Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL

IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.     

Prevention of graft-versus-host disease by anti IL-7Ralpha antibody

Graft-versus-host disease (GVHD) continues to be a serious complication that limits the success of allogeneic bone marrow transplantation (BMT). Using IL-7-deficient murine models, we have previously shown that IL-7 is necessary for the pathogenesis of GVHD. In the present study, we determined whether GVHD could be prevented by antibody-mediated blockade of IL-7 receptor alpha (IL-7Ralpha) signaling. C57/BL6 (H2K(b)) recipient mice were lethally irradiated and underwent cotransplantation with T-cell-depleted (TCD) BM and lymph node (LN) cells from allogeneic BALB/c (H2K(d)) donor mice. Following transplantation, the allogeneic BMT recipients were injected weekly with either anti-IL-7Ralpha antibody (100 mug per mouse per week) or PBS for 4 weeks. Anti-IL-7Ralpha antibody treatment significantly decreased GVHD-related morbidity and mortality compared with placebo (30% to 80%). IL-7Ralpha blockade resulted in the reduction of donor CD4(+) or CD8(+) T cells in the periphery by day 30 after transplantation. Paradoxically, the inhibition of GVHD by anti-IL-7Ralpha antibody treatment resulted in improved long-term thymic and immune function. Blockade of IL-7R by anti-IL-7Ralpha antibody resulted in elimination of alloreactive T cells, prevention of GVHD, and improvement of donor T-cell reconstitution.     

IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin

Regulatory T cells (Tregs) are crucial for the induction and maintenance of self-tolerance and are present in peripheral tissues such as skin and gut under normal, noninflamed conditions. We report isolation and expansion of the Treg population resident in normal human skin. Cutaneous Tregs expressed high levels of CD25, L-selectin, GITR, FOXP3, and intracellular CTLA-4, low levels of CD69, and high levels of the skin-homing addressins CLA, CCR4, and CCR6. Skin Tregs suppressed the proliferation of CD25(lo) T cells from the same skin sample in response to CD3 and CD28 antibodies. Suppression was dependent on cell contact and not affected by neutralizing antibodies to interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Surprisingly, cutaneous Tregs proliferated in an antigen-independent manner when cultured in contact with dermal fibroblasts and IL-15, conditions similar to those found in chronically inflamed skin. We hypothesize that local proliferation of Tregs may occur within inflamed skin and could serve as a brake for cutaneous inflammation as well as a mechanism for the homeostatic proliferation of natural Tregs that has been observed within intact organisms.     

Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells

Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4?CD25?Foxp3? regulatory T-cell (Treg) proliferation and function in vitro. We investigated whether the combination of RAPA plus IL-2 could impact acute GVHD induction after bone marrow transplantation (BMT). RAPA plus IL-2 resulted in improved survival and a reduction in acute GVHD lethality associated with an increased expansion of donor type CD4?Foxp3? Tregs and reduced CD4?CD25? conventional T cells (Tcons). RAPA plus IL-2, but not either drug alone, increased both expansion of donor natural Tregs and conversion of induced Tregs from donor CD25? Tcons while IL-2 alone increased conversion of Tregs from CD25? Tcon. RAPA plus IL-2 treatment resulted in less production of IFN-γ and TNF, cytokines known to be important in the initiation of acute GVHD. These studies indicate that the pharmacologic stimulation of T cells with IL-2 and the suppression of Tcon proliferation with RAPA result in a selective expansion of functional Tregs and suppression of acute GVHD.     

Costimulatory molecule-targeted immunotherapy of cutaneous graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Current therapies for cGVHD reduce symptoms but are not cures. The B10.D2-->Balb/c (H-2(d)) minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD, was used in this study. We demonstrated that a single injection of an agonistic monoclonal antibody (mAb) against CD137, a member of the tumor necrosis factor receptor superfamily, reverses skin fibrosis, ulceration, and alopecia, a dominant feature of cGVHD (cutaneous GVHD), ultimately improving general health conditions. The reversal is associated with markedly reduced CD4(+) T-cell cytokines and increased apoptosis of donor CD4(+) T cells. The Fas pathway is required for ameliorating cutaneous GVHD by anti-CD137 mAb. Taken together, these data indicate that the anti-CD137 mAb has a therapeutic effect on cutaneous GVHD by removing donor CD4(+) T cells that cause cutaneous GVHD. Thus, our study demonstrates an agonistic mAb, specific for a costimulatory molecule, as a possible target for therapeutic intervention in cutaneous GVHD.     

Complement-induced regulatory T cells suppress T-cell responses but allow for dendritic-cell maturation

Concurrent activation of the T-cell receptor (TCR) and complement regulator CD46 on human CD4+ T lymphocytes induces Tr1-like regulatory T cells that suppress through IL-10 secretion bystander T-cell proliferation. Here we show that, despite their IL-10 production, CD46-induced T-regulatory T cells (Tregs) do not suppress the activation/maturation of dendritic cells (DCs). DC maturation by complement/CD46-induced Tregs is mediated through simultaneous secretion of GM-CSF and soluble CD40L, factors favoring DC differentiation and reversing inhibitory effects of IL-10. Thus, CD46-induced Tregs produce a distinct cytokine profile that inhibits T-cell responses but leaves DC activation unimpaired. Such "DC-sparing" Tregs could be desirable at host/environment interfaces such as the gastrointestinal tract where their specific cytokine profile provides a mechanism that ensures unresponsiveness to commensal bacteria while maintaining reactivity to invading pathogens.     

IL-6 blockade inhibits the induction of myelin antigen-specific Th17 cells and Th1 cells in experimental autoimmune encephalomyelitis

The development of Th17 cells is a key event in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). Previous studies have demonstrated that an IL-6-dependent pathway is involved in the differentiation of Th17 cells from naïve CD4-positive T cells in vitro. However, the role of IL-6 in vivo in the development of Th17 cells in EAE has remained unclear. In the present study, we found that IL-6 blockade by treatment with an anti-IL-6 receptor monoclonal antibody (anti-IL-6R mAb) inhibited the development of EAE and inhibited the induction of myelin oligodendrocyte glycoprotein (MOG) peptide-specific CD4-positive, CD8-positive, and Th17 T cells, in inguinal lymph nodes. Thus, the protective effect of IL-6 blockade in EAE is likely to be mediated via the inhibition of the development of MOG-peptide-specific Th17 cells and Th1 cells, which in turn leads to reduced infiltration of T cells into the CNS. These findings indicate that anti-IL-6R mAb treatment might represent a novel therapy for human MS.     

Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.     

Anti-IL6-receptor-alpha (tocilizumab) does not inhibit human monocyte-derived dendritic cell maturation or alloreactive T-cell responses

Significant comorbidites and lethality complicate GVHD and its treatment. Targeting the cytokine milieu may improve GVHD control; and IL6 is an attractive candidate, given its role in dendritic cell activation and T-cell differentiation. Tocilizumab is a humanized mAb to IL6-receptor-α (IL6R-α), which is Food and Drug Administration-approved for treatment of rheumatoid arthritis. Mouse transplant models have demonstrated that IL6 blockade also improves GVHD scores and survival. Definitive immunologic effects of IL6 inhibition have not emerged given inconsistent alterations in regulatory T cells (Tregs) and suppression of T-cell proliferation. Despite on-target suppression of IL6R-α signaling in human monocyte-derived dendritic cells (moDCs) and T cells, our data show no effect on moDC maturation/activation, alloreactive T-cell proliferation, Treg expansion, or allogeneic Th1/Th17 responses in vitro. These findings merit attention in any clinical trials of tocilizumab for GVHD prevention or treatment and provide a rationale for evaluating more specific inhibitors of downstream JAK2/STAT3 signaling as well.     

Cell-autonomous role of TGFβ and IL-2 receptors in CD4(+) and CD8(+) inducible regulatory T-cell generation during GVHD

FoxP3(+) regulatory T cells (Tregs) suppress GVHD while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from the expansion of preexisting natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor Tconvs in the transplantation recipient. Using an MHC-mismatched model of acute GVHD, in the present study we found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Experiments using various combinations of T cells from wild-type and FoxP3-deficient mice suggested that both preexisting donor nTregs and the generation of iTregs in the recipient mice contribute to protection against GVHD. Surprisingly, CD8(+)FoxP3(+) T cells represented approximately 70% of the iTreg pool. These CD8(+)FoxP3(+) T cells shared phenotypic markers with their CD4(+) counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4(+) and CD8(+) Tregs appeared to be protective against GVHD-induced lethality and required IL-2 and TGFβ receptor expression for their generation. These data illustrate the complex makeup of the donor-derived FoxP3(+) Treg pool in allogeneic recipients and their potential role in protection against GVHD.     

Both IL-12p70 and IL-23 are synthesized during active Crohn's disease and are down-regulated by treatment with anti-IL-12 p40 monoclonal antibody

BACKGROUND: Interleukin (IL)-12p70 and IL-23 are key T helper-1 (TH1) cytokines that drive the inflammation seen in numerous models of intestinal inflammation. These molecules contain an identical p40 chain that is bound to a p35 chain in IL-12 and a p19 chain in IL-23, making both potentially susceptible to modulation by an anti-IL-12p40 monoclonal antibody (mAb). METHODS: In the present study, we sought to determine whether active inflammation in Crohn's disease (CD) is associated with the increased synthesis of both of these cytokines and whether patients treated with an anti-IL-12p40 mAb down-regulate IL-23 as well as IL-12p70 as previous reported. RESULTS: To this end we initially determined that IL-12p70 secretion by control and CD antigen-presenting cells (macrophages) in lamina propria mononuclear populations is optimized by stimulation with CD40L and interferon-gamma. In subsequent studies using these stimulation conditions we found that patients with CD manifested both increased IL-12p70 and IL-23 secretion before anti-IL-12p40 mAb treatment and normal levels of secretion of these cytokines following cessation of treatment. Antigen-presenting cells in lamina propria mononuclear cells from ulcerative colitis patients, in contrast, produced only baseline levels of IL-23. Finally, we found that IL-23-induced T cell production of IL-17 and IL-6 are also greatly reduced after antibody treatment. The latter data are parallel to those from previous studies showing that anti-IL-12p40 down-regulates IFN-gamma and tumor necrosis factor-alpha secretion. CONCLUSIONS: We conclude that CD but not ulcerative colitis is associated with high levels of both IL-12p70 and IL-23 secretion as well as the secretion of downstream effector cytokines, and that this cytokine production is down-regulated following administration of IL-12p40 mAb.     

Role of IL-16 in delayed-type hypersensitivity reaction

Interleukin (IL)-16 is a chemoattractant cytokine for CD4(+) leukocytes. Because delayed-type hypersensitivity (DTH) reaction is mediated by T helper 1 (Th1) cells and CD4(+) T cells can be chemoattracted by IL-16, we have investigated the involvement of IL-16 in the DTH reaction. Immunohistochemical analysis revealed the IL-16 expression in infiltrating cells and epithelial cells in the DTH footpads. The IL-16 expression was also detected intracellularly in the infiltrating cells. In addition, markedly increased production of IL-16 was detected in the DTH footpad extracts, but not in the control footpad extracts, by an enzyme-linked immunosorbent assay and also by Western blot analysis. The DTH footpad extracts exhibited a strong chemoattractant activity toward splenic T cells, which was significantly inhibited by the inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in the migration assay. Furthermore, treatment of sensitized mice in vivo with the anti-IL-16 neutralizing mAb significantly suppressed the footpad swelling induced by an antigen challenge, together with decreased infiltration of leukocytes including not only CD4(+) T cells but also CD8(+) T cells and macrophages into the DTH footpads. Decreased production of macrophage inflammatory protein 1alpha was also observed in the DTH footpad extracts by the mAb treatment. These results suggest that IL-16 plays an important role in the recruitment of leukocytes-presumably including antigen-specific Th1 cells, which secrete cytokines and chemokines mediating the following hypersensitivity reaction after activation by the interaction with Langerhans cells carrying the antigen-for the elicitation of DTH response. (Blood. 2000;95:2869-2874)     

Anti-IL-6 antibodies suppress myeloid cell production and the generation of CFU-c in long-term bone marrow cultures.

Interleukin 6 (IL-6) is one of several hemopoietic growth factors produced by stromal cell lines derived from the adherent layer of long-term bone marrow cultures (LTBMCs). To evaluate the potential role of IL-6 in stromal cell-dependent myelopoiesis, we established LTBMCs and verified that IL-6 mRNA is transcribed by heterogeneous adherent cell layers and that IL-6 protein is present in culture supernatants. Established LTBMCs were then depleted of IL-6 by using a specific neutralizing monoclonal antibody (mAb). Cultures treated for 2-3 weeks with anti-IL-6 mAb showed decreased production of maturing myeloid cells and colony-forming progenitor cells (colony-forming units in culture, CFU-c) but not stem cells (spleen colony-forming units, CFU-s). In parallel experiments, it was also found that the addition of IL-6 to LTBMCs stimulated a marked increase in total cell production, CFU-c, and day-8 CFU-s. In sum, it appears that endogenous production of IL-6, although limiting, is essential for the normal level of myelopoiesis associated with stromal cell function in LTBMCs.     

Paradoxical effects of interleukin-18 on the severity of acute graft-versus-host disease mediated by CD4+ and CD8+ T-cell subsets after experimental allogeneic bone marrow transplantation

Administration of exogenous interleukin-18 (IL-18) regulates experimental acute graft-versus-host disease (GVHD) in a Fas-dependent manner when donor CD4(+) T cells are required for mortality after experimental allogeneic bone marrow transplantation (BMT). However, CD4(+) and CD8(+) T cells can induce acute GVHD after clinical allogeneic BMT, and the role of IL-18 in CD8(+)-mediated acute GVHD is unknown. We, therefore, determined the role of IL-18 in GVHD mediated by CD4(+) or CD8(+) T cells across major histocompatibility complex (MHC) class II- and class I-disparate allogeneic BMT, respectively. Administering IL-18 significantly increased survival in CD4(+)-mediated GVHD but reduced survival in CD8(+)-mediated GVHD. This increase in deaths was associated with significantly greater clinical, biochemical, and histopathologic parameters of GVHD damage and was independent of Fas expression on donor T cells. Administering IL-18 significantly enhanced allospecific cytotoxic function and expansion of CD8(+) cells. Endogenous IL-18 was critical to GVHD mediated by CD8(+) donor T cells because IL-18 receptor-deficient donors caused significantly less GVHD but exacerbated CD4(+)-mediated, GVHD-related death. Furthermore, administering anti-IL-18 monoclonal antibody significantly reduced CD8(+)-mediated, GVHD-related death. Together these findings demonstrate that IL-18 has paradoxical effects on CD4(+) and CD8(+) cell-mediated GVHD.     

Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion

OBJECTIVE: Interleukin-17 (IL-17) is a proinflammatory cytokine that is expressed in the synovium of rheumatoid arthritis (RA) patients. This T cell cytokine is implicated in the initiation phase of arthritis. However, the role of IL-17 during the effector phase of arthritis has still not been identified; this was the objective of the present study. METHODS: Mice with collagen-induced arthritis (CIA) were treated with polyclonal rabbit anti-murine IL-17 (anti-IL-17) antibody-positive serum or normal rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and treated with anti-IL-17 antibody or control serum. Arthritis was monitored visually, and joint pathology was examined radiologically and histologically. Systemic IL-6 levels were measured by enzyme-linked immunosorbent assay, and local synovial IL-1 and receptor activator of NF-kappaB ligand (RANKL) expression was analyzed using specific immunohistochemistry. RESULTS: Treatment with a neutralizing anti-IL-17 antibody after the onset of CIA significantly reduced the severity of CIA. Radiographic analysis revealed marked suppression of joint damage in the knee and ankle joints. Histologic analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after anti-IL-17 antibody therapy. Systemic IL-6 levels were significantly reduced after anti-IL-17 antibody treatment. Moreover, fewer IL-1beta-positive and RANKL-positive cells were detected in the synovium after treatment with neutralizing IL-17. Interestingly, initiation of anti-IL-17 antibody therapy during a later stage of CIA, using mice with higher clinical arthritis scores, still significantly slowed the progression of the disease. CONCLUSION: IL-17 plays a role in early stages of arthritis, but also later during disease progression. Systemic IL-6 was reduced and fewer synovial IL-1-positive and RANKL-positive cells were detected after neutralizing endogenous IL-17 treatment, suggesting both IL-1-dependent and IL-1-independent mechanisms of action. Our data strongly indicate that IL-17 neutralization could provide an additional therapeutic strategy for RA, particularly in situations in which elevated IL-17 may attenuate the response to anti-tumor necrosis factor/anti-IL-1 therapy.     

Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance

We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSF prevented GVHD in the B6 --> B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSF (survival, 75% versus 11%, P <.001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent fashion in vitro. T cells from peg-G-CSF-treated IL-10(-/-) donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10(-/-) donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT.     

Inhibition of IL-32 activation by α-1 antitrypsin suppresses alloreactivity and increases survival in an allogeneic murine marrow transplantation model

Interleukin (IL)-32 was originally identified in natural killer cells and IL-2-activated human T lymphocytes. As T cells are activated in allogeneic transplantation, we determined the role of IL-32 in human mixed lymphocyte cultures (MLCs) and GVHD. In allogeneic MLCs, IL-32 increased two-fold in responding T cells, accompanied by five-fold increases of TNFα, IL-6, and IL-8. After allogeneic hematopoietic cell transplantation, IL-32 mRNA levels in blood leukocytes were statistically significantly higher in patients with acute GVHD (n = 10) than in serial samples from patients who did not develop acute GVHD (n = 5; P = .02). No significant changes in IL-32 levels were present in patients with treated (n = 14) or untreated (n = 8) chronic GVHD, compared with healthy controls (n = 8; P = .5, and P = .74, respectively). As IL-32 is activated by proteinase-3 (PR3), we determined the effect of the serine protease inhibitor α-1 antitrypsin (AAT) on IL-32 levels and showed suppression of IL-32 and T-lymphocyte proliferation in MLCs. In an MHC-minor antigen disparate murine transplant model, preconditioning and postconditioning treatment with AAT resulted in attenuation or prevention of GVHD and superior survival compared with albumin-treated controls (80% vs 44%; P = .04). These findings suggest that AAT modulates immune and inflammatory functions and may represent a novel approach to prevent or treat GVHD.     

Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation

The granulocyte colony-stimulating factor (G-CSF) and Flt-3 receptor agonist progenipoietin-1 (ProGP-1) has potent effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to minor and major histocompatibility antigens in the well-described B6 --> B6D2F1 SCT model. ProGP-1, G-CSF, or control diluent was administered to donor B6 mice. ProGP-1 expanded all cell lineages in the spleen, and unseparated splenocytes from these animals produced large amounts of interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) whereas the expression of T-cell adhesion molecules was diminished. Transplantation survival was 0%, 50%, and 90% in recipients of control-, G-CSF-, and ProGP-1-treated allogeneic donor splenocytes, respectively (P <.0001). Donor pretreatment with ProGP-1 allowed a 4-fold escalation in T-cell dose over that possible with G-CSF. Donor CD4 T cells from allogeneic SCT recipients of ProGP-1 splenocytes demonstrated an anergic response to host antigen, and cytokine production (interferon gamma [IFNgamma], IL-4, and IL-10) was also reduced while CD8 T-cell cytotoxicity to host antigens remained intact. Neither CD11c(hi) DCs nor CD11c(dim)/B220(hi) DCs from ProGP-1-treated animals conferred protection from GVHD when added to control spleen. Conversely, when equal numbers of purified T cells from control-, G-CSF-, or ProGP-1-treated allogeneic donors were added to allogeneic T-cell-depleted control spleen, survival at day 60 was 0%, 15%, and 90%, respectively (P <.0001). The improved survival in recipients of ProGP-1 T cells was associated with reductions in systemic tumor necrosis factor alpha generation and GVHD of the gastrointestinal tract. We conclude that donor pretreatment with ProGP-1 is superior to G-CSF for the prevention of GVHD after allogeneic SCT, primarily due to incremental affects on T-cell phenotype and function.