The magnitude of germinal center reactions is restricted by a fixed number of preexisting niches.

Antibody affinity maturation occurs in the germinal center (GC), a highly dynamic structure that arises upon antigen stimulation and recedes after infection is resolved. While the magnitude of the GC reaction is highly fluctuating and depends on antigens or pathological conditions, it is unclear whether GCs are assembled ad hoc in different locations or in preexisting niches within B cell follicles. We show that follicular dendritic cells (FDCs), the essential cellular components of the GC architecture, form a predetermined number of clusters. The total number of FDC clusters is the same on several different genetic backgrounds and is not altered by immunization or inflammatory conditions. In unimmunized and germ-free mice, a few FDC clusters contain GC B cells; in contrast, immunization or autoimmune milieu significantly increases the frequency of FDC clusters occupied by GC B cells. Excessive occupancy of GC niches by GC B cells after repeated immunizations or in autoimmune conditions suppresses subsequent antibody responses to new antigens. These data indicate that the magnitude of the GC reaction is restricted by a fixed number of permissive GC niches containing preassembled FDC clusters. This finding may help in the future design of vaccination strategies and in the modulation of antibody-mediated autoimmunity.

HMGB1 Mediates Anemia of Inflammation in Murine Sepsis Survivors.

Patients surviving sepsis develop anemia, but the molecular mechanism is unknown. Here we observed that mice surviving polymicrobial gram-negative sepsis develop hypochromic, microcytic anemia with reticulocytosis. The bone marrow of sepsis survivors accumulates polychromatophilic and orthochromatic erythroblasts. Compensatory extramedullary erythropoiesis in the spleen is defective during terminal differentiation. Circulating tumor necrosis factor (TNF) and interleukin (IL)-6 are elevated for 5 d after the onset of sepsis, and serum high-mobility group box 1 (HMGB1) levels are increased from d 7 until at least d 28. Administration of recombinant HMGB1 to healthy mice mediates anemia with extramedullary erythropoiesis and significantly elevated reticulocyte counts. Moreover, administration of anti-HMGB1 monoclonal antibodies after sepsis significantly ameliorates the development of anemia (hematocrit 48.5 ± 9.0% versus 37.4 ± 6.1%, p < 0.01; hemoglobin 14.0 ± 1.7 versus 11.7 ± 1.2 g/dL, p < 0.01). Together, these results indicate that HMGB1 mediates anemia by interfering with erythropoiesis, suggesting a potential therapeutic strategy for anemia in sepsis.

Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.

The goal of cancer immunotherapy is the generation of an effective, stable, and self-renewing antitumor T-cell population. One such approach involves the use of high-affinity cancer-specific T-cell receptors in gene-therapy protocols. Here, we present the generation of functional tumor-specific human T cells in vivo from genetically modified human hematopoietic stem cells (hHSC) using a human/mouse chimera model. Transduced hHSC expressing an HLA-A*0201-restricted melanoma-specific T-cell receptor were introduced into humanized mice, resulting in the generation of a sizeable melanoma-specific naïve CD8(+) T-cell population. Following tumor challenge, these transgenic CD8(+) T cells, in the absence of additional manipulation, limited and cleared human melanoma tumors in vivo. Furthermore, the genetically enhanced T cells underwent proper thymic selection, because we did not observe any responses against non-HLA-matched tumors, and no killing of any kind occurred in the absence of a human thymus. Finally, the transduced hHSC established long-term bone marrow engraftment. These studies present a potential therapeutic approach and an important tool to understand better and to optimize the human immune response to melanoma and, potentially, to other types of cancer.

Single mutations in HIV integrase confer high-level resistance to raltegravir in primary human macrophages.

CD4(+) T cells and macrophages are the primary target cells for HIV in vivo, and antiretroviral drugs can vary in their ability to inhibit the infection of these different cell types. Resistance pathways to the HIV integrase inhibitor raltegravir have previously been investigated in T cells. Primary raltegravir resistance mutations, most often at integrase amino acid position 148 or 155, afford some resistance to the drug. The acquisition of pathway-specific secondary mutations then provides higher-level resistance to viruses infecting T cells. We show here that during macrophage infection, the presence of a single primary raltegravir resistance mutation (Q148H, Q148R, N155H, or N155S) is sufficient to provide resistance to raltegravir comparable to that seen in viruses expressing both primary and secondary mutations in costimulated CD4(+) T cells. These data implicate macrophages as a potential in vivo reservoir that may facilitate the development of resistance to raltegravir. Notably, the newer integrase inhibitor MK-2048 effectively suppressed the infection of all raltegravir-resistant viruses in both T cells and macrophages, indicating that more recently developed integrase inhibitors are capable of inhibiting infection in both major HIV cellular reservoirs, even in patients harboring raltegravir-resistant viruses.

Simulated microgravity impairs leukemic cell survival through altering VEGFR-2/VEGF-A signaling pathway.

Motile cells capable of undergoing transendothelial migration, such as hematopoietic and leukemic cells, have been shown to sense and respond to a decrease in their surrounding gravity. In this study, we investigated the effects of microgravity on human leukemic cell proliferation and expression of receptors that control cell survival, such as the tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2). VEGFR-2 is shuttled between the nucleus and membrane, and through an autocrine activation of its ligand, VEGF-A, conveys signals that control cell survival. Autocrine or paracrine stimulation of VEGFR-2 facilitates localization of this receptor from the membrane to the nucleus--a process that results in increased survival of the leukemic cells. Here, we provide evidence that the mechanical forces altered by simulated microgravity localize and maintain VEGFR-2 in the membrane, and also block VEGF-A expression. This interferes with the shuttling of VEGFR-2 to the nucleus, resulting in a decrease in signaling and enhanced leukemic cell death. These data suggest that microgravity modulates cell survival through altering the cellular trafficking and activation state of tyrosine kinase receptors. This study has potential implications for understanding the regulation of receptor biology in pathophysiology, particularly VEGFR trafficking, thereby providing for the development of appropriate therapeutic strategies to abrogate intracrine stimulation triggered by VEGFR internalization.