Emerging translational strategies and challenges for enhancing regulatory T cell therapy for graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for many types of cancer. Genetic disparities between donor and host can result in immune-mediated attack of host tissues, known as graft versus host disease (GVHD), a major cause of morbidity and mortality following HSCT. Regulatory CD4+ T cells (Tregs) are a rare cell type crucial for immune system homeostasis, limiting the activation and differentiation of effector T cells (Teff) that are self-reactive or stimulated by foreign antigen exposure. Adoptive cell therapy (ACT) with Treg has demonstrated, first in murine models and now in patients, that prophylactic Treg infusion can also suppress GVHD. While clinical trials have demonstrated Treg reduce severe GVHD occurrence, several impediments remain, including Treg variability and practical need for individualized Treg production for each patient. Additionally, there are challenges in the use of in vitro expansion techniques and in achieving in vivo Treg persistence in context of both immune suppressive drugs and in lymphoreplete patients being treated for GVHD. This review will focus on 3 main translational approaches taken to improve the efficacy of tTreg ACT in GVHD prophylaxis and development of treatment options, following HSCT: genetic modification, manipulating TCR and cytokine signaling, and Treg production protocols. In vitro expansion for Treg ACT presents a multitude of approaches for gene modification to improve efficacy, including: antigen specificity, tissue targeting, deletion of negative regulators/exhaustion markers, resistance to immunosuppressive drugs common in GVHD treatment. Such expansion is particularly important in patients without significant lymphopenia that can drive Treg expansion, enabling a favorable Treg:Teff ratio in vivo. Several potential therapeutics have also been identified that enhance tTreg stability or persistence/expansion following ACT that target specific pathways, including: DNA/histone methylation status, TCR/co-stimulation signaling, and IL-2/STAT5 signaling. Finally, this review will discuss improvements in Treg production related to tissue source, Treg subsets, therapeutic approaches to increase Treg suppression and stability during tTreg expansion, and potential for storing large numbers of Treg from a single production run to be used as an off-the-shelf infusion product capable of treating multiple recipients.

Retinoic acid signaling acts as a rheostat to balance Treg function.

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner.

PD-L1 signaling selectively regulates T cell lymphatic transendothelial migration.

Programmed death-1 (PD-1) and its ligand PD-L1 are checkpoint molecules which regulate immune responses. Little is known about their functions in T cell migration and there are contradictory data about their roles in regulatory T cell (Treg) function. Here we show activated Tregs and CD4 effector T cells (Teffs) use PD-1/PD-L1 and CD80/PD-L1, respectively, to regulate transendothelial migration across lymphatic endothelial cells (LECs). Antibody blockade of Treg PD-1, Teff CD80 (the alternative ligand for PD-L1), or LEC PD-L1 impairs Treg or Teff migration in vitro and in vivo. PD-1/PD-L1 signals through PI3K/Akt and ERK to regulate zipper junctional VE-cadherin, and through NFκB-p65 to up-regulate VCAM-1 expression on LECs. CD80/PD-L1 signaling up-regulates VCAM-1 through ERK and NFκB-p65. PD-1 and CD80 blockade reduces tumor egress of PD-1high fragile Tregs and Teffs into draining lymph nodes, respectively, and promotes tumor regression. These data provide roles for PD-L1 in cell migration and immune regulation.

Treg tissue stability depends on lymphotoxin beta-receptor- and adenosine-receptor-driven lymphatic endothelial cell responses.

Regulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αß and LEC LTß receptor (LTßR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTßR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.

Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion.

BACKGROUND AIMS: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. RESULTS: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. DISCUSSION: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.

First-in-human phase 1 trial of induced regulatory T cells for graft-versus-host disease prophylaxis in HLA-matched siblings.

Human CD4+25- T cells cultured in interleukin 2 (IL-2), rapamycin, and transforming growth factor ß (TGFß) along with anti-CD3 monoclonal antibody-loaded artificial antigen-presenting cells generate FoxP3+ induced regulatory T cells (iTregs) with potent suppressive function. We performed a phase 1, single-center, dose-escalation study to determine the safety profile of iTregs in adults with high-risk malignancy treated with reduced-intensity conditioning and mobilized peripheral blood stem cells (PBSCs) from HLA-identical sibling donors. Sixteen patients were enrolled and 14 were treated (2 productions failed to meet desired doses). One patient each received 3.0 × 106/kg, 3.0 × 107/kg, and 3.0 × 108/kg iTregs with corresponding T-conventional-to-iTreg ratios of 86:1, 8:1, and 1:2. After 3 patients received 3.0 × 108/kg in the presence of cyclosporine (CSA) and mycophenolate mofetil (MMF) with no dose-limiting toxicities, subsequent patients were to receive iTregs in the presence of sirolimus/MMF that favors Foxp3 stability based on preclinical modeling. However, 2 of 2 developed grade 3 acute graft-versus-host disease (GVHD), resulting in suspension of the sirolimus/MMF. An additional 7 patients received 3.0 × 108/kg iTregs with CSA/MMF. In the 14 patients treated with iTregs and CSA/MMF, there were no severe infusional toxicities with all achieving neutrophil recovery (median, day 13). Of 10 patients who received 3.0 × 108/kg iTregs and CSA/MMF, 7 had no aGVHD, 2 had grade 2, and 1 had grade 3. Circulating Foxp3+ iTregs were detectable through day 14. In summary, iTregs in the context of CSA/MMF can be delivered safely at doses as high as 3 × 108/kg. This trial was registered at www.clinicaltrials.gov as #NCT01634217.

Repurposing a novel anti-cancer RXR agonist to attenuate murine acute GVHD and maintain graft-versus-leukemia responses.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.

ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

Author Correction: Human in vitro-induced regulatory T cells display Dlgh1 dependent and PKC-θ restrained suppressive activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The lymph node stromal laminin α5 shapes alloimmunity.

Lymph node stromal cells (LNSCs) regulate immunity through constructing lymphocyte niches. LNSC-produced laminin α5 (Lama5) regulates CD4+ T cells but the underlying mechanisms of its functions are poorly understood. Here we show that depleting Lama5 in LNSCs resulted in decreased Lama5 protein in the LN cortical ridge (CR) and around high endothelial venules (HEVs). Lama5 depletion affected LN structure with increased HEVs, upregulated chemokines, and cell adhesion molecules, and led to greater numbers of Tregs in the T cell zone. Mouse and human T cell transendothelial migration and T cell entry into LNs were suppressed by Lama5 through the receptors α6 integrin and α-dystroglycan. During immune responses and allograft transplantation, depleting Lama5 promoted antigen-specific CD4+ T cell entry into the CR through HEVs, suppressed T cell activation, and altered T cell differentiation to suppressive regulatory phenotypes. Enhanced allograft acceptance resulted from depleting Lama5 or blockade of T cell Lama5 receptors. Lama5 and Lama4/Lama5 ratios in allografts were associated with the rejection severity. Overall, our results demonstrated that stromal Lama5 regulated immune responses through altering LN structures and T cell behaviors. This study delineated a stromal Lama5-T cell receptor axis that can be targeted for immune tolerance modulation.

Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration.

Regulatory T cells (Tregs) express high levels of cell surface lymphotoxin alpha beta (LTα1ß2) to activate the LT beta receptor (LTßR) on the lymphatic endothelial cells (LECs), modulating LEC adhesion molecules, intercellular junctions, and chemokines. We demonstrate a role for Tregs through this pathway to condition the permissiveness of lymphatic endothelia for transendothelial migration (TEM), thus gating leukocyte traffic. Human Tregs share the same property with murine Tregs. Activation of TLR2 on Tregs during inflammation specifically augments LTα1ß2-LTßR signaling, which further enhances the permissiveness of LECs to facilitate TEM. The conditioning of endothelia may promote the resolution of inflammation by directing leukocytes out of tissues to lymphatic vessels and draining lymph nodes (dLNs). Thus, Tregs interact with lymphatic endothelia under homeostasis and inflammation and dictate endothelial permissiveness and gating mechanisms for subsequent leukocyte migration through endothelial barriers.

Distinct Regulatory and Effector T Cell Metabolic Demands during Graft-Versus-Host Disease.

Despite graft-versus-host disease (GVHD) prophylactic agents, the success and wider utilization of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by GVHD. Increasing donor graft regulatory T cell (Treg):effector T cell (Teff) ratios can substantially reduce GVHD in cancer patients, but pre-HSCT conditioning regimens and GVHD create a challenging inflammatory environment for Treg stability, persistence, and function. Metabolism plays a crucial role in T cell and Treg differentiation, and development of effector function. Although glycolysis is a main driver of allogeneic T cell-driven GVHD, oxidative phosphorylation is a main driver of Treg suppressor function. This review focuses on recent advances in our understanding of Treg metabolism in the context of GVHD, and discusses potential therapeutic applications of Tregs in the prevention or treatment of GVHD in cancer patients.

Mitochondrial Integrity Regulated by Lipid Metabolism Is a Cell-Intrinsic Checkpoint for Treg Suppressive Function.

Regulatory T cells (Tregs) subdue immune responses. Central to Treg activation are changes in lipid metabolism that support their survival and function. Fatty acid binding proteins (FABPs) are a family of lipid chaperones required to facilitate uptake and intracellular lipid trafficking. One family member, FABP5, is expressed in T cells, but its function remains unclear. We show that in Tregs, genetic or pharmacologic inhibition of FABP5 function causes mitochondrial changes underscored by decreased OXPHOS, impaired lipid metabolism, and loss of cristae structure. FABP5 inhibition in Tregs triggers mtDNA release and consequent cGAS-STING-dependent type I IFN signaling, which induces heightened production of the regulatory cytokine IL-10 and promotes Treg suppressive activity. We find evidence of this pathway, along with correlative mitochondrial changes in tumor infiltrating Tregs, which may underlie enhanced immunosuppression in the tumor microenvironment. Together, our data reveal that FABP5 is a gatekeeper of mitochondrial integrity that modulates Treg function.

Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1-3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

CD4 T cell sphingosine 1-phosphate receptor (S1PR)1 and S1PR4 and endothelial S1PR2 regulate afferent lymphatic migration.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) regulate migration of lymphocytes out of thymus to blood and lymph nodes (LNs) to efferent lymph, whereas their role in other tissue sites is not known. Here, we investigated the question of how these molecules regulate leukocyte migration from tissues through afferent lymphatics to draining LNs (dLNs). S1P, but not other chemokines, selectively enhanced human and murine CD4 T cell migration across lymphatic endothelial cells (LECs). T cell S1PR1 and S1PR4, and LEC S1PR2, were required for migration across LECs and into lymphatic vessels and dLNs. S1PR1 and S1PR4 differentially regulated T cell motility and vascular cell adhesion molecule-1 (VCAM-1) binding. S1PR2 regulated LEC layer structure, permeability, and expression of the junction molecules VE-cadherin, occludin, and zonulin-1 through the ERK pathway. S1PR2 facilitated T cell transcellular migration through VCAM-1 expression and recruitment of T cells to LEC migration sites. These results demonstrated distinct roles for S1PRs in comodulating T cell and LEC functions in migration and suggest previously unknown levels of regulation of leukocytes and endothelial cells during homeostasis and immunity.

Rorc restrains the potency of ST2+ regulatory T cells in ameliorating intestinal graft-versus-host disease.

Soluble stimulation-2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2+ Tregs were thymus derived and predominantly localized to the intestine. ST2-/- Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2-/- versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type 1 and increased IL-4/IL-10-producing type 2 T cells. Cotransfer of ST2+ Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33-stimulated Tregs (TregIL-33) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that TregIL-33 has potential as a cellular therapy avenue for preventing GVHD.

The lineage stability and suppressive program of regulatory T cells require protein O-GlcNAcylation.

Regulatory T (Treg) cells control self-tolerance, inflammatory responses and tissue homeostasis. In mature Treg cells, continued expression of FOXP3 maintains lineage identity, while T cell receptor (TCR) signaling and interleukin-2 (IL-2)/STAT5 activation support the suppressive effector function of Treg cells, but how these regulators synergize to control Treg cell homeostasis and function remains unclear. Here we show that TCR-activated posttranslational modification by O-linked N-Acetylglucosamine (O-GlcNAc) stabilizes FOXP3 and activates STAT5, thus integrating these critical signaling pathways. O-GlcNAc-deficient Treg cells develop normally but display modestly reduced FOXP3 expression, strongly impaired lineage stability and effector function, and ultimately fatal autoimmunity in mice. Moreover, deficiency in protein O-GlcNAcylation attenuates IL-2/STAT5 signaling, while overexpression of a constitutively active form of STAT5 partially ameliorates Treg cell dysfunction and systemic inflammation in O-GlcNAc deficient mice. Collectively, our data demonstrate that protein O-GlcNAcylation is essential for lineage stability and effector function in Treg cells.

Induction of immunosuppressive functions and NF-κB by FLIP in monocytes.

Immunosuppression is a hallmark of tumor progression, and treatments that inhibit or deplete monocytic myeloid-derived suppressive cells could promote anti-tumor immunity. c-FLIP is a central regulator of caspase-8-mediated apoptosis and necroptosis. Here we show that low-dose cytotoxic chemotherapy agents cause apoptosis linked to c-FLIP down-regulation selectively in monocytes. Enforced expression of c-FLIP or viral FLIP rescues monocytes from cytotoxicity and concurrently induces potent immunosuppressive activity, in T cell cultures and in vivo models of tumor progression and immunotherapy. FLIP-transduced human blood monocytes can suppress graft versus host disease. Neither expression of FLIP in granulocytes nor expression of other anti-apoptotic genes in monocytes conferred immunosuppression, suggesting that FLIP effects on immunosuppression are specific to monocytic lineage and distinct from death inhibition. Mechanistically, FLIP controls a broad transcriptional program, partially by NF-κB activation. Therefore, modulation of FLIP in monocytes offers a means to elicit or block immunosuppressive myeloid cells.