Lisbeth A. Welniak

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

IFN-γ mediates CD4 + T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy

Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (TH1) cytokines such as interferon-γ (IFN-γ). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-γ. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr−/− mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-γ responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.

Expansion of LTC-ICs and Maintenance of p21 and BCL-2 Expression in Cord Blood CD34+/CD38− Early Progenitors Cultured over Human MSCs as a Feeder Layer

Allogeneic transplantation with umbilical cord blood (UCB) is limited in adult recipients by a low CD34+ cell dose. Clinical trials incorporating cytokine‐based UCB in vitro expansion have not demonstrated significant shortening of hematologic recovery despite substantial increases in CD34+ cell dose, suggesting loss of stem cell function. To sustain stem cell function during cytokine‐based in vitro expansion, a feeder layer of human mesenchymal stem cells (MSCs) was incorporated in an attempt to mimic the stem cell niche in the marrow microenvironment. UCB expansion on MSCs resulted in a 7.7‐fold increase in total LTC‐IC output and a 3.8‐fold increase of total early CD34+ progenitors (CD38−/HLA‐DR−). Importantly, early CD34+/CD38−/HLA‐DR− progenitors from cultures expanded on MSCs demonstrated higher cytoplasmic expression of the cell‐cycle inhibitor, p21cip1/waf1, and the antiapoptotic protein, BCL‐2, compared with UCB expanded in cytokines alone, suggesting improved maintenance of stem cell function in the presence of MSCs. Moreover, the presence of MSCs did not elicit UCB lymphocyte activation. Taken together, these results strongly suggest that the addition of MSCs as a feeder layer provides improved conditions for expansion of early UCB CD34+/CD38−/HLA‐DR− hematopoietic progenitors and may serve to inhibit their differentiation and rates of apoptosis during short‐term in vitro expansion.

Synergistic Anti-Tumor Responses After Administration of Agonistic Antibodies to CD40 and IL-2: Coordination of Dendritic and CD8+ Cell Responses

In cancer, the coordinate engagement of professional APC and Ag-specific cell-mediated effector cells may be vital for the induction of effective antitumor responses. We speculated that the enhanced differentiation and function of dendritic cells through CD40 engagement combined with IL-2 administration to stimulate T cell expansion would act coordinately to enhance the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of an agonist Ab to CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent rechallenge in the majority of treated mice. The combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cell and CD8+ T cell number in advanced tumor-bearing mice compared with either agent administered singly. The antitumor effects of anti-CD40 and IL-2 were found to be dependent on CD8+ T cells, IFN-γ, IL-12 p40, and Fas ligand. CD40 stimulation and IL-2 may therefore be of use to promote antitumor responses in advanced metastatic cancer.

Recombinant Human Growth Hormone Promotes Hematopoietic Reconstitution after Syngeneic Bone Marrow Transplantation in Mice

Recombinant human growth hormone (rhGH) was administered to mice after syngeneic bone marrow transplantation (BMT) to determine its effect on hematopoietic reconstitution. BALB/c mice were given 10 μg intraperitoneal injections of rhGH every other day for a total of 10 injections following syngeneic BMT. Mice that received rhGH exhibited significant increases in total hematopoietic progenitor cell content (colony‐forming unit‐culture) in both bone marrow and spleen. Erythroid cell progenitor content (burst‐forming unit‐erythroid) was also significantly increased after rhGH treatment. Analysis of peripheral blood indicated that administration of rhGH resulted in significant increases in the rate of white blood cell and platelet recovery. Granulocyte marker 8C5+ cells were also increased in the bone marrow and spleens of treated mice. Red blood cell, hematocrit, and hemoglobin levels were increased at all time points after rhGH treatment. No significant pathologic effects or weight gain were observed in mice receiving repeated injections of 10 μg rhGH. Thus, rhGH administration after syngeneic BMT promoted multilineage hematopoietic reconstitution and may be of clinical use for accelerating hematopoiesis after autologous BMT.

Opposing roles of interferon-gamma on CD4+ T cell-mediated graft-versus-host disease: Effects of conditioning

Although alloreactive T cells are required for the induction of graft-versus-host disease (GVHD), other factors can influence outcome in murine models of the disease. Lethal total body irradiation (TBI) conditioning regimens followed by reconstitution with allogeneic lymphohematopoietic cells results in the generation of donor anti-host cytotoxic T lymphocyte (CTL)-mediated solid organ (gut, liver, skin) destruction. In contrast, donor anti-host CTL-mediated hematopoietic failure is the primary cause of morbidity following sublethal TBI. To determine the role of interferon (IFN)-gamma in graft-versus-host reactions against hematopoietic and solid organ targets, we used IFN-gamma knockout mice as donors in both lethal TBI and bone marrow transplantation (BMT) rescue and sublethal TBI models. In this report, we show that CD4+ T cells from IFN-gamma knockout (KO) mice resulted in accelerated GVHD after lethal TBI/BMT using a single major histocompatibility class II mismatch model. In marked contrast, the use of these same IFN-gamma KO CD4+ donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. In these recipients, severe anemia, bone marrow aplasia, and intestinal lesions were observed in the presence but not the absence of donor-derived IFN-gamma. Administration of anti-IFN-gamma antibodies to sublethally irradiated recipients of wild-type donor cells confirmed the role of IFN-gamma depletion in CD4+ T cell-mediated GVHD. In conclusion, the extent of conditioning markedly affects the role of IFN-gamma in GVHD lesions mediated by CD4+ T cells. In models using sublethal TBI, the absence of IFN-gamma is protective from GVHD, whereas in lethal TBI situations, the loss is deleterious.

Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.

Neuroendocrine hormones such as growth hormone and prolactin are integral members of the immunological cytokine network

Neuroendocrine hormones such as growth hormone (GH) and prolactin (PRL) have been demonstrated to accelerate the recovery of the immune response after chemotherapy and bone marrow transplantation and to enhance the restoration of immunity in individuals infected with HIV and in normal individuals with compromised immune systems associated with aging. As the mechanism of action of these hormones has been elucidated, it has become clear that they are integral members of the immunological cytokine/chemokine network and share regulatory mechanisms with a wide variety of cytokines and chemokines. The members of this cytokine network induce and can be regulated by members of the suppressor of cytokine signaling (SOCS) family of intracellular proteins. In order to take advantage of the potential beneficial effects of hormones such as GH or PRL, it is essential to take into consideration the overall cytokine network and the regulatory effects of SOCS proteins.

2015 proceedings of the National Heart, Lung, and Blood Institute's State of the Science in Transfusion Medicine symposium

On March 25 and 26, 2015, the National Heart, Lung, and Blood Institute sponsored a meeting on the State of the Science in Transfusion Medicine on the National Institutes of Health (NIH) campus in Bethesda, Maryland, which was attended by a diverse group of 330 registrants. The meetings goal was to identify important research questions that could be answered in the next 5 to 10 years and which would have the potential to transform the clinical practice of transfusion medicine. These questions could be addressed by basic, translational, and/or clinical research studies and were focused on four areas: the three “classical” transfusion products (i.e., red blood cells, platelets, and plasma) and blood donor issues. Before the meeting, four working groups, one for each area, prepared five major questions for discussion along with a list of five to 10 additional questions for consideration. At the meeting itself, all of these questions, and others, were discussed in keynote lectures, small‐group breakout sessions, and large‐group sessions with open discourse involving all meeting attendees. In addition to the final lists of questions, provided herein, the meeting attendees identified multiple overarching, cross‐cutting themes that addressed issues common to all four areas; the latter are also provided. It is anticipated that addressing these scientific priorities, with careful attention to the overarching themes, will inform funding priorities developed by the NIH and provide a solid research platform for transforming the future practice of transfusion medicine.

CTL-Promoting Effects of CD40 Stimulation Outweigh B Cell-Stimulatory Effects Resulting in B Cell Elimination and Disease Improvement in a Murine Model of Lupus

CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA→F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.