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The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.

Studies in mice have shown that proinflammatory Th17 cells can cause acute graft-versus-host disease (aGVHD) related tissue damage; however, whether they play a role in human aGVHD remains unclear. In a prospective study, we measured the proportion of Th17 cells in the blood and skin of patients at the onset of aGVHD. We found no difference in the proportion or amount of IL-17 produced by T cells in the blood of patients with aGVHD (n = 20) compared with time-matched patients without GVHD (n = 14). Moreover, Th17 cells were not increased in the skin of patients with cutaneous aGVHD (n = 7) compared with healthy controls (n = 10). In contrast, we found significantly more interferon-γ–producing T cells in the skin of patients with aGVHD compared with controls. These data support the long-standing paradigm that tissue localized interferon-γ–producing cells are the perpetrators of aGVHD.

Forkhead box P3 (FOXP3) is constitutively expressed by CD4+CD25hi regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)–FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4+T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Rα (CD25). We investigated whether altered T-cell–receptor (TCR)–mediated signaling in pure populations of ex vivo human CD4+CD25+ Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR–mediated activation and lack of cytokine production. CD4+CD25+ Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+ Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+ Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+ Treg cells.

The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3-binding subunit (alpha(IL-3)) and a larger beta chain-beta(c), or, in the mouse, beta(c) or its close relative beta(IL-3). There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of alpha(IL-3) and beta(IL-3), but is, rather, the formation of a beta-beta homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from alpha(IL-3), beta(c) or beta(IL-3), and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of alpha(IL-3) and beta(IL-3). Homodimers of either alpha(IL-3) or beta(IL-3), alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of alpha(IL-3) and beta(IL-3).

This report shows that interleukin-4 (IL-4), which plays a key role in regulating immune responses, fails to support cellular growth. We investigated whether this failure of IL-4 to promote growth was because of its unique inability to activate the Ras/Raf/Erk pathway. Consistent with other reports, expression in Ba/F3, a factor-dependent hematopoietic cell line, of either activated Q61KN-Ras or a hormone-inducible activated Raf-1, resulted in suppression of apoptosis but not in long-term growth. However, in the presence of IL-4, Ba/F3 cells that expressed either Q61KN-Ras or activated Raf-1 grew continuously at a rate comparable with that stimulated by IL-3. Investigation of the biochemical events associated with the stimulation of long-term growth showed that, as expected, the presence of activated Raf-1 resulted in an increased activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) but not of c-jun N-terminal kinase/stress-activated protein kinase (JNK). However, surprisingly, if IL-4 was present, cells expressing active Raf-1 exhibited increases in JNK activity. These observations point to a novel mechanism for JNK activation involving synergy between Raf-1 and pathways activated by IL-4 and suggest that in hematopoietic cells proliferation is correlated not only with “mitogen activated” ERK activity, but also with JNK activity.

Several recent reports have suggested that in vitro exposure of CD4+ T cells to rabbit antithymocyte globulin (rATG), which is commonly used to prevent and treat graft-versus-host disease and allograft rejection, is an effective method to induce CD4+CD25+FOXP3+ T regulatory cells (Tregs). We and others, however, have shown that FOXP3 is also expressed in activated T cells. We therefore investigated whether the induction of FOXP3 expression by rATG resulted in a stable population of suppressive Tregs. We found that exposure of peripheral blood mononuclear cells (PBMCs) or conventional T cells to rATG resulted in induction of transient rather than stable expression of CD25 and FOXP3. Furthermore, rATG-treated T effector cells acquired neither an immunosuppressive profile of cytokine production nor suppressive capacity, even at the time of maximal FOXP3 expression. These findings indicate that the notion that rATG can be used to induce Tregs in vitro for cellular therapy in vivo should be re-evaluated.

T regulatory cells (Tregs) play a pivotal role in promoting and maintaining tolerance. Several subsets of Tregs have been identified but, to date, the best characterized are the CD4+FOXP3+ Tregs (FOXP3+Tregs), thymic-derived or induced in the periphery, and the CD4+ IL-10-producing T regulatory type 1 (Tr1) cells. In the past decade much effort has been dedicated to develop methods for the in vitro induction and expansion of FOXP3+Tregs and of Tr1 cells for Treg-based cell therapy to promote and restore tolerance in T-cell mediated diseases, and for expanding antigen (Ag)-specific Tregs in vivo. FOXP3+Tregs constitutively express high levels of CD25 and of the transcription factor FOXP3. FOXP3+Tregs are distinguished from activated CD4+ T cells by the low expression of CD127, and by the DNA demethylation of a specific region of the FOXP3 gene called Treg-specific demethylated region (TSDR). FOXP3+Tregs suppress effector T-cell responses through cell-to-cell contact-dependent mechanisms and suppression requires activation via TCR and is IL-2 dependent. In vitro protocols to expand FOXP3+Tregs for adoptive transfer in vivo have been established. We demonstrated that rapamycin permits the in vitro expansion of FOXP3+Tregs while impairing the proliferation of non-Tregs. Moreover, rapamycin-expanded FOXP3+Tregs maintain their regulatory phenotype in a proinflammatory environment and Th17 cells do not expand in the presence of rapamycin. Despite the progress in FOXP3+Tregs expansion protocols, adoptive transfer of FOXP3+Tregs in humans remains a difficult experimental procedure due to the ability to expand a sufficient number of Ag-specific FOXP3+Tregs in vitro. To propagate a homogenous population of FOXP3+Tregs we developed a lentiviral vector (LV)-based strategy to ectopically express FOXP3 in CD4+ T cells. This method results in the development of suppressive cells that are super-imposable to FOXP3+Tregs. Conversion of effector T cells into FOXP3+Tregs upon LV-mediated gene transfer of wild-type FOXP3 was also obtained in CD4+T cells from immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. We also developed a LV platform, which selectively targets expression of the transgene in hepatocytes, to induce tolerance to self or exogenous Ags. Using this approach we showed that systemic administration of LV encoding for the gene of interest leads to the induction of Ag-specific FOXP3+ Tregs, which mediate tolerance even in pre-immunized hosts. Tr1 cells are identified by their cytokine profile (IL-10+TGF-b+IL-4-IL-17-). Tr1 cells express transiently FOXP3 upon activation; but FOXP3 expression never reaches the high levels characteristic of FOXP3+Tregs. Tr1 cell differentiation and function is independent of FOXP3 since suppressive Tr1 cells can be isolated or generated from peripheral blood of IPEX patients. Tr1 cells were first discovered in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant (HSCT). Since their discovery, Tr1 cells have proven to be important in mediating tolerance in several immune-mediated diseases. The immuno-regulatory mechanisms of Tr1 cells have been studied over the years thanks to the possibility to generate these cells in vitro. Tr1 cells suppress T-cell responses via the secretion of IL-10 and TGF-β and by the specific killing of myeloid APC via Granzyme B and perforin. Tr1 cells can be induced in vitro in an Ag-specific manner in the presence of IL-10 or of DC-10. Proof-of-principle clinical trials in allogeneic HSCT demonstrated the safety of Treg-based cell therapy with these polarized Tr1 cells. We are currently planning a phase I/II trial using in vitro polarized Tr1 cells with DC-10 in patients after kidney transplantation. An alternative strategy for the induction of high numbers of human Tr1 cells is the LV-mediated gene transfer of human IL-10 into conventional CD4+ T cells. Stable ectopic expression of IL-10 leads to the differentiation of homogeneous populations of Tr1-like cells displaying potent suppressive functions both in vitro and in vivo. A major hurdle, which limited the studies and the clinical use of Tr1 cells, was the lack of specific biomarkers. By gene expression profiling of human Tr1 cell clones we identified two surface markers (CD49b and LAG-3), which are stably and selectively co-expressed on murine and human Tr1 cells induced in vitro or in vivo. The co-expression of CD49b and LAG-3 enables the isolation of highly suppressive Tr1 cells from in vitro IL-10-polarized Tr1 cells and allows tracking of Tr1 cells in peripheral blood of patients who developed tolerance after allogeneic HSCT. The identification of CD49b and LAG-3 as Tr1-specific biomarkers will facilitate the study of Tr1 cells in vivo in healthy and pathological conditions and the use of Tr1 cells for forthcoming therapeutic interventions. In conclusion, Tregs play a key role in maintaining immunological homeostasis in the periphery. Several open questions regarding FOXP3+ Tregs or Tr1 cell-based therapy in humans remain: how long do Tregs survive after transfer? Is their phenotype stable in pathological conditions and inflammatory environments? Is their mechanism of suppression in vivo Ag-specific? Carefully designed and standardized future clinical protocols reflecting a concerted action among different investigators will help to address these questions and to advance the field. Disclosures: No relevant conflicts of interest to declare.

Abstract 1944 Background: The insidious onset and heterogeneous nature of chronic GVHD (cGVHD) impedes its diagnosis and treatment. Donor and graft characteristics, including those which lead to variations in specific cell populations infused at the time of transplantation, may predict the onset of cGVHD. Methods: This study evaluated graft lymphocyte populations of donors enrolled on the prospective Phase III Canadian BMT Group (CBMTG) clinical trial “A Randomized Multicentre Study Comparing G-CSF Mobilized Peripheral Blood and G-CSF Stimulated Bone Marrow in Patients Undergoing Matched Sibling Transplantation for Hematologic Malignancies (CBMTG 0601)”. Proportions of specific lymphocytes in donor grafts were associated with cGVHD in the recipient using logistic regression. Associations were significant if the corresponding p-value was less than 0.05 and the difference between the medians of the two groups was 50% lower than or twice as high as the control. Analyses were performed on G-CSF peripheral blood and bone marrow as a single population due to a data lock on the donor source randomization pending longer follow up. Recipients were considered cGVHD positive if diagnosed with extensive cGVHD and cGVHD negative if free of extensive cGVHD for a minimum of 12 months post transplant. Relapsed recipients and those who died without previous diagnosis of GVHD were excluded from analyses. Cell phenotypes and cytokine production of lymphocytes in the donor grafts were analyzed by flow cytometry in a set of panels that enabled identification of distinct NK, T and B cell subsets. After an initial analysis of 40 samples, a subset of immune populations, which significantly associated with recipient outcome, were analysed in an additional 20 samples. Results: Analyses of cGVHD- vs. cGVHD+ showed the following associations: higher proportions of IFNg-producing T helper cells and CD56bright NK cells in donor grafts were associated with a lack of cGVHD (p Conclusions: We have identified an IFNg-producing CD4+ T cell, an NK cell, and an immature B cell population in donor grafts that impact the development of cGVHD in the recipient. CD56bright NK cells are characteristically weakly cytotoxic but efficient producers of IFNg. The protective function of IFNg is supported by previous results from our lab showing that PBMCs of transplant recipients who did not develop cGVHD have higher levels of IFNg mRNA after stimulation (Rozmus et al, 2011). Donor IFNg polymorphisms, as well as graft source (bone marrow vs. peripheral blood), may cause differences in the immune cell proportions of donor grafts. The discovery that immature B cells are associated only with de novo cGVHD lends support to the concept that cGVHD following acute disease may differ biologically from de novo cGVHD. To better understand the role of these cells in disease pathology, more detailed functional assays of these lymphocyte subsets are required. All values are expressed as medians (first – third percentile range). *acute GVHD present, chronic GVHD absent (A+C-) recipients were excluded from the multivariate analyses due to low numbers (n=2). The values are expressed as percentages of the following lymphocytes: CD56bright NK cells; total CD3−, IFNg-producing T cells; total CD3+ and immature B cells; total CD19+. Disclosures: No relevant conflicts of interest to declare.

Acute graft versus host disease (aGVHD) following haematopoietic stem cell transplantation (HCT) occurs when donor T cells infused with the graft recognise and react to histo-incompatible recipient antigens causing tissue damage. Historically, the inflammatory response in aGVHD was attributed to alloreactive CD4+ T helper and CD8+ cytotoxic T cells and alterations in cytokine production. Recently, a new CD4+ T cell subset, characterised by IL-17 production has been identified. TH17 cells produce high levels of proinflammatory cytokines, including IL-17A, IL-17F, and IL-22, and have been implicated in solid organ rejection and more recently a number of murine studies suggest that Th17 cells play a role in the development of aGVHD. It is well known that FOXP3+ regulatory T cells (Tregs) are critical for the maintenance of self-tolerance, and control the immune response to alloantigens. Murine studies have shown that adoptive transfer of these cells can prevent acute GVHD whereas selective depletion leads to an increased severity. In humans, Tregs also appear to control acute GVHD as they occur at a lower frequency in the peripheral blood patients with aGVHD compared to patients without GVHD. These findings have led to active interest into the use of these cells to prevent or decrease GVHD following allogeneic HCT. It has been reported that in vitro, Th17 cells are resistant to Treg cell mediated suppression of proliferation and IL-17 production, suggesting that the effector functions of Th17 cells might not be susceptible to Treg-cell-mediated inhibition. If true, this would suggest that Treg-based therapies might not be effective at limiting Th17-cell-mediated tissue damage. However, there is currently no evidence regarding whether Treg cells affect the phenotype or function of Th17 cells in tissues. Understanding the interactions between suppressive Tregs and pro-inflammatory T effectors in tissues that are targets of aGVHD, such as the skin, is critical to better define the potential of Tregs as adoptive therapy for the prevention or treatment of aGVHD. In order to address this question, we developed two methods to generate human Th17 cells, one based on over-expression of RORC2 and the other on sorting CCR4+CCR6+CD4+ T cells. We found that ectopic expression of RORC induces a cytokine and chemokine receptor profile analogous to in vivo differentiated Th17 cells. Although expression of RORC2 made CD4+ T cells resistant to Treg-cell mediated suppression of proliferation and IL-17 production, production of IFN-g, TNF-a and IL-6 could be suppressed in these Th17-like cells. In order to further delineate the functional consequence of the interaction between Treg and Th17 cells in tissues we developed a modified the human skin explant model that involves culture of 4 mm punch biopsies of skin with ex vivo Th17 cells (CCR4+CCR6+CD4+ T cells), RORC2 transduced CD4+ T cells, or controls, in the presence or absence of Treg and grading the graft-versus-host reactivity (grades I–IV) histopathologically. Preliminary data suggest that Th17 cells cause significant tissue destruction in this skin explant model, and experiments are ongoing to determine whether Treg cells can counteract these effects.

Human CD4 + CD25 + regulatory T cells (T R ) have the ability to suppress T cell responses and have recently been shown to play a critical role in peripheral tolerance and regulation of immune responses. CD4 + CD25 + T R are anergic, phenotypically CD25 +bright and express high levels of the transcription factor FOXP3. Peripheral blood T R are rare and must be highly enriched for their suppressor function to be detected in vitro . This, combined with the lack of a unique marker that distinguishes them from activated T cells, makes it difficult to study T R function and evaluate their therapeutic potential. Current methods for isolating T R are cumbersome and time-consuming, and generally require three steps: Ficoll™ density centrifugation to isolate mononuclear cells; subsequent immunomagnetic T cell enrichment; and finally FACS sorting. The objective of this study was to develop a more simple technique to isolate highly purified T R from whole blood without using FACS sorting. Three steps were reduced to two by replacing the Ficoll™ separation and immunomagnetic T cell enrichment with a single antibody-mediated buoyant density centrifugation (RosetteSep ® ) to enrich CD4 T cells directly from whole blood. A cocktail of bi-specific antibodies was used to selectively bind unwanted cells to red cells causing them to pellet when centrifuged over Ficoll™. Purified CD4 T cells were recovered at the plasma-Ficoll™ interface and then separated using EasySep ® column-free magnetic separation to select CD25 expressing cells. The EasySep ® separation conditions were optimized for maximal CD4 + CD25 +bright T cell purity and recovery. The purified cells were analyzed for CD25, GITR, CD62L, HLA-DR and CTLA-4 expression. FOXP3 mRNA levels in purified CD4 + CD25 + T cell fractions were compared to corresponding CD4 + CD25 neg T cell fractions using quantitative PCR. As T R are anergic and therefore not responsive to TCR stimulation, the purified CD4 + CD25 + T cell fractions were assessed for anergy in response to anti-CD3/CD28 coated beads. Suppression activity of purified CD4 + CD25 + T cells was assessed by measuring their ability to reduce the proliferative response of CD4 + CD25 neg T cells to CD3/CD28 beads. T cell proliferation was quantified by measuring dilution of the fluorescent dye CFSE with flow cytometry. Purified CD4 + CD25 + T cell suspensions were 94 ± 3% (n=6) CD4 + CD25 + , 83 ± 7% (n=6) CD4 + CD25 +bright , 89 ± 2% (n=4) GITR + and CD62L + (92%, 95%; n=2). The purified cell suspensions were also highly enriched for CTLA-4 and HLA-DR expressing cells. FOXP3 mRNA levels in the purified CD4 + CD25 + T cell fractions from two donors were found to be 64 and 124 times higher than for the corresponding CD4 + CD25 neg T cell fractions. The purified CD4 + CD25 + T cells displayed low or undetectable proliferative responses (n=4) to CD3/CD28 beads suggesting most cells in the purified fractions were anergic. Mixing purified CD4 + CD25 + T cells with CD4 + CD25 neg T cells at a ratio of 1:1 almost completely eliminated detectable CD4 + CD25 neg T cell proliferation (95 ± 3 % reduction, n=3), and suppression of proliferation continued to be detected when cells were mixed at a ratio of 1:10 (25 ± 6% reduction, n=3). In conclusion, combining RosetteSep ® CD4 T cell enrichment with EasySep ® CD25 positive selection yields highly pure CD4 + CD25 +bright / FOXP3 + T R in less time and with fewer steps than current isolation methods.