Chromosomally-Encoded Yersinia pestis Type III Secretion Effector Proteins Promote Infection in Cells and in Mice

Yersinia pestis, the causative agent of plague, possesses a number of virulence mechanisms that allows it to survive and proliferate during its interaction with the host. To discover additional infection-specific Y. pestis factors, a transposon site hybridization (TraSH)-based genome-wide screen was employed to identify genomic regions required for its survival during cellular infection. In addition to several well-characterized infection-specific genes, this screen identified three chromosomal genes (y3397, y3399, and y3400), located in an apparent operon, that promoted successful infection. Each of these genes is predicted to encode a leucine-rich repeat family protein with or without an associated ubiquitin E3 ligase domain. These genes were designated Yersinia leucine-rich repeat gene A (ylrA), B (ylrB), and C (ylrC). Engineered strains with deletions of y3397 (ylrC), y3399 (ylrB), or y3400 (ylrA), exhibited infection defects both in cultured cells and in the mouse. C-terminal FLAG-tagged YlrA, YlrB, and YlrC were secreted by Y. pestis in the absence but not the presence of extracellular calcium and deletions of the DNA sequences encoding the predicted N-terminal type III secretion signals of YlrA, YlrB, and YlrC prevented their secretion, indicating that these proteins are substrates of the type III secretion system (T3SS). Further strengthening the connection with the T3SS, YlrB was readily translocated into HeLa cells and expression of the YlrA and YlrC proteins in yeast inhibited yeast growth, indicating that these proteins may function as anti-host T3S effector proteins

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-β. 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

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

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

•Post-transplantation cyclophosphamide (PTCy) and regulatory T cell (Treg) treatment for graft-versus-host disease prophylaxis improves natural killer (NK) cell reconstitution after MHC-matched allogeneic hematopoietic stem cell transplantation.•Effector function of the NK cell compartment is augmented after PTCy or Treg treatment. 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

In Situ Manipulation of Tregs Via Combined IL-2 and TNFRSF25 Signaling Pre- and Post-Transplant: A Novel Approach to Regulate GVHD By Marked Expansion of CD4.sup.+Foxp3.sup.+ T Cells

Academi

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

TNFRSF25 and CD25 Stimulation Expands Tregs and ILC2s in the GI Tract: Recipient Modulation Pre-HSCT

Our lab has shown that using a 2-pathway strategy of stimulating TNFRSF25 and CD25 with a TL1A-Ig fusion protein (FP) and low dose IL-2, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased. Tregs and innate lymphoid cells (ILCs) are critical to maintaining immune homeostasis and generating tolerance in the GI tract. In addition to a highly diverse population of Tregs, the GI also contains various populations of ILCs, recently shown to modulate GVHD (Bruce, 2017). ILCs express CD25 as well as TNFRSF25 and respond individually to IL-2 and TL1A administration respectively. Here we asked how our 2-pathway (FP+IL-2) approach affected Tregs and ILCs in the GI of healthy mice. First, we examined Tregs from B6-Nur77GFP or B6-Nur77GFPFoxP3RFP mice to assess the Treg TcR signaling status in different tissues. Mice were administered FP, IL-2, or FP+IL-2 over 1 wk. The frequency of Treg/CD4 in the lamina propria (LP) of the large intestine (LI) reached levels >60% (1A). This elevation included FoxP3+RORyt− and FoxP3+RORyt+ Tregs. In contrast to our previous findings in the LHC, treatment with FP alone elevated levels to the same extent as the FP+IL-2 in the LI/LP, whereas IL-2 treatment alone had only a modest effect on elevating the frequency of Tregs (1A), suggesting that the activation status of Tregs differs based on compartmental location. The frequency of GFP+ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LHC (<35%) (1C). An independent experiment verified these findings and found that only conjunctival Tregs had a similar activation (GFP) phenotype (1D). Interestingly, without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon (1B). Next, we evaluated FP+IL-2 treated B6-Nur77GFP mice. Tregs - but not CD8 T-cells in the LHC had elevated GFP by frequency and MFI vs untreated mice (1E). Notably, we also found that FP+IL-2 treatment in vivo increases the relative frequency of both ILC2s and ILC3s (1F). Regarding ILC2s, FP+IL-2 increased their frequency greater than either TNFRSF25 or CD25 stimulation alone in the GI tract (1G). We then asked if our FP+IL-2 approach could be used to augment these populations as a potential strategy to protect HSCT recipients. FP+IL-2 stimulation prior to TBI in BALB/c mice significantly augmented Tregs and ILC2s in the GI tract for at least 1 wk post-irradiation (2A,2B). In total, these findings demonstrate 1) basal Treg activation status differs depending on the compartment and mucosal Tregs express highest TcR activation, 2) FP alone vs. FP+IL-2 stimulation results in equivalent expansion of GI Tregs, 3) only FP+IL-2 effectively expand ILC2s and 4) pre-TBI FP+IL-2 treatment results in elevated Treg and ILC2 levels post-irradiation. Ongoing experiments are examining GI GVHD and the role of Tregs/ILCs in TNFRSF25 +/- CD25 stimulated aHSCT recipients

The Location of CD4+FoxP3+ Cells with Regard to CD25 and TNFRSF25 Receptor Signals Matters: Different Requirements for GI and Peripheral Tregs

Regulatory T cells (Tregs) are critical to maintaining immune homeostasis and generating tolerance in the gastrointestinal (GI) tract. GI complications play a prominent role following allogeneic HSCT (aHSCT) and we are interested in manipulating GI Tregs to regulate GVHD. Previously our lab has shown that using a two-pathway strategy of stimulating the TNFRSF25 and CD25 receptors with a TL1A-Ig fusion protein (FP) and IL-2low dose, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased (Wolf, BBMT 2017; Copsel, BBMT 2018). As a consequence of microbes and food antigens, Treg populations in the GI have a relative highly diverse composition compared to the lymphohematopoietic Treg compartment. This includes, but is not limited to, the presence of a stable and suppressive FoxP3+RORyt+ Treg population. Additionally, the GI tract contains various populations of innate lymphoid cells (ILCs) that interact with Tregs. ILCs express CD25 as well as TNFRSF25, and respond individually to IL-2 and TL1A administration respectively (Danny, JCI 2017; Verneris, Blood 2013). Based on the extensive differences between the lymphohematopoietic and GI Treg compartments, we evaluated how our two-pathway strategy may be differentially affecting the levels and activation status of Tregs in the GI tract. To address this question, we initially utilized B6-Nur77GFP mice, where GFP expression occurs with activation of the Nur77 promoter. However, since Nur77 is only produced following TcR engagement and not inflammatory signals, the strength of TcR stimulation in all T cell populations can be monitored by GFP levels. We first examined Tregs from 2-pathway treated B6-Nur77GFP mice and subsequently generated B6-Nur77GFPFoxP3RFP mice to readily assess the TcR signaling status of Tregs in different compartments. Mice were systemically administered FP, IL-2, or FP + IL-2 over a 1 wk period. The frequency of Tregs (FoxP3+CD4+) / Tcon (CD4+FoxP3-) in the lamina propria (LP) of the large intestine (LI) reached levels >60%. (1A). This elevation of Treg / Tcon frequency included FoxP3+RORyt- Tregs as well as FoxP3+RORyt+ double positive Treg populations. In contrast to our previous findings in the LHC, treatment with TL1A-Ig FP alone elevated levels to the same extent as the combination of FP + IL-2 (1A). Importantly, IL-2 treatment alone - as reported in the LHC - again had only a modest effect on elevating the frequency of Tregs in the LI/LP (1A). These observations suggest that the activation status of Tregs may differ based on the compartmental location. To asses activation status, we evaluated 2-pathway treated B6-Nur77GFP mice. Tregs in the LN/spleen had elevated frequency of GFP+ Tregs and higher GFP MFI than untreated mice. This elevated TcR stimulation was present in peripheral Tregs - but not CD8 - T cells (1B). Without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon cells (1B). The frequency of GFP+ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LN/spleen (<35%) (1C). In an independent experiment, we verified these findings and learned that only conjunctival Tregs demonstrated similarly increased frequency of GFP+ Tregs with elevated GFP MFI (1D). We also found that TL1A-Ig + IL-2 treatment in vivo increases the relative frequency of both innate ILC2 and ILC3 lymphoid populations (2A). The combination also increased the frequency of ILC2 cells greater than either reagent alone in the GI tract (2B). In total, our results objectively validate selective Treg vs. Tcon targeting via TNFRSF25. These findings also demonstrate that basal Treg activation status differs depending on the compartment. Notably, Tregs in mucosal vs. LHC tissue expressed higher TcR activation levels. Such Tregs have the potential for co-stimulation via TNFRSF25. Moreover, since IL-2 is required to maintain Tregs under both basal and activated conditions, our findings suggest a local source of IL-2 is present to maintain GI Tregs. ILC3 cells are recently reported to generate IL-2 (Zhou L, Nature 2019). We hypothesize that TNFRSF25 stimulation in ILC3 cells results in a local source of IL-2 which can account for our finding that TL1A-Ig alone vs. TL1A-Ig + IL-2 stimulation results in equivalent levels of GI Tregs. Ongoing experiments are examining how GI GVHD in aHSCT recipients are affected by TNFRSF25 +/- CD25 Treg/ILC stimulation. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding

Sequential Cyclophosphamide and Trametinib Improve Clinical Graft Versus Host Disease and Survival in Murine Hematopoietic Stem Cell Transplant

Ongoing acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (alloSCT) causes significant morbidity and mortality with no standard therapy except corticosteroids, which are broadly immunosuppressive and toxic. Cyclophosphamide (Cy) given day 3 and day 4 is effective for prophylaxis of aGVHD but has proven ineffective when given later as in ongoing aGVHD in both murine and human studies. We previously demonstrated that the MEK inhibitor trametinib (Tram) is effective for aGVHD prophylaxis in murine models of aGVHD through the novel mechanism of inhibition of naïve T cells (CD44−CD62L+). Our hypothesis is that ongoing aGVHD could be better inhibited by sequential Cy and post-Cy Tram inhibition (to prevent naïve T cells from re-initiating alloreactivity) without compromising graft-versus-malignancy (GVM) responses. Mice were administered sequential Cy and Tram post SCT starting day 12. Efficacy of treatment was assessed in a murine model of MHC-mismatched alloSCT. Overall survival was assessed and clinical GVHD was scored three times weekly. Peripheral blood, spleen, and lymph node cells phenotypes were determined by flow cytometry. GVM was also assessed with A20 luciferase/YFP (A20luc/YFP) lymphoma cells imaged with in vivo imaging system (IVIS). Overall survival following sequential Cy and Tram was identical to that seen with T cell depleted bone marrow alone and significantly superior to treatment using either single-agent Cy or Tram (p<0.05). Clinical GVHD scores were improved by sequential Cy and Tram compared to treatment with either single agent Cy (p=0.002) or single agent Tram (day 38 p=0.03). Timing of initiation of sequential Cy and Tram starting day 12 was significantly better than in the GVHD control group (p=0.002) and also significantly better than after later initiation at day 19 (p=0.005). Initiation of treatment on day 6 resulted in 100% mortality by day 15. Higher doses of Trametinib at 0.3mg/kg were significantly better than the GVHD control (p=0.006). Analysis of peripheral CD4+ and CD8+ T cells demonstrated a more physiologic CD4+CD8+ ratio and a more normal distribution of naïve and effector T cells in the sequential Cy and Tram within CD4+ and CD8+ T cell subsets. Sequential Cy and Tram did not adversely affect peripheral CD4+FOXP3+ regulatory T cells. Using A20 luciferase/YFP (A20luc/YFP) lymphoma cells in a transplant model demonstrated that GVM was still effective with sequential Cy and Tram. Combination cyclophosphamide and trametinib demonstrates superior overall survival and limits GVHD severity without affecting regulatory T cells or GVM in murine allogeneic stem cell models. Utilizing Cy and Tram are readily translatable and support the notion that clinical approaches can be developed in the future