IntroductionGraft-versus-host disease (GVHD) remains an important complication after allogeneic bone marrow transplantation (BMT). Despite broadly reactive pharmacologic agents, GVHD is not uniformly avoided and immunosuppression may cause malignancy recurrence or immunodeficiency. Selective GVHD preventive approaches retaining a graft-versus-leukemia (GVL) effect are needed.Interleukin-21 (IL-21) is produced by CD4 ϩ T cells (especially T helper 17 [Th17]-producing cells) and natural killer T (NKT) cells 1 and signals through the IL-2␥c and IL-21R complex. IL-21R is expressed on hematopoietic and epithelial cells and promotes the activation, differentiation, maturation, or expansion of NK cells, B cells, CD8 ϩ and CD4 ϩ T cells, dendritic cells, and macrophages, resulting in anticancer activity. 2-5 IL-21 facilitates autoimmunity in some [6][7][8] but not all 9,10 experimental models by supporting immunoglobulin production and Th17 cell-mediated pathogenesis.Because IL-21 augments Th17 cell differentiation, indirect evidence for the role of IL-21 in GVHD pathogenesis may be derived from such GVHD studies. Whereas IL-17 and Th17 cells reduce GVHD mediated by CD4 ϩ and CD8 ϩ donor T cells, 11 Th17 cells accelerated GVHD mediated exclusively by CD4 ϩ T cells. 12 Naive CD4 ϩ T cells skewed toward a Th17 phenotype in vitro have been used to demonstrate that Th17 cells contribute to GVHD pathogenesis, especially involving the skin and lung. 13 IL-21 has been described variably as an inhibitor 14 or enhancer 15 of Th1 differentiation. IL-21 supports Th17 cell survival at the expense of regulatory T cells (Tregs), which are reciprocally controlled by Th17 cells. 16 By inhibiting naive T-cell conversion into CD4 ϩ 25 ϩ FoxP3 ϩ regulatory T cells (termed inducible Tregs, iTregs), 17,18 limiting the suppression of T-effectors (Teffs) by Tregs, and augmenting Th17 responses, 19,20 IL-21 may increase GVHD lethality.The present studies were conducted to delineate the influence of IL-21 on GVHD and GVL and to elucidate the mechanisms associated with the observed biologic effects. We show that blocking or abrogating the IL-21 signaling pathway reduced acute GVHD mortality and tissue damage in the small intestine and the colon associated with decreased frequencies of interferon ␥ (IFN␥)-producing tissue-resident donor T cells in the colonic lamina propria (LP). At the same time FoxP3-expressing Tregs, which were virtually absent in the presence of IL-21, were found at relatively high frequencies at the site of inflammation in the colon and the small intestine in the absence of IL-21. These data, which are the first to demonstrate an in vivo role for IL-21 in iTreg generation, suggested a causative role of iTregs in GVHD attenuation. This was confirmed using Teffs incapable of generating iTregs. Despite acute GVHD attenuation, we show that GVL can occur in the absence of IL-21. Lastly, we show that the perforin and IL-21 pathways are nonredundant in the context of both the GVHD and GVL settings. Methods MiceC57BL/6 (H-2 b , term...

Myeloablative conditioning before bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T-cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen-induced TEC damage directly contributes to slow thymopoietic recovery after BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given before conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BM transplant recipients. Therefore, we investigated whether the addition of a pharmacologic inhibitor, PFT-␤, to transiently inhibit p53 during radiotherapy could spare TECs from radiation-induced damage in congenic and allogeneic BMTs. IntroductionAllogeneic bone marrow transplantation (BMT) is used to treat malignant and nonmalignant disorders. 1,2 Chemoradiotherapy conditioning preceding donor graft infusion damages thymic stroma, severely delaying peripheral CD4 ϩ and CD8 ϩ T-cell reconstitution. [2][3][4][5][6] Thus, BM transplant recipients are at increased risk of opportunistic fungal and viral infections. 7 Thymic stroma is composed of a 3-dimensional matrix of thymic epithelial cells (TECs), fibroblasts, macrophages, dendritic cells (DCs), and mesenchymal cells. 8 Critical signals are supplied by TECs to developing thymocytes directing their thymic ingress, 9,10 survival, 11,12 trafficking, 11 selection, 13 and export. 11 Conditioning depletes TECs, impairing T-cell production for prolonged periods of time after BMT. [14][15][16][17] Restoring thymic function would speed peripheral T-cell recovery after BMT.Developing thymocytes can be distinguished by their CD4 and CD8 cell surface expression. CD4 Ϫ CD8 Ϫ (double-negative, DN) thymocytes mature to become CD4 ϩ CD8 ϩ (double-positive, DP) thymocytes and undergo positive selection on cortical TECs (cTECs). DP thymocytes continue their maturation into CD4 ϩ or CD8 ϩ (single-positive, SP) thymocytes and migrate into the thymic medulla where negative selection is mediated by medullary TECs (mTECs) and medullary DCs. 8 SP thymocytes complete their maturation in the medulla and are exported into the periphery as mature T cells. 13 Keratinocyte growth factor (KGF) is a fibroblast growth factor family member that controls cell migration, proliferation, and differentiation in epithelial tissues. 18 Endogenous KGF is upregulated in mucosal tissues after injury, and exogenous KGF enhances protection/repair of epithelial cells in models of chemoradiotherapy-induced injury. 18 KGF is approved by the Food and Drug Administration for prevention of oral mucositis associated with high-dose chemoradiotherapy and hematopoietic stem cell transplantation. [19][20][21] KGF is produced by thymic mesenchymal cells and binds exclusively to FGFR2-IIIb expressed by TECs. 15,22 KGF is a potent mitogen for TECs. 23 KGF pretreatment prevents thymic injury and prolonged T-cell deficiency in murine BMT models. 15,[23][24][25][26][27] KGF facilitates alloe...

Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that IntroductionCytokines regulate survival, proliferation, and differentiation of various immune cells, and those using the ␥ c chain play a major role in lymphocyte homeostasis. Interleukin-2 (IL-2), IL-7, and IL-15 interactions with their receptors are essential for normal B-and T-cell development and peripheral T-cell homeostasis and proliferation. [1][2][3] The major pathways activated on ␥ c receptor activation are Jak/Stat, MAPK, and PI3K. 4 Although these signals lead to changes in cell proliferation, differentiation, and survival in several T-cell lineages, the transcription factor Stat5 is particularly important for CD4 ϩ CD25 ϩ FoxP3 ϩ regulatory T-cell (Treg) development and homeostasis in mice and humans. [5][6][7][8] T-cell specific deficiency in both homologs, Stat5a and Stat5b, resulted in significantly decreased numbers of Tregs, as well as CD8 ϩ , CD4 ϩ , and ␥␦ T-cell numbers in the thymus and in the periphery in mice. 7,9 Conversely, transgenic (TG) expression of a constitutively active form of Stat5b (Stat5b-CA) in T and B cells resulted in an increase in Tregs, CD8 ϩ , CD4 ϩ , ␥␦, and natural killer T-cell numbers. 5 Deletion or overexpression of Stat5 affected CD8 ϩ T cells more than CD4 ϩ T cells, with the exception of Tregs. CD8 ϩ T cells in Stat5b-CA TG mice were increased far more than CD4 ϩ T cells and exhibited a memory phenotype, whereas most CD4 ϩ T cells maintained a naive phenotype. The majority of CD4 ϩ CD25 ϩ T cells from Stat5b-CA TG mice expressed FoxP3 and suppressed wild-type (WT) effector T-cell (Teff) responses in vitro. On T-cell receptor (TCR) stimulation, in vitro proliferation of CD4 ϩ T cells was lower in Stat5b-CA TG versus WT mice, although because Treg-depleted CD4 ϩ T-cell responders were not used, the influence of Stat5b-CA expression on Teffs could not be derived from these studies. Naive CD4 ϩ CD25 Ϫ CD45RB high T cells from Stat5b-CA TG mice showed enhanced homeostatic proliferation when transferred into WT recipients 10 and, in contrast to WT CD4 ϩ CD25 Ϫ CD45RB high T cells, this proliferation was major histocompatibility complex (MHC) class II-and IL-15-independent, suggesting deregulation of homeostatic proliferation by Stat5b-CA in naive CD4 ϩ T cells. 10 Previous studies have not reported the in vivo function of Stat5b-CA TG Tregs. We show that Stat5b-CA TG Tregs suppressed graft-versus-host disease (GVHD) lethality more efficiently than WT Tregs. Surprisingly, Stat5b-CA TG CD4 ϩ CD25 Ϫ Teffs also had a reduced GVHD lethality capacity compared with WT Teffs. Nonetheless, Stat5b-CA TG Teffs retained a graft-versusleukemia (GVL) effect. These results indicate, for the first time, that Stat5 overexpression in CD4 ϩ T cells plays a major role in Treg expansion and potency in vivo. Moreover, we provide the first evidence that Stat5 overexpression in CD4 ϩ T cells regulates Th2 cytokine production in vivo that is associated with a reduction in G...

Chemotherapy or chemoradiotherapy conditioning regimens required for bone marrow transplantation (BMT) cause significant morbidity and mortality as a result of insufficient immune surveillance mechanisms leading to increased risks of infection and tumor recurrence. Such conditioning causes host stromal cell injury, impairing restoration of the central (thymus) and peripheral (spleen and lymph node) T cell compartments and slow immune reconstitution. The chemokine, CCL21, produced by host stromal cells, recruits T- and B-cells that provide lymphotoxin mediated instructive signals to stromal cells for lymphoid organogenesis. Moreover, T- and B-cell recruitment into these sites is required for optimal adaptive immune responses to pathogens and tumor antigens. Previously, we reported that CCL21 was markedly reduced in secondary lymphoid organs of transplanted animals. Here, we utilized adenoviral CCL21 gene transduced dendritic cells (DC/CCL21) given by footpad injections as a novel approach to restore CCL21 expression in secondary lymphoid organs post-transplant. CCL21 expression in secondary lymphoid organs reached levels of naïve controls and resulted in increased T cell trafficking to draining lymph nodes (LNs). An increase in both lymphoid tissue inducer cells and the B cell chemokine CXCL13 known to be important in LN formation was observed. Strikingly, only mice vaccinated with DC/CCL21 loaded with bacterial, viral or tumor antigens and not recipients of DC/control adenovirus loaded cells or no DCs had a marked increase in the systemic clearance of pathogens (bacteria; virus) and leukemia cells. Because DC/CCL21 vaccines have been tested in clinical trials for patients with lung cancer and melanoma, our studies provide the foundation for future trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens.