During hematopoiesis, stem cell proliferation is dependent on expression of the D-type cyclins. However, little is known about how each cyclin D contributes to the development of specific hematopoietic lineages. Here, analysis of Ccnd1(-/-), Ccnd2(-/-), Ccnd3(-/-) and Ccnd2(-/-)Ccnd3(-/-) mice showed that cyclin D3 was uniquely required for the development of pre-B cells. Transcription of Ccnd3 was dependent on expression of the common gamma-chain. In contrast, expression of the pre-B cell receptor and activation of 'downstream' signaling pathways prevented proteasome-mediated degradation of cyclin D3. Cyclin D3 has a key function in B cell development by integrating cytokine and pre-B cell receptor-dependent signals to expand the pool of pre-B cells that have successfully rearranged immunoglobulin heavy chain.

A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing antibodies (bNAbs). This has proved to be challenging, however. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated “knock-in” mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the accompanying study, 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms including receptor editing, clonal deletion and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1−/− background 4E10HL mice had virtually no serum immunoglobulins of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of an MPER-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly-described bNAb 10E8.

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.

Challenge studies following passive immunization with neutralizing antibodies suggest that an HIV vaccine could be efficacious were it able to elicit broadly neutralizing antibodies (bNAbs4). To better understand the requirements for activation of B cells producing bNAbs, we generated cell lines expressing bNAbs or their germline-reverted versions (gl-bNAbs) as BCRs. We then tested the abilities of the bNAb-expressing cells to recognize HIV pseudovirions and vaccine candidate proteins by binding and activation assays. The results suggest that HIV Env antigen-expressing, infection-competent virions are poorly recognized by high affinity bNAb-expressing cells, as measured by the inability of antigens to induce rapid increases in intracellular calcium levels. Other antigen forms appear to be highly stimulatory: in particular, soluble gp140 trimers and a multimerized, scaffolded epitope protein. Virions failed to efficiently activate bNAb-expressing B cells owing to delayed or inefficient BCR recognition, most likely caused by the low density of Env spikes. Importantly, B cells carrying gl-bNAb BCRs were not stimulated by any of the tested vaccine candidates. These data provide insight into why many HIV immunogens, and natural HIV infections, fail to rapidly stimulate bNAb responses and suggest that bNAb-expressing cell lines might be useful tools in evaluation of vaccine antigens for infectious diseases. As soluble Env trimers or multimerized scaffolded epitopes are best at activating B cell expressing bNAbs, these antigenic forms should be considered as preferred vaccine components, though they should be modified to better target naïve gl-bNAb B cells.

In the majority of HIV-1 infected individuals, the adaptive immune response drives virus escape resulting in persistent viremia and a lack of immune-mediated control. The expression of negative regulatory molecules such as PD-1 during chronic HIV infection provides a useful marker to differentiate functional memory T cell subsets and the frequency of T cells with an exhausted phenotype. In addition, cell-based measurements of virus persistence equate with activation markers and the frequency of CD4 T cells expressing PD-1. High-level expression of PD-1 and its ligands PD-L1 and - L2 are found on hematopoietic and non-hematopoietic cells, which are regulated by chronic antigen stimulation, Type 1 and Type II interferons (IFNs), and homeostatic cytokines. In HIV infected subjects, PD-1 levels on CD4 and CD8 T cells continue to remain high following combination anti-retroviral therapy (cART). System biology approaches have begun to elucidate signal transduction pathways regulated by PD-1 expression in CD4 and CD8 T cell subsets that become dysfunctional through chronic TCR activation and PD-1 signaling. In this review, we summarize our current understanding of transcriptional signatures and signal transduction pathways associated with immune exhaustion with a focus on recent work in our laboratory characterizing the role of PD-1 in T cell dysfunction and HIV pathogenesis. We also highlight the therapeutic potential of blocking PD-1-PD-L1 and other immune checkpoints for activating potent cellular immune responses against chronic viral infections and cancer.

Broadly neutralizing antibodies (bNAbs) against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic (“knock-in”) mice expressing, in the physiological immunoglobulin heavy (H) and light (L) chain loci, two well-studied bNAbs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the accompanying paper. Here, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV Env gp140 trimers and to BCR ligands, but only weakly to HIV pseudovirions. Upon transfer to wild type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, while generating abundant precursors and antibodies with affinity for Env, were unable to rapidly generate neutralizing antibodies, highlighting the importance of developing antigen forms that better focus responses to neutralizing epitopes. b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding non-protective specificities.

To analyze B lymphocyte central tolerance in a polyclonal immune system, mice were engineered to express a superantigen reactive to IgM of allotype b (IgMb). IgMb/b mice carrying superantigen were severely B cell lymphopenic, butsmall numbers of Bcells matured. Their sera contained low levels of IgG andoccasionally high levels of IgA. In bone marrow, immature B cells were normal in number, but internalized IgM and had a unique gene expression profile, compared to thoseexpressing high levels of surface IgM, including elevated recombinase activator gene expression. A comparable B cell population was defined in wild-type bone marrows, with an abundance suggesting that at steady state ∼20% of normal developing B cells are constantly encountering autoantigens in situ. In superantigen-expressing mice, as well as in mice carrying the 3H9 anti-DNA Ig heavy chain transgene, or 3H9 H along with mutation in the murine kappa deleting element RS, IgM internalization was correlated with CD19 downmodulation. CD19low bone marrow cells from 3H9;RS−/− mice were enriched in light chains that promote DNA binding. Our results suggest that central tolerance and attendant light chain receptor editing affect a large fraction of normal developing B cells.IgHa/b mice carrying the superantigen had a ∼50% loss in follicular B cell numbers, suggesting that escape from central tolerance by receptor editing from one IgH allele to another was not a major mechanism. IgMb superantigen hosts reconstituted withexperimental bone marrow were demonstrated to be useful in revealing pathways involved in central tolerance.

The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody-drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments-squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. .