Divino Deoliveira

The Glucose Transporter Glut1 Is Selectively Essential for CD4 T Cell Activation and Effector Function

CD4 T cell activation leads to proliferation and differentiation into effector (Teff) or regulatory (Treg) cells that mediate or control immunity. While each subset prefers distinct glycolytic or oxidative metabolic programs in vitro, requirements and mechanisms that control T cell glucose uptake and metabolism in vivo are uncertain. Despite expression of multiple glucose transporters, Glut1 deficiency selectively impaired metabolism and function of thymocytes and Teff. Resting T cells were normal until activated, when Glut1 deficiency prevented increased glucose uptake and glycolysis, growth, proliferation, and decreased Teff survival and differentiation. Importantly, Glut1 deficiency decreased Teff expansion and the ability to induce inflammatory disease in vivo. Treg cells, in contrast, were enriched in vivo and appeared functionally unaffected and able to suppress Teff, irrespective of Glut1 expression. These data show a selective in vivo requirement for Glut1 in metabolic reprogramming of CD4 T cell activation and Teff expansion and survival.

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.

Growth Hormone Mitigates against Lethal Irradiation and Enhances Hematologic and Immune Recovery in Mice and Nonhuman Primates

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 µg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

CD62L− memory T cells enhance T-cell regeneration after allogeneic stem cell transplantation by eliminating host resistance in mice

A major challenge in allogeneic hematopoietic cell transplantation is how to transfer T-cell immunity without causing graft-versus-host disease (GVHD). Effector memory T cells (CD62L(-)) are a cell subset that can potentially address this challenge because they do not induce GVHD. Here, we investigated how CD62L(-) T cells contributed to phenotypic and functional T-cell reconstitution after transplantation. On transfer into allogeneic recipients, CD62L(-) T cells were activated and expressed multiple cytokines and cytotoxic molecules. CD62L(-) T cells were able to deplete host radioresistant T cells and facilitate hematopoietic engraftment, resulting in enhanced de novo T-cell regeneration. Enhanced functional immune reconstitution was demonstrated in CD62L(-) T-cell recipients using a tumor and an influenza virus challenge model. Even though CD62L(-) T cells are able to respond to alloantigens and deplete host radioresistant immune cells in GVHD recipients, alloreactive CD62L(-) T cells lost the reactivity over time and were eventually tolerant to alloantigens as a result of prolonged antigen exposure, suggesting a mechanism by which CD62L(-) T cells were able to eliminate host resistance without causing GVHD. These data further highlight the unique characteristics of CD62L(-) T cells and their potential applications in clinical hematopoietic cell transplantation.

Novel mechanism of rapamycin in GVHD: increase in interstitial regulatory T cells

Rapamycin (RAPA) is an immunosuppressive drug that prevents and treats graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplant (HCT). One possible mechanism for its efficacy is induction of tolerance, through increased number or enhanced survival of regulatory T cells. In our experiments, B10.D2 BM and splenocytes were injected into lethally irradiated BALB/cJ recipients. The mice received i.p. injections of either RAPA or vehicle control on days 1–28. There was a significant survival advantage in RAPA-treated mice. Evaluation of the skin biopsies showed a dense cellular infiltrate in RAPA-treated mice. Further characterization of these cells revealed a higher percentage of regulatory T cells characterized by FoxP3-positive cells in high-dose RAPA-treated mice as compared with controls on day 30. This effect appears to be dose dependent. When peripheral blood analysis for FoxP3-positive cells was performed, there was no significant difference observed in the RAPA-treated mice as compared with control mice. These data show a novel mechanism of rapamycin in GVHD, accumulation of regulatory T cells in the GVHD target tissue: the skin.

An ear punch model for studying the effect of radiation on wound healing

Purpose: Radiation and wound combined injury represents a major clinical challenge because of the synergistic interactions that lead to higher morbidity and mortality than either insult would produce singly. The purpose of this study was to develop a mouse ear punch model to study the physiological mechanisms underlying radiation effects on healing wounds. Materials and methods: Surgical wounds were induced by a 2 mm surgical punch in the ear pinnae of MRL/MpJ mice. Photographs of the wounds were taken and the sizes of the ear punch wounds were quantified by image analysis. Local radiation to the ear was delivered by orthovoltage X-ray irradiator using a specially constructed jig that shields the other parts of body. Results: Using this model, we demonstrated that local radiation to the wound area significantly delayed the healing of ear punch wounds in a dose-dependent fashion. The addition of sublethal whole body irradiation (7 Gy) further delayed the healing of ear punch wounds. These results were replicated in C57BL/6 mice; however, wound healing in MRL/MpJ mice was accelerated. Conclusions: These data indicate that the mouse ear punch model is a valuable model to study radiation and wound combined injury.

Long-Term In Vivo Imaging of Multiple Organs at the Single Cell Level

Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D) plus time) information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.