High-affinity Antibody-secreting Cells Research Articles

Rescuing pathogen-specific memory B-cell from PBMC of prior Zika virus-infected individuals

Immunological memory, a fundamental immune system mechanism, is instrumental in long-term protection. Successful vaccines can elicit and sustain immunological memory against pathogens for the long term. Memory B cells (MBC) are key players in secondary responses due to their longevity and rapid differentiation into high-affinity antibody-secreting cells upon second antigen exposure. However, the availability of circulating MBCs is limited. Here we describe a protocol, which presents a straightforward and practical method for activating and expanding Zika virus (ZIKV) specific MBC. PBMCs collected from individuals who had been infected with ZIKV two years prior were cultured by supplementing with IL-2 and R848, a TLR-7/8 agonist, and then pulsed with inactivated virus. After seven days, this stimulation led to a notable rise in virus-specific functional MBC, as evidenced by a significant increase in the production of anti-ZIKV IgG. Importantly, the ZIKV pulse did not induce changes in the PBMC culture of individuals without a history of ZIKV infection. These findings demonstrate that virus-specific MBC can be expanded in vitro, even using PBMC cultures from individuals infected years before. Therefore, our protocol is a practical and effective tool for studies that require a larger number of human MBCs from previously infected individuals that are functional and specific to the pathogen under investigation.

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation.

The delay in parasite-specific B cell development leaves people in malaria endemic areas vulnerable to repeated Plasmodium infections. Here, we investigated the role of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a molecule involved in the generation of antigen-specific antibody secreting cells, in host response to non-lethal Plasmodium yoelii infection. We found that TACI deficiency not only resulted in higher peak parasitemia levels in P. yoelii challenged mice, but also led to a delay in parasite clearance and anti-P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-119]) protein and anti-rMSP-119 and anti-P. yoelii IgG antibody development. There was also a delay in the generation of splenic high affinity antibody secreting cells that recognize rMSP-119 protein as compared to wild-type mice. Interestingly, coinciding with the delay in parasite clearance there was a delay in the resolution of T follicular helper (TFH) cell and germinal center (GC) B cell responses in TACI -/- mice. The persistence of TFH and GC B cells is likely a result of enhanced interaction between TFH and GC B cells because inducible costimulator ligand (ICOSL) expression was significantly higher on TACI -/- GC B cells than wild-type cells. The difference in the kinetics of GC reaction appeared to also impact the emergence of plasma cells (PC) because there was a delay in the generation of TACI -/- mice PC. Nevertheless, following the recovery from P. yoelii infection, TACI -/- and wild-type mice were both protected from a rechallenge infection. Establishment of protective B cell response was responsible for the resolution of parasitemia because B cells purified from recovered TACI -/- or wild-type mice were equally protective when introduced to naïve wild-type mice prior to P. yoelii challenge. Thus, despite the increased susceptibility of TACI -/- mice to P. yoelii infection and a delay in the development of protective antibody levels, TACI -/- mice are able to clear the infection and resist rechallenge infection.

The LFA1 ligand, CD54, is not required for the differentiation of TFH cells or germinal center formation

Abstract In germinal centers (GC), B cells undergo cellular proliferation and affinity maturation, and ultimately differentiate into either high-affinity antibody-secreting cells (ASCs) or memory B cells. GC B cells present antigen to T follicular helper (TFH) cells in order to elicit survival and proliferation signals. The integrin, Lymphocyte Function-Associated Antigen 1 (LFA1), is required for this process, as mice lacking LFA1 or treated with LFA1 blocking antibodies fail to form GCs or TFH cells. We hypothesized that the counter-receptor for LFA1, ICAM-1 or CD54, would be similarly required for GC formation and TFH differentiation. To test this hypothesis, we infected CD54-deficient (KO) and wild-type (WT) mice with influenza virus (PR8) and enumerated ASCs and GC B cells on days 7, 9, and 14. We found that KO mice had more GC B cells than WT mice on days 9 and 14, but observed no difference in the number of ASCs or TFH cells. To determine if this result was intrinsic to the B cell lineage, we generated 50:50 (KO:WT) bone marrow chimeras. Following reconstitution, we infected the mice with PR8 and assayed donor-specific GC B cells and ASCs on days 9 and 14. On day 9, we found that GC B cells were highly enriched for KO cells, whereas the ASCs were highly enriched for WT cells. In contrast, TFH cells were represented equally in WT and KO populations. On day 14, we found that GC B cells were still highly enriched for KO cells, whereas there was no significant difference between WT and KO ASCs at this time. These results lead us to the surprising conclusion that, although the loss of LFA1 leads to the failure of TFH and GC responses, the loss of CD54 does not affect the TFH compartment and actually improves GC B cell responses.

Transcription Factor STAT3 and Type I Interferons Are Corepressive Insulators for Differentiation of Follicular Helper and T Helper 1 Cells

Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4(+) Tcells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of IFN-inducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper 1 (Th1) cell-like effector phenotype. Conversely, IFN-αβ receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4(+) Tcells. These data suggest mutually repressive roles for STAT3 andtype I IFN signaling pathways in the differentiation of Tfh cells following viral infection.

Characterization of memory B cells responsible for affinity maturation of anti- (4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies

We searched for memory B cells responsible for high-affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody production by C57BL/6 mice immunized with NP-chicken γ-globulin (CGG), using flow cytometry. We first prepared transfectants expressing B-cell antigen receptor (BCR) of known affinity as a memory B-cell model as well as NP-allophycocyanin (APC) of different NP valences, NP(lo), NP(med) and NP(hi). We then used the latter as probes capable of distinguishing BCR affinities: NP(lo)-APC bound to BCRs with an affinity higher than 3.4 × 10(6) M(-1), while NP(med)-APC bound to those with a higher than germline affinity. B cells capable of binding to NP(lo)-APC appeared in spleens on day 14 post-immunization, and harbored Tyr95 (Tyr95 type) as well as a mutation from Trp33 to Leu. B cells with BCRs harboring Gly95 (Gly95 type) appeared only in the NP(med)-APC-binding fraction on day 56 and in the NP(lo)-APC-binding fraction on day 77, indicating that this long duration was necessary for Gly95 type B cells to acquire high affinity and to become a member of the group of memory B cells with high affinity. Administration of NP-CGG on day 77 caused little change in the proportion of the Gly95 type in NP(lo)-APC-binding B cells in the following 2 weeks but brought about an increase in the number of high-affinity antibody-secreting cells (ASC), suggesting that the memory B-cell compartment established was maintained at a later stage and supplied high-affinity ASCs. The relationship between these Gly95 type memory B cells and ASCs is discussed.

Aged B lymphocytes retain their ability to express surface markers but are dysfunctional in their proliferative capability during early activation events

BackgroundAgeing is associated with dysfunction in the humoral response leading to decreased protection against infectious diseases. Defects in T cell function due to age have been well characterized but it is unclear if dysfunctions in antibody responses are due to deficiencies in a helper environment or intrinsic B cell defects. Previous studies from our laboratory have shown that aged B lymphocytes are able to differentiate into high affinity antibody-secreting cells at a frequency similar to their young counterparts. However, expansion of B cells in vivo was reduced in aged animals when compared to young.MethodsTo further investigate the cause of this reduced expansion, we have now examined early activation events of aged B cells in response to anti-CD40 monoclonal antibody (mAb) and lipopolysaccharide (LPS) stimulation in vitro. To do this spleen cells were harvested from young, middle-aged and aged quasi-monoclonal (QM) mice and cultured in complete RPMI for 24 and 48 hours. Cultures contained either LPS or anti-CD40 mAb and murine IL-4. Cells were collected and analyzed using flow cytometry. To examine the proliferative capacity of aged B cells spleen cells were collected as before and cultured in 96 well microtiter plates with either LPS or anti-CD40 mAb and murine IL-4 for 24 hours. Tritiated thymidine ([3H]-Tdr) was added to each well and incubated for another 24 hours after which cells were collected and analyzed using a scintillation counter.ResultsResting aged B cells exhibited similar levels of CD40 expression when compared to young cells and efficiently up-regulated CD86 and CD69 and also down-regulated CD38 upon stimulation. However, aged B cells proliferated less than young B cells and showed a consistent, but not statistically significant, reduction in their ability to form blast cells.ConclusionAged B cells exhibited a reduced response in some early activation events but produced at least a partial response in all cases. Thus, therapeutic intervention may be possible, despite intrinsically different responses in aged B cells.

Intraclonal competition inhibits the formation of high-affinity antibody-secreting cells.

Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when preexisting clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1-8i(+/-) mice, which feature a high frequency of B cells with nitrophenyl (NP)-binding specificity, respond to NP-haptenated proteins with the production of NP-specific Abs, but affinity maturation is impaired due to insufficient generation of high-affinity Ab-producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naive, wild-type recipients. Remarkably, when 10(4) B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 10(6) B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation.

Ontogeny of B-lymphocyte function: XII. Evidence that the ability to generate memory cells precedes the ability to produce antibody-secreting cells

Abstract Using a cell transfer system it was shown that lymphoid cells of B.C-9 mice mature to be able to generate a primary response of high magnitude and high affinity to dinitrophenylated γ-globulin at about 4 weeks of age. Irradiated mice reconstituted with lymphoid cells from B.C-9 or C57BL/6 donors younger than 4 weeks of age fail to produce high-affinity plaque-forming cells and their serum antibody responses were of low magnitude. Nevertheless such recipients give adult-like, high-affinity, high-magnitude secondary responses when assayed at both the plaque-forming cell and serum antibody levels. The antibody produced by the reconstituted mice, in both the primary and secondary responses, was shown to be of donor cell origin in transfers between allotype congenic pairs. Thus, naive, immature lymphoid cells, although not capable of giving rise to high-affinity antibody-secreting cells in a primary response, are capable of giving rise to high-affinity memory B cells which in turn can give an adult-like, high-affinity secondary response following boosting.

Aberrant maturational characteristics of the immune responses of NZB mice to autologous and heterologous erythrocyte antigens

The maturational characteristics of the humoral immune responses of C3H and NZB mice to autologous and heterologous erythrocyte antigens were investigated. Clonal selection of antibody-secreting B lymphocytes was examined at the plaque-forming cell level of analysis of changes in mean antibody affinity and the heterogeneity of binding affinities. The primary immune response of C3H mice to SRBC exhibited a progressive temporal increase in mean relative antibody affinity and a concomitant restriction in the heterogeneity of binding affinities consistent with clonal selection and restriction of B lymphocytes to high affinity antibody-secreting cells. By contrast, the anti-SRBC immune response of NZB mice displayed aberrant maturational characteristics with a progressive decrease in mean relative antibody affinity but also clonal restriction with selection of clones of cells secreting low affinity antibodies. The spontaneous autoimmune responses of NZB mice to autologous erythrocyte surface autoantigens X and HB were different from the response to heterologous erythrocytes in that there was neither a progressive change in mean relative binding affinity nor evidence of progressive clonal restriction. Although the precise mechanisms responsible for the aberrant selection and derepression of B lymphocyte clones in NZB mice have not been identified, the very nature of the aberration suggests the existence of one or more defects which may be intrinsic to the B lymphocytes of NZB mice.