+) T-cell survival. Interestingly, transfer of CD11c(+) dendritic cells (DCs), but not of CD4(+) T or CD19(+) B cells, from VAG539-treated long-term tolerant hosts into mice that recently underwent transplantation resulted in donor (C57Bl/6)-specific graft acceptance and in a significantly higher frequency of splenic CD4(+)CD25(+)Foxp3(+) Tr cells. Furthermore, the transfer of CD4(+)CD25(+) T cells from these mice into mice that recently underwent transplantation promoted graft acceptance. Similarly, cell therapy with in vitro VAF347-treated bone marrow-derived mature DCs prevented islet graft rejection, and reduced OVA-specific T-cell responses in OVA-immunized mice. Collectively, our data indicate that AhR activation induces islet allograft-specific tolerance through direct as well as DC-mediated effects on Tr-cell survival and function.     

WASp verprolin homology, cofilin homology, and acidic region domain-mediated actin polymerization is required for T cell development

All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASpdeltaVCA) in WAS(-/-) mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor alphabeta expression and a consequent failure to generate single-positive CD4(+) and CD8(+) T cells. These latter defects, which are not observed in WAS(-/-) mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization.     

Targeting of antigens to B cells augments antigen-specific T-cell responses and breaks immune tolerance to tumor-associated antigen MUC1

B cells are antibody (Ab)-secreting cells as well as potent antigen (Ag)-presenting cells that prime T-cell activation, which evokes great interest in their use for vaccine development. Here, we targeted ovalbumin (OVA) to B cells via CD19 and found that a single low dose of anti-CD19-OVA conjugates, but not isotype mAb-OVA, stimulated augmented CD4 and CD8 T-cell proliferation and expansion. Administration of TLR9 agonist CpG could significantly enhance long-term T-cell survival. Similar results were obtained when the tumor-associated Ag MUC1 was delivered to B cells. MUC1 transgenic (Tg) mice were previously found to lack effective T-cell help and produce low-titer of anti-MUC1 Abs after vaccination. Targeting MUC1 to B cells elicited high titer of anti-MUC1 Abs with different isotypes, predominantly IgG2a and IgG2b, in MUC1 Tg mice. The isotype switching of anti-MUC1 Ab was CD4 dependent. In addition, IFN-gamma-producing CD8 T cells and in vivo cytolytic activity were significantly increased in these mice. The mice also showed significant resistance to MUC1(+) lymphoma cell challenge both in the prophylactic and therapeutic settings. We conclude that Ags targeting to B cells stimulate CD4 and CD8 T-cell responses as well as Th-dependent humoral immune responses.     

Increased TSLP availability restores T- and B-cell compartments in adult IL-7 deficient mice

Interleukin 7 (IL-7) plays a crucial role in adult lymphopoiesis, while in fetal life its effect can be partially compensated by TSLP. Whether adult hematopoietic progenitor cells are unresponsive to TSLP or whether TSLP is less available in adult microenvironments is still a matter of debate. Here, we show that increased TSLP availability through transgene (Tg) expression fully restored lymphopoiesis in IL-7-deficient mice: it rescued B-cell development, increased thymic and splenic cellularities, and restored double-negative (DN) thymocytes, alphabeta and gammadelta T-cell generation, and all peripheral lymphoid compartments. Analysis of bone marrow chimeras demonstrated that hematopoietic progenitor cells from adult wild-type mice efficiently differentiated toward B- and T-cell lineages in lethally irradiated IL-7 deficient mice provided TSLP Tg was expressed in these mice. In vitro, TSLP promoted the differentiation of uncommitted adult bone marrow progenitors toward B and T lineages and the further differentiation of DN1 and DN2 thymocytes. Altogether, our results show that adult hematopoietic cells are TSLP responsive and that TSLP can sustain long-term adult lymphopoiesis.     

IgG1+ ovalbumin-specific B-cell transnuclear mice show class switch recombination in rare allelically included B cells

We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an IgG1(+) ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1(-/-) mice have exclusively IgG1 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin of nearly all isotypes 3' of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of IgM(+)IgG1(+) double producer B cells that represent <1% of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and IgG3.