Cascalho, Marília

Abstract Although T cells and B cells can respond immunologically to some tumors, whether and how they collaborate to control tumor growth is incompletely understood. To address this question we introduced a foreign antigen (HIV envelope), as a model for a tumor antigen, linked to C3d (AgC3d) in a tumor cell line and tested the fate of that line in isogeneic mice. AgC3d-positive tumors invoked antigen-specific antibodies and T cell-mediated IFN gamma secretion. Tumors expressing AgC3d grew more slowly than non-transfected cells and mice with tumors expressing AgC3d survived longer than mice with tumor alone. Slower growth of tumors was due to immunity directed against the viral antigen since AgC3d-positive tumors grew as fast as AgC3d-negative tumors in immune-deficient RAG-2 gammac-deficient hosts. Immunity and control of tumor growth required C3d since tumors expressing the antigen without C3d grew slower than tumors expressing AgC3d. Anti-tumor immunity depended on CR2 since tumors implanted in mice treated with CR2-Ig grew more rapidly than tumors grown in the absence of CR2-Ig. Control of tumors was not the direct result of C3d interaction with B cell CR2 as AgC3d-positive tumors grew slower and in some cases regressed in B cell deficient (JH-/-) mice than in wild type mice. Our findings suggest that C3d enables antigen specific responses by T cells and this response may be regulated by B cells.

Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNF receptor superfamily member 13B ( TNFRSF13B ) variants, which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells’ functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of those with antibody-mediated rejection (AMR) but fewer than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses, we investigated alloimmunity and rejection in mice. Cardiac allografts in Tnfrsf13b- mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b -deficient mice were not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased “natural” IgM and compromised complement regulation, leading to complement deposition in allografted hearts and autogenous kidneys. Thus, WT TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhance morbidity associated with immune responses.

Children with severe heart disease early in life may undergo cardiac transplantation in infancy. Attendant to that procedure, the thymus is removed in whole or part and mature T cells are depleted. These manipulations should cause the T cell repertoire to be severely contracted. Surprisingly, however, those who undergo cardiac transplantation in infancy are not immunodeficient suggesting that the T cell compartment adapts in early life to absence of the thymus and depletion of T cells. Whether the “adapted” T cell compartment provides optimal help for antibody production and development and/or maintenance of B cell memory is not known. To answer that question we studied mice subjected to removal of the thymus and/or T cell depletion. The number and phenotype of T cells in adult thymectomized mice was comparable to controls. Thymectomy did not perturb the development of B cells or T‐independent type 2 antibody responses. In contrast, thymectomy did profoundly impair B cell memory as depicted by decreased production of long‐lived antibody secreting cells in the bone marrow, and decreased antibody affinity maturation. Antibodies produced by thymectomized mice were encoded by heavy chain genes with CDR3 regions longer by 2 or 3 amino‐acids than the sham operated controls. Our findings indicate an unprecedented role for the adult thymus in B cell memory and in the selection of the peripheral B cell repertoire.

TNFRSF13B encodes the transmembrane activator and CAML interactor (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned that the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we investigated how a common human dominant-negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono- or biallelic A144E variants of tnrsf13B , corresponding to A181E, exhibited a striking resistance to pathogenicity and transmission of Citrobacter rodentium , a murine pathogen that models enterohemorrhagic Escherichia coli , and resistance was principally owed to natural IgA deficiency in the intestine. In WT mice with gut IgA and in mutant mice reconstituted with enteric IgA obtained from WT mice, IgA induces LEE expression of encoded virulence genes, which confer pathogenicity and transmission. Taken together, our results show that C . rodentium and most likely other enteric organisms appropriated binding of otherwise protective antibodies to signal induction of the virulence program. Additionally, the high prevalence of TNFRSF13B dominant-negative variants reflects balancing selection.

Abstract Background: Classic reports on lymphocyte development hold that B and T cells develop independently. This concept derives in part from the observation that patients with pure B cell immunodeficiency and hypogammaglobulinemia have a normal thymus and T cell numbers. Our recent findings however challenge this concept. We found that T cell development depends not only on the interaction of T cell precursors with thymic epithelial cells but also on other cells. Here we report that those other cells are B cells. Aims: The purpose of this study was to determine whether B cells drive T cell development and TCR diversification in the thymus. Methods: We compared the number of sub-populations of thymocytes and TCR repertoire diversity in B-cell deficient and B-cell proficient mice and in B cell deficient mice following immunoglobulin (Ig) injections. Total leucocytes numbers were determined with a Coulter counter and numbers of thymocytes sub-populations were calculated by flow cytometry analysis. TCR repertoire diversity was measured by a novel method based on hybridization of TCR Vβ specific cRNA on a gene chip platform. Results: In B-cell deficient mice the number of thymocytes was four times reduced and TCR Vβ chain diversity was up to one million times lower compared with wild type mice. Numbers and diversity were restored by treatment of the mice with gamma globulin (see table). Conclusions: T cell development and diversification is driven by B cells. Mice Number of total thymocytes (mean ± standard deviation) p Value β V TCR diversity of thymocytes (median; min.-max.) p Value C57BL/6 (wild mice) 1.3 x 108 ± 5.1 x 107N=7 4.7 x 106; 1.0 x 105 − 1.1 x 108N=5 JH−/− (B cell immunodeficient mice) 3.1 x 107 ± 1.7 x107N=7 0.002 5.9 x 102; 3.6 x 102 − 1.1 x 103N=5 0.0002 JH−/− treated with Ig 3.9 x 107 ± 1.4 x106N=2 0.20 1.1 x 105; 2.7 x 100 − 7.7 x 105N=4 0.08

Abstract Activation-induced cytidine deaminase (AID) is required for Ig class switch recombination, a process that introduces DNA double-strand breaks in B cells. We show in this study that AID associates with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) promoting cell survival, presumably by resolving DNA double-strand breaks. Wild-type cells expressing AID mutants that fail to associate with DNA-PKcs or cells deficient in DNA-PKcs or 53BP1 expressing wild-type AID accumulate γH2AX foci, indicative of heightened DNA damage response. Thus, AID has two independent functions. AID catalyzes cytidine deamination that originates DNA double-strand breaks needed for recombination, and it promotes DNA damage response and cell survival. Our results thus resolve the paradox of how B cells undergoing DNA cytidine deamination and recombination exhibit heightened survival and suggest a mechanism for hyperIgM type II syndrome associated with AID mutants deficient in DNA-PKcs binding.

Abstract 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 Vβ diversity of thymocytes in mice that lack B cells and Ig is reduced to 6 × 102 from wild-type values of 1.1 × 108; in mice with oligoclonal B cells, the TCR Vβ 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.

Abstract The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin VH17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had ∼2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, VHT/λ2 B cells significantly predominated over VHT/λ1 B cells in MZ-(VHT/λ1:VHT/λ2 ≈ 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of λ2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the VHT/λ2 B cells were further augmented by doubling the VHT gene, but dampened by the dysfunction of Bruton’s tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between VHT/λ1 and VHT/λ2 IgM mAbs revealed a polyreactive nature of the VHT/λ2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

Abstract Cytidine deamination of nucleic acids underlies diversification of Ig genes and inhibition of retroviral infection, and thus, it would appear to be vital to host defense. The host defense properties of cytidine deamination require two distinct but homologous cytidine deaminases—activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G. Although cytidine deamination has clear benefits, it might well have biological costs. Uncontrolled cytidine deamination might generate misfolded polypeptides, dominant-negative proteins, or mutations in tumor suppressor genes, and thus contribute to tumor formation. How cytidine deaminases target a given nucleic acid substrate at specific sequences is not understood, and what protects cells from uncontrolled mutagenesis is not known. In this paper, I shall review the functions and regulation of activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G, and speculate about the basis for site specificity vis-à-vis generalized mutagenesis.

Abstract The quasi-monoclonal mouse has limited B cell diversity, whose major (∼80%) B cell Ag receptors are comprised of the knockin VH 17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/λ1 and VHT/λ2 IgM were equally produced, VHT/λ2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded γ-chains could form λ1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/λ1 anti-pNP IgG may not be due to the incompatibility between the γ-chains and the λ1-chain, but may be explained by the fact that VHT/λ1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/λ2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.