Minnelide suppresses GVHD and enhances survival while maintaining GVT responses.

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

Regulatory T Cell Amelioration of Graft-versus-Host Disease following Allogeneic/Xenogeneic Hematopoietic Stem Cell Transplantation Using Mobilized Mouse and Human Peripheral Blood Donors.

The present studies examined experimental transplant outcomes using mobilized peripheral blood from mice and humans together with FoxP3+Treg cells. Donor mice were treated with filgrastim and / or plerixafor and their peripheral blood (PB) displayed significant elevations in hematopoietic stem and progenitor populations. Some of these PB donors were concurrently administered a Treg expansion strategy consisting of a TL1A-Ig fusion protein low dose rIL-2. A significant increase (4-5x) in the frequency Tregs occurred during mobilization. C3H.SW PB was collected from mobilized and Treg unexpanded ("TrUM") or mobilized and Treg expanded ("TrEM") donors and transplanted into MHC-matched B6 (H2b) recipients. Recipients of TrEM, exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients against leukemia levels. Next, huTregs (CD4+CD25+CD127lo) from a healthy human PB mobilized donor were expanded ex-vivo prior to transplant into NSG/ NOD-scid IL2Rgammanull mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of lethality and clinical xGVHD scores. Notably, post-transplant, PB huTregs levels remained elevated and the frequency of huCD4+Tconv and CD8+ cells was diminished supporting the improved xGVHD outcomes. These findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced GVHD following "MUD" and MHC-mismatched mouse HSCT without loss of GVL activity. Moreover, utilizing ex-vivo expanded huTregs from a mobilized PB donor and added back to donor PB ameliorated xGVHD. In total, these studies support the notion that in vivo or ex-vivo manipulation of donor Tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes.

Recipient Tregs: Can They Be Exploited for Successful Hematopoietic Stem Cell Transplant Outcomes?

Human and mouse CD4+FoxP3+ T cells (Tregs) comprise non-redundant regulatory compartments which maintain self-tolerance and have been found to be of potential therapeutic usefulness in autoimmune disorders and transplants including allogeneic hematopoietic stem cell transplantation (allo-HSCT). There is substantial literature interrogating the application of donor derived Tregs for the prevention of graft versus host disease (GVHD). This Mini-Review will focus on the recipient's Tregs which persist post-transplant. Although treatment in patients with low dose IL-2 months post-HSCT are encouraging, manipulating Tregs in recipients early post-transplant is challenging, in part likely an indirect consequence of damage to the microenvironment required to support Treg expansion of which little is understood. This review will discuss the potential for manipulating recipient Tregs in vivo prior to and after HSCT (fusion proteins, mAbs). Strategies that would circumvent donor/recipient peripheral blood harvest, cell culture and ex-vivo Treg expansion will be considered for the translational application of Tregs to improve HSCT outcomes.

Improved NK Cell Recovery Following Use of PTCy or Treg Expanded Donors in Experimental MHC-Matched Allogeneic HSCT.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by graft- versus-host disease (GVHD), which causes immune dysfunction and further delays immune reconstitution through its effects on primary and secondary lymphoid organs. Treatments to prevent GVHD and improve immune recovery following allo-HSCT are needed. Post-transplantation cyclophosphamide (PTCy) is a well-established and clinically widely used method for GVHD prophylaxis after HLA-matched as well as haploidentical allo-HSCT, as well as a promising strategy in the setting of mismatched unrelated donor allo-HSCT. Recently, regulatory T cells (Tregs), a critical subset for immune homeostasis and tolerance induction, have been evaluated for use as GVHD prophylaxis in experimental models and clinical trials. Natural killer (NK) cells are one of the first lymphoid populations to reconstitute following allo-HSCT and are important mediators of protective immunity against pathogens, and are also critical for limiting post-transplantation relapse of hematologic cancers. Several reports have noted that a delay in NK cell recovery may occur following experimental mouse allo-HSCT as well as after clinical allo-HSCT. Here we examined how 2 treatment strategies, PTCy and donor expanded Tregs (TrED), in experimental MHC-matched allo-HSCT affect NK recovery. Our experiments show that both strategies improved NK cell numbers, with PTCy slightly better than TrED, early after allo-HSCT (1 month) compared with untreated allo-HSCT recipients. Importantly, NK cell IFN-γ production and cytotoxic function, as reflected by CD107 expression as well as in vivo killing of NK-sensitive tumor cells, were improved using either PTCy or TrED versus control allo-HSCT recipients. In conclusion, both prophylactic treatments were found to be beneficial for NK recovery and NK cell function following MHC-matched minor antigen-mismatched experimental allo-HSCT. Improved NK recovery could help provide early immunity toward tumors and pathogens in these transplant recipients.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after ("post-transplant," PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular "immune privilege." PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

STING differentially regulates experimental GVHD mediated by CD8 versus CD4 T cell subsets.

The stimulator of interferon genes (STING) pathway has been proposed as a key regulator of gastrointestinal homeostasis and inflammatory responses. Although STING reportedly protects against gut barrier damage and graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched allogeneic hematopoietic stem cell transplantation (aHSCT), its effect in clinically relevant MHC-matched aHSCT is unknown. Studies here demonstrate that STING signaling in nonhematopoietic cells promoted MHC-matched aHSCT-induced GVHD and that STING agonists increased type I interferon and MHC I expression in nonhematopoietic mouse intestinal organoid cultures. Moreover, mice expressing a human STING allele containing three single-nucleotide polymorphisms associated with decreased STING activity also developed reduced MHC-matched GVHD, demonstrating STING's potential clinical importance. STING-/- recipients experienced reduced GVHD with transplant of purified donor CD8+ T cells in both MHC-matched and MHC-mismatched models, reconciling the seemingly disparate results. Further examination revealed that STING deficiency reduced the activation of donor CD8+ T cells early after transplant and promoted recipient MHC class II+ antigen-presenting cell (APC) survival. Therefore, APC persistence in STING pathway absence may account for the increased GVHD mediated by CD4+ T cells in completely mismatched recipients. In total, our findings have important implications for regulating clinical GVHD by targeting STING early after aHSCT and demonstrate that an innate immune pathway has opposing effects on the outcome of aHSCT, depending on the donor/recipient MHC disparity.

The promise of CD4+FoxP3+ regulatory T-cell manipulation in vivo: applications for allogeneic hematopoietic stem cell transplantation.

CD4+FoxP3+ regulatory T cells (Tregs) are a non-redundant population critical for the maintenance of self-tolerance. Over the past decade, the use of these cells for therapeutic purposes in transplantation and autoimmune disease has emerged based on their capacity to inhibit immune activation. Basic science discoveries have led to identifying key receptors on Tregs that can regulate their proliferation and function. Notably, the understanding that IL-2 signaling is crucial for Treg homeostasis promoted the hypothesis that in vivo IL-2 treatment could provide a strategy to control the compartment. The use of low-dose IL-2 in vivo was shown to selectively expand Tregs versus other immune cells. Interestingly, a number of other Treg cell surface proteins, including CD28, CD45, IL-33R and TNFRSF members, have been identified which can also induce activation and proliferation of this population. Pre-clinical studies have exploited these observations to prevent and treat mice developing autoimmune diseases and graft-versus-host disease post-allogeneic hematopoietic stem cell transplantation. These findings support the development of translational strategies to expand Tregs in patients. Excitingly, the use of low-dose IL-2 for patients suffering from graft-versus-host disease and autoimmune disease has demonstrated increased Treg levels together with beneficial outcomes. To date, promising pre-clinical and clinical studies have directly targeted Tregs and clearly established the ability to increase their levels and augment their function in vivo Here we review the evolving field of in vivo Treg manipulation and its application to allogeneic hematopoietic stem cell transplantation.

Superior immune reconstitution using Treg-expanded donor cells versus PTCy treatment in preclinical HSCT models.

Posttransplant cyclophosphamide (PTCy) has been found to be effective in ameliorating acute graft-versus-host disease (GVHD) in patients following allogeneic hematopoietic stem cell transplantation (aHSCT). Adoptive transfer of high numbers of donor Tregs in experimental aHSCT has shown promise as a therapeutic modality for GVHD regulation. We recently described a strategy for in vivo Treg expansion targeting two receptors: TNFRSF25 and CD25. To date, there have been no direct comparisons between the use of PTCy and Tregs regarding outcome and immune reconstitution within identical groups of transplanted mice. Here, we assessed these two strategies and found both decreased clinical GVHD and improved survival long term. However, recipients transplanted with Treg-expanded donor cells (TrED) exhibited less weight loss early after HSCT. Additionally, TrED recipients demonstrated less thymic damage, significantly more recent thymic emigrants, and more rapid lymphoid engraftment. Three months after HSCT, PTCy-treated and TrED recipients showed tolerance to F1 skin allografts and comparable immune function. Overall, TrED was found superior to PTCy with regard to weight loss early after transplant and initial lymphoid engraftment. Based on these findings, we speculate that morbidity and mortality after transplant could be diminished following TrED transplant into aHSCT recipients, and, therefore, that TrED could provide a promising clinical strategy for GVHD prophylaxis.

Very Low Numbers of CD4+ FoxP3+ Tregs Expanded in Donors via TL1A-Ig and Low-Dose IL-2 Exhibit a Distinct Activation/Functional Profile and Suppress GVHD in a Preclinical Model.

Regulatory T cells (Tregs) are essential for the maintenance of tolerance and immune homeostasis. In allogeneic hematopoietic stem cell transplantation (aHSCT), transfer of appropriate Treg numbers is a promising therapy for the prevention of graft-versus-host disease (GVHD). We have recently reported a novel approach that induces the marked expansion and selective activation of Tregs in vivo by targeting tumor necrosis factor receptor superfamily 25 (TNFRSF25) and CD25. A potential advance to promote clinical application of Tregs to ameliorate GVHD and other disorders would be the generation of more potent Treg populations. Here we wanted to determine if very low doses of Tregs generated using the "2-pathway" stimulation protocol via TL1A-Ig fusion protein and low-dose IL-2 (targeting TNFRSF25 and CD25, respectively) could be used to regulate preclinical GVHD. Analysis of such 2-pathway expanded Tregs identified higher levels of activation and functional molecules (CD103, ICOS-1, Nrp-1, CD39, CD73, il-10, and tgfb1) versus unexpanded Tregs. Additionally, in vitro assessment of 2-pathway stimulated Tregs indicated enhanced suppressor activity. Notably, transplant of extremely low numbers of these Tregs (1:6 expanded Tregs/conventional T cells) suppressed GVHD after an MHC-mismatched aHSCT. Overall, these results demonstrate that 2-pathway stimulated CD4+ FoxP3+ Tregs were quantitatively and qualitatively more functionally effective than unexpanded Tregs. In total, the findings in this study support the notion that such 2-pathway stimulated Tregs may be useful for prevention of GVHD and ultimately promote more widespread application of aHSCT in the clinic.

BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT.

A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-ß. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6 → BALB/c) was performed using in vivo expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial strategy can suppress pre-clinical GVHD and posit, in vivo EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses.

Marked in Vivo Donor Regulatory T Cell Expansion via Interleukin-2 and TL1A-Ig Stimulation Ameliorates Graft-versus-Host Disease but Preserves Graft-versus-Leukemia in Recipients after Hematopoietic Stem Cell Transplantation.

Regulatory T cells (Tregs) are critical for self-tolerance. Although adoptive transfer of expanded Tregs limits graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), ex vivo generation of large numbers of functional Tregs remains difficult. Here, we demonstrate that in vivo targeting of the TNF superfamily receptor TNFRSF25 using the TL1A-Ig fusion protein, along with IL-2, resulted in transient but massive Treg expansion in donor mice, which peaked within days and was nontoxic. Tregs increased in multiple compartments, including blood, lymph nodes, spleen, and colon (GVHD target tissue). Tregs did not expand in bone marrow, a critical site for graft-versus-malignancy responses. Adoptive transfer of in vivo-expanded Tregs in the setting of MHC-mismatched or MHC-matched allogeneic HSCT significantly ameliorated GVHD. Critically, transplantation of Treg-expanded donor cells facilitated transplant tolerance without GVHD, with complete sparing of graft-versus-malignancy. This approach may prove valuable as a therapeutic strategy promoting transplantation tolerance.

Cloning, expression, and functional characterization of TL1A-Ig.

TNF superfamily member 15 (TL1A) is the ligand for TNFR superfamily (TNFRSF)25. We previously reported that TNFRSF25 stimulation with an agonist Ab, 4C12, expands pre-existing CD4(+)Foxp3(+) regulatory T cells (Tregs) in vivo. To determine how the physiological ligand differs from the Ab, we generated a soluble mouse TL1A-Ig fusion protein that forms a dimer of TL1A trimers in solution with an apparent molecular mass of 516 kDa. In vitro, TL1A-Ig mediated rapid proliferation of Foxp3(+) Tregs and a population of CD4(+)Foxp3(-) conventional T cells. TL1A-Ig also blocked de novo biogenesis of inducible Tregs and it attenuated the suppressive function of Tregs. TNFRSF25 stimulation by TL1A-Ig in vivo induced expansion of Tregs such that they increased to 30-35% of all CD4(+) T cells in the peripheral blood within 5 d of treatment. Treg proliferation in vivo was dependent on TCR engagement with MHC class II. Elevated Treg levels can be maintained for at least 20 d with daily injections of TL1A-Ig. TL1A-Ig-expanded Tregs expressed high levels of activation/memory markers KLRG1 and CD103 and were highly suppressive ex vivo. TL1A-Ig-mediated Treg expansion in vivo was protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of conventional T cells to Tregs in the lung and blocking eosinophil exudation into the bronchoalveolar fluid. Thus, TL1A-Ig fusion proteins are highly active and tightly controllable agents to stimulate Treg proliferation in vivo, and they are uniquely able to maintain high levels of expanded Tregs by repeated administration.

T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination.

TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4(+) and CD8(+) T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4(+)Foxp3(+); Tregs) and conventional T cells (CD4(+)Foxp3(-) or CD8(+)Foxp3(-); Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8(+)-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8(+) T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4(+) T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4(+) Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4(+) Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.

Tumor antigen specific iTreg accumulate in the tumor microenvironment and suppress therapeutic vaccination.

Tumor specific antigens (TSA) provide an opportunity to mobilize therapeutic immune responses against cancer. To evade such responses, tumor development in immunocompetent hosts is accompanied by acquisition of both active and passive mechanisms of immune suppression, including recruitment of CD4+FoxP3+ regulatory T cells (Treg). Thymic derived Treg (nTreg) may recognize self-antigens in the tumor microenvironment, while peripherally induced Treg (iTreg) may preferentially recognize the same TSA which provide an opportunity for therapeutic immunity from peripheral T cells. In this study we provide a systematic analysis of nTreg and iTreg accumulation in the tumor microenvironment (TME) at the cellular level. iTreg accumulation to the TME was influenced by the abundance of a known TSA, and in the absence of a known TSA intratumoral Treg displayed a unique TCR repertoire from peripheral Treg. In vivo suppression assays demonstrate that cognate-antigen matched iTreg are more potent suppressors of CD4+ than are polyclonal iTreg or nTreg, but were unable to suppress CD8+ T cell proliferation. Suppression occurred only locally at the site of immunization, and correlated with decreased expression of CD80 and CD86 on CD11c positive cells. Although established tumors facilitated the induction of TSA-specific iTreg, these iTreg suppressed CD4+ T cell accumulation only locally to the TME. Tumor mediated suppression of CD8+ T cell immunity appeared independent of TSA-specific iTreg.

Tregs expanded in vivo by TNFRSF25 agonists promote cardiac allograft survival.

BACKGROUND: Thymic-derived Foxp3(+)CD4(+) regulatory T cells (Tregs)-also called natural Tregs-are critical for the induction and maintenance of transplantation tolerance. Using an agonistic tumor necrosis factor-receptor super family (TNFRSF) 25 antibody, clone 4C12, we showed that TNFRSF25 is a powerful regulator of Treg proliferation-mediating expansion of natural Tregs in vivo. In the present study, we investigate the role of Tregs expanded in vivo by TNFRSF25 on cardiac allograft survival in a mouse model of fully major histocompatibility complex-mismatched ectopic heart transplants. METHODS: C57BL/6 mice were treated with 20 µg of TNFRSF25 agonist 4C12 4 days before heterotopic allogeneic heart transplantation. The survival of the graft was monitored daily by abdominal palpation until the cessation of cardiac contraction. The severity of immune rejection was evaluated by histopathology. Infiltration of inflammatory cells and Tregs into the graft were characterized by flow cytometry. The expression of cytokines and other regulatory proteins was measured by quantitative real-time polymerase chain reaction. RESULTS: Treatment with 4C12 resulted in expansion of Tregs to 30%-35% of CD4(+) cells and was associated with a significant prolongation of median graft survival from 8 days to 17 days (P=0.0049). On day 7 after transplantation, the time point when controls reject the graft, the transplants of 4C12-treated animals beat strongly and showed increased accumulation of Foxp3(+) Tregs within the graft and decreased infiltration of inflammatory cells. CONCLUSIONS: TNFRSF25 agonists expand Tregs in vivo and delay allograft rejection.

Therapeutic Treg expansion in mice by TNFRSF25 prevents allergic lung inflammation.

TNF receptor superfamily member 25 (TNFRSF25; also known as DR3, and referred to herein as TNFR25) is constitutively and highly expressed by CD4(+)FoxP3(+) Tregs. However, its function on these cells has not been determined. Here we used a TNFR25-specific agonistic monoclonal antibody, 4C12, to study the effects of TNFR25 signaling on Tregs in vivo in mice. Signaling through TNFR25 induced rapid and selective expansion of preexisting Tregs in vivo such that they became 30%-35% of all CD4(+) T cells in the peripheral blood within 4 days. TNFR25-induced Treg proliferation was dependent upon TCR engagement with MHC class II, IL-2 receptor, and Akt signaling, but not upon costimulation by CD80 or CD86; it was unaffected by rapamycin. TNFR25-expanded Tregs remained highly suppressive ex vivo, and Tregs expanded by TNFR25 in vivo were protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of effector T cells to Tregs in the lung, suppressing IL-13 and Th2 cytokine production, and blocking eosinophil exudation into bronchoalveolar fluid. Our studies define what we believe to be a novel mechanism for Treg control and important functions for TNFR25 in regulating autoaggression that balance its known role in enhancing autoimmunity.

HIV Nef enhances Tat-mediated viral transcription through a hnRNP-K-nucleated signaling complex.

Although dispensable in vitro, HIV Nef enables high-level viral replication in infected hosts by an as yet unexplained mechanism. Previously, we proposed that Nef functionally cooperates with the viral transactivator Tat by derepressing the viral promoter via a Nef-associated kinase complex (NAKC). Here, we demonstrate that hnRNP-K, a host factor thought to facilitate crosstalk between kinases and gene expression, interacts with Nef and, as part of NAKC, nucleates Nef-interacting kinases, including Lck, PKCdelta, and PI-3 kinase, leading to Lck and Erk1/2 activation. This strongly increased HIV transcription, which depended on Tat and the NF-kB motif in the viral promoter, but not on NF-kB activation. Depletion of hnRNP-K in a Jurkat model of HIV latency increased Erk1/2 activity and greatly augmented HIV reactivating stimuli. We conclude that hnRNP-K coordinates membrane signaling with transcriptional derepression through Erk1/2 and is targeted by HIV to enable Tat-mediated transcription.