John Treml

The BLyS Family: Toward a Molecular Understanding of B Cell Homeostasis

The B Lymphocyte Stimulator (BLyS) family of ligands and receptors regulates humoral immunity by controlling B lymphocyte survival and differentiation. Herein, we review the ligands and receptors of this family, their biological functions, and the biochemical processes through which they operate. Pre-immune B lymphocytes rely on BLyS signaling for their survival, whereas antigen experienced B lymphocytes generally interact more avidly with a homologous cytokine, A Proliferation Inducing Ligand (APRIL). The molecular basis for signaling via the three BLyS family receptors reveals complex interplay with other B lymphocyte signaling systems, affording the integration of selective and homeostatic processes. As our understanding of this system advances, molecular targets for manipulating humoral immunity in both health and disease should be revealed.

Homeostatic control of B lymphocyte subsets

Lymphocyte homeostasis poses a multi-faceted biological puzzle, because steady pre-immune populations must be maintained at an acceptable steady state to yield effective protection, despite stringent selective events during their generation. In addition, activated, memory and both short- and long-term effectors must be governed by independent homeostatic mechanisms. Finally, advancing age is accompanied by substantial changes that impact the dynamics and behavior of these pools, leading to cumulative homeostatic perturbations and compensation. Our laboratory has focused on the overarching role of BLyS family ligands and receptors in these processes. These studies have led to a conceptual framework within which distinct homeostatic niches are specified by BLyS receptor signatures, which define the BLyS family ligands that can afford survival. The cues for establishing these receptor signatures, as well as the downstream survival mechanisms involved, are integrated with cell extrinsic inputs via cross talk among downstream mediators. A refined understanding of these relationships should yield insight into the selection and maintenance of B cell subsets, as well as an appreciation of how homeostatic mechanisms may contribute to immunosenescence.

Adjuvant properties of listeriolysin O protein in a DNA vaccination strategy

The use of infectious agents as vaccine adjuvants has shown utility in both prophylactic and therapeutic vaccinations. Listeria monocytogenes has been used extensively as a vaccine vehicle due to its ability to initiate both CD4+ and CD8+ immune responses. Previous work from this laboratory has used transgenic Listeria to deliver vaccine constructs. A chimeric protein composed of tumor antigen and a non-hemolytic variant of the Listeria protein, listeriolysin O (LLO), has demonstrated effective tumor protection beyond that of antigen alone expressed in the same system. To address the question of how fusion with LLO improves vaccine efficacy, we constructed a number of DNA plasmid vaccines to isolate this effect in the absence of other endogenous Listeria effects. Here we have analyzed the ability of these vaccines to induce the regression of previously established tumors. A vaccine strategy using DNA vaccines bearing the tumor antigen either alone or in combination with LLO in addition to plasmids encoding MIP-1α and GM-CSF was examined. Further, LLO was used either as a chimera or in a bicistronic construct to address the importance of fusion between these elements. Notably, the strategies employing both chimeric and bicistronic vaccines were effective in reducing tumor burden suggesting that LLO can act as an adjuvant that does not require fusion with the tumor antigen to mediate its effect.

Analysis of the Individual Contributions of Igα (CD79a)- and Igβ (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation

The individual contribution of Igα and Igβ for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igα and Igβ participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igα and Igβ under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igα or Igβ and Igα/Igβ heterodimers with targeted Tyr→Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2−/− primary pro-B cell lines and in gene transfer experiments in the μMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igα/Igβ ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igα and Igβ chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.

Manipulating B cell homeostasis: a key component in the advancement of targeted strategies.

Understanding the homeostatic mechanisms governing lymphocyte pools achieves critical importance as lymphocyte-targeted therapies expand in use and scope. The primacy of B lymphocyte stimulator (BLyS) family ligands and receptors in governing B lymphocyte homeostasis has become increasingly clear in recent years, affording insight into novel opportunities and potential pitfalls for targeted B cell therapeutics. Interclonal competition for BLyS-BR3 interactions determines the size of naíve B cell pools and can regulate the stringency of selection applied as cells complete maturation. Thus one of the predicted consequences of ablative therapies targeting primary pools is relaxed negative selection. This suggests that BLyS levels and B cell reconstitution rates may serve useful prognostic roles and that BLyS itself might be targeted to circumvent relapse. Alternatively, manipulations that allow rare, minimally autoreactive specificities to survive and mature may lead to opportunities in cases where antibody-based vaccine development has heretofore been unsuccessful. BLyS family ligands and receptors also play a role in activated and memory B cell pools, suggesting they might likewise be targeted to promote or delete particular antigen-experienced subpopulations in a similar way.

Direct targeting of genetically modified tumour cells to FcγRI triggers potent tumour cytotoxicity

Expression of the type I receptor for Fc domain of immunoglobulin (Ig)G (FcγRI or CD64) is restricted to myeloid effector cells, such as monocytes, macrophages and a subset of dendritic cells. Previous work has indicated a role for FcγRI in antibody‐dependent phagocytosis and lysis of tumour cells. We hypothesised that tagging of tumour cells with an anti‐FcγRI single chain Fv (sFv) may facilitate targeting to this receptor on effector cells, thereby initiating tumour cytotoxicity. A vector encoding the sFv for an FcγRI‐specific antibody (H22), linked to the transmembrane domain of platelet‐derived growth factor was constructed. Transfected tumour cells expressed high surface levels of functional H22‐sFv, which greatly enhanced susceptibility for phagocytosis and lysis by monocytes and macrophages. The expression of H22‐sFv evoked the ability of tumour cells to directly activate monocytes, as evidenced by phosphorylation of mitogen‐activated protein kinase and secretion of the inflammatory cytokines interleukin (IL)‐1β, tumour necrosis factor‐α and IL‐6. Moreover, growth of tumour cells in mice expressing H22‐sFv was profoundly delayed (or absent) in transgenic mice expressing human FcγRI. These results demonstrated that tumour cells can be readily modified to activate cell effector mechanisms, a strategy that may be useful for in vivo targeting in patients.