Pre-Transplant CSF-1 Therapy Expands the Recipient Macrophage Pool and Modulates Graft Versus Host Disease After Allogeneic Hematopoietic Cell Transplantation

Abstract Abstract 242 Host antigen presenting cells are critical to activate allo-reactive T cells and to initiate acute graft versus host disease (GVHD) after allogeneic bone marrow transplantation (BMT). However, while the role of host dendritic cells (DCs) in GVHD has been established, the contribution of host macrophages to GVHD has not been clearly addressed. In this study we revisited the role of host macrophages in GVHD. To this end we used an anti-CSF-1R monoclonal antibody (aCSF1R) to reduce macrophages but not DC numbers in lymphoid organs. We treated recipient C57BL/6 mice with αCSF1R on days -5 to -3 followed on day 0 by TBI and i.v. injection of BM cells and splenocytes isolated from MHC-mismatched BALB/c donor. We found that host macrophages that persist in the spleen and LN of recipient mice were severely reduced by αCSF1R, whereas host DCs remained unaffected by the treatment. We also found that αCSF1R-treatement significantly enhanced GVHD morbidity and mortality (Figure left panel) after allogeneic BMT, enhanced donor T cell expansion in recipient spleen, LN and liver, and increased IFN-gamma and TNF-alfa sera levels compared to mice treated with control IgG. Similar results were obtained when low dose Lip-Clod was administered 10 days prior to transplant in order to deplete macrophages in lymphoid tissues but not host DC whose half-life in lymphoid tissues does not exceed three days allowing them to recover to normal at the time of transplant. Our results revealed that in contrast to a previous report in which higher Lip-Clod doses administered 7 and 2 days prior to transplant led to the depletion of both DCs and macrophages and improved GVHD, low dose Lip-Clod administered 10 days prior to transplant, depleted host macrophages but not DCs and aggravated GVHD. We also explored the mechanisms by which host macrophages could potentially control donor T cell expansion induced by host DC after allo-HCT. We found that host macrophages isolated from allogeneic recipients inhibited the proliferation of allo-reactive T cells co-cultured with host C57BL/6 DC partly through a cell-cell contact pathway. Host macrophages were also able to engulf and reduce alloreactive T cells after few hrs of culture. Consistently, 18 hrs after allo-HCT, the number of donor T cells accumulating in the recipient spleen and mesenteric LNs were higher in Ab-treated mice compared to the control groups and CFSE-labeled donor T cells were engulfed by splenic macrophages during the first day of transplant and prior to the initiation of donor T cell proliferation. These results suggest that host macrophages limit the expansion of donor T cells through their ability to engulf donor T cell and to inhibit donor T cell proliferation induced by host DC stimulation. Further supporting the importance of host macrophages in regulating the development of GVHD, we found that pre-transplant administration of the cytokine CSF-1 increased the number of spleen macrophages, limited the expansion of donor allo-reactive T cells and improved GVHD outcome (Figure right panel). Altogether, we showed that in contrast to host DCs, host macrophages modulate GVHD through their ability to limit donor T cell activation and expansion. In addition, our data also identify pre-transplant CSF-1 injection as a novel clinical strategy for the prevention of GVHD in patient candidate for allo-HCT. Disclosures: No relevant conflicts of interest to declare.

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CD11b+gr-1+ myeloid Derived Suppressor Cells (MDSC) In Normal Bone Marrow Suppress T Cell Proliferation and Enhance T-Regulatory Function Via a IL10-, IL4- and IDO-Independent Mechanism

Blood ◽

10.1182/blood.v116.21.4801.4801 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4801-4801 ◽

Cited By ~ 1

Author(s):

Parvin Forghani ◽

Wayne Harris ◽

jian-Ming Li ◽

M.R. Khorramizadeh ◽

Edmund Waller

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Regulatory Function ◽

T Cell Proliferation ◽

Suppressive Activity ◽

Ifn Γ

Abstract Abstract 4801 MDSC have been described as an important negative regulators of autologous anti-cancer immune responses. Considering the important role of MDSC in immune regulation in allogenic stem cell and organ transplantation, we undertook an investigation of the mechanism(s) by which MDSC inhibit T–cell activation and proliferation, and tested the hypothesis that local cytokine secretion or IDO activity is required for suppression of T-cell proliferation. Two separate populations CD11bhiGr-1hi and CD11bhi Gr-1int were isolated by high-speed FACS from lineage- BM antigen presenting cells (C57 & BALB/c mice). Both MDSC subsets had potent capacity for in–vitro suppression of CD4+ and CD8+ T cells proliferation in response to anti-CD3/anti-CD28 beads and Con A. A ratio of 0.5/1 MDSC: T-cells were sufficient to inhibit >66% control levels of T-cell proliferation. MDSC isolated from transgenic mice that had been “knocked-out” for IFN-γ and IDO had equivalent suppressive activity as MDSC from wild-type donors. Addition of saturating concentrations of anti IL-10 and IL-4 MAb, or in combination with anti- IFN-γ MAb did not abrogate MDSC-suppressive activity. Ex-vivo culture of MDSC with mitogen-activated T-cells generated two—fold more Fox-p3 T-reg compared with cultures of T cell plus mitogen. Data will be presented regarding the novel role of MDSC involving in the homeostasis regulation of normal T-cell activation and proliferation in non-tumor-bearing mice. Disclosures: No relevant conflicts of interest to declare.

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Donor Requirements For CD4+CD25+FoxP3+ Regulatory T Cells Capable Of Suppressing CD4+ and CD8+ Conventional T Cell Proliferation and Graft Versus Host Disease

Blood ◽

10.1182/blood.v122.21.4484.4484 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4484-4484 ◽

Cited By ~ 1

Author(s):

Antonio Pierini ◽

Lucrezia Colonna ◽

Maite Alvarez ◽

Dominik Schneidawind ◽

Byung-Su Kim ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Animal Models ◽

Graft Versus Host Disease ◽

Third Party ◽

T Cell Proliferation ◽

Graft Versus Host

Adoptive transfer of CD4+CD25+FoxP3+ regulatory T cells (Tregs) prevents graft versus host disease (GvHD) in several animal models and following allogeneic hematopoietic cell transplantation (HCT) in clinical trials. In these models donor derived Tregs have been mainly used as they share the same major histocompatibility complex (MHC) with conventional CD4+ and CD8+ T cells (Tcons) that are primarily responsible for GvHD onset and persistence. Third-party derived Tregs are a promising alternative tool for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. In this study we explored MHC disparities between Tregs and Tcons in HCT to evaluate the impact of these different cell populations in GvHD prevention and survival after transplant. Methods and Results We evaluated the ability of highly purified Treg to suppress proliferation of C57BL/6 (H-2b) Tcons following exposure to irradiated splenocytes from BALB/C (H-2d) mice in vitro in a mixed lymphocyte reaction (MLR). Either donor derived C57BL/6 (H-2b) or third party FVB (H-2q) Tregs suppressed Tcon proliferation at the Treg/Tcon ratios of 1:2 and 1:4. The same Treg population effectively suppressed different MHC derived Tcons where BALB/C (H-2d) or FVB (H-2q, third-party) Tcons were incubated with irradiated splenocytes from C57BL/6 (H-2b) mice and were effectively suppressed with BALB/C (H-2d) Tregs. In the MLR, third-party Tregs present the same activation molecule expression patterns as MHC matched Tregs: CTLA4 and LAG3 expression is enhanced after stimulation with interleukin-2 (IL-2) and anti-CD3/CD28 beads, while MHC class II molecule expression is increased after 3-4 days of culture with Tcons and irradiated splenocytes. Furthermore third-party and MHC matched Tregs express the same levels of interleukin-10 (IL-10). We translated these results to in vivo studies in animal models. In these studies T cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) mice was injected into lethally irradiated (total body irradiation, 8 Gy) BALB/C (H-2d) recipient mice. 2 days later GvHD was induced by injecting luc+ donor derived Tcons (1x106/mouse). Using this model GvHD was evaluated following the adoptive transfer of freshly isolated CD4+CD25+FoxP3+ Tregs derived from BALB/C (H-2d, host type), C57BL/6 (H-2b, donor type), FVB (H-2q, third-party) or BALB/B (H-2b, minor mismatched with the donor, major mismatched with the host) mice at the different Treg/Tcon ratios of 1:1, 1:2 and 1:4. As expected, donor Tregs exerted the strongest dose dependent GvHD protection (p = 0.028), while host Tregs did not improve mouse survival (p = 0.58). Third-party and minor mismatched with the donor Tregs improved mouse survival (third-party and minor mismatched with the donor respectively, p = 0.028 and p = 0.17) but mice had worse GvHD score profiles (both p< 0.001) and could not recover their weight as well as mice treated with donor Tregs (both p< 0.001). In vivoTcon bioluminescent imaging confirmed these results showing a reduced Tcon proliferation in mice treated with donor, third-party and minor mismatched with the donor Tregs, the first exerting the strongest effect (after 6 weeks of observation, p< 0.001). Conclusions Our studies indicate that MHC disparities between Tregs and Tcons do not represent an insurmountable barrier for Treg function. In vitro and in vivo data strongly suggest that Tregs can suppress Tcon proliferation without requiring MHC matching. In vivo GvHD prevention efficiency was affected by MHC disparities with donor derived Treg being the most effective, however, third party Treg also resulted in GvHD attenuation. These studies indicate that both donor and third party Treg could be effective in clinical application raising the possibility of screening and banking Treg for use. Further, these studies highlight the need for activation of the Treg on host tissues to effectively suppress conventional T cell proliferation and GvHD induction. Disclosures: No relevant conflicts of interest to declare.

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β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

Blood ◽

10.1182/blood-2019-123139 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1915-1915 ◽

Cited By ~ 1

Author(s):

Hemn Mohammadpour ◽

Joseph L. Sarow ◽

George L. Chen ◽

Cameron R. MacDonald ◽

Umesh Sharma ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Adrenergic Receptor ◽

Graft Versus Host ◽

Graft Versus Leukemia ◽

Donor T Cells ◽

Β2 Adrenergic Receptor ◽

Ifn Γ

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P< 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P<0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

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Effect of In Vivo Azacitidine on GvHD and Gvl: Mechanistic Studies

Blood ◽

10.1182/blood.v116.21.2537.2537 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2537-2537

Author(s):

Jaebok Choi ◽

Julie Ritchey ◽

Jessica Su ◽

Julie Prior ◽

Edward Ziga ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Leukemia Cell ◽

T Cell Proliferation ◽

Cell Trafficking ◽

Alloreactive T Cells ◽

Donor T Cells

Abstract Abstract 2537 Introduction: Regulatory T cells (Tregs) have been shown to mitigate graft-versus-host disease (GvHD) while preserving the beneficial graft-versus-leukemia (GvL) effect in animal models of allogeneic bone marrow transplantation (BMT). However, three major obstacles prevent their use in human clinical trials: the low numbers of Tregs, loss of suppressor activity following in vitro expansion, and the lack of Treg-specific markers to purify expanded Tregs. The locus of the Foxp3 gene, the master regulator of Tregs, is unmethylated and expressed only in Tregs. We have recently reported that the hypomethylating agent azacitidine (AzaC) induces FOXP3 expression in non-Tregs, converting them into Tregs in vitro and in vivo when administered after allogeneic BMT completely mitigating GvHD without abrogating GvL (Choi, et al Blood 2010). Three possible mechanisms for these effects include: 1) AzaC induces FOXP3+ Tregs, which in turn mitigate GvHD without abrogating GvL by regulating alloreactive donor T cells, 2) AzaC directly suppresses the proliferation of alloreactive donor T cells reducing GvHD, 3) AzaC alters donor T cell trafficking to GvHD target organs to prevent GvHD without altering interaction of donor T cells with recipient leukemia or trafficking of leukemic cells. Methods: Balb/c (CD45.2+, H-2Kd) were lethally irradiated one day prior to injection of T cell-depleted BM cells isolated from B6 (CD45.1+, H-2Kb) and luciferase-expressing A20 leukemia cells derived from Balb/c. Allogeneic donor T cells isolated from B6 (CD45.2+, H-2Kb) were given 11 days after BMT. AzaC (2 mg/kg) was administrated subcutaneously every other day (4 doses total) starting 4 days after T cell injection. In vivo bioluminescence imaging (BLI) was performed to assess leukemia cell localization. For T cell proliferation/trafficking analyses, Balb/c were lethally irradiated one day prior to injection of T cell-depleted BM cells isolated from B6 (CD45.1+). Allogeneic donor T cells isolated from B6 (CD45.2+) were transduced with Click Beetle Red luciferase and were given 11 days after BMT, followed by AzaC treatment as described above. BLI was performed to track the donor T cells. Results: While neither T cell or leukemia cell trafficking was affected by the AzaC treatment, proliferation of donor T cells was significantly reduced compared to mice treated with PBS. The observed reduced T cell proliferation is not likely due to the direct effect of AzaC on T cells since the AzaC treatment preserved GvL activity comparable with the PBS control group. In addition, T cells isolated from both AzaC and PBS groups were equally reactive against third party antigen presenting cells, based on mixed lymphocyte reactions and cytotoxic T lymphocyte killing assays. These data along with our previous report demonstrating that the AzaC treatment increases Tregs in vivo strongly suggest that the therapeutic effect of AzaC on GvHD and GvL are mediated by the AzaC-induced Tregs which preferentially target alloreactive T cells while preferentially sparing anti-tumor T cells. Currently, secondary transplantation of Treg-depleted/replete T cells isolated from AzaC/PBS-treated recipient mice is underway to further confirm that donor T cells in the AzaC-treated mice are fully functional and that alloresponses of donor T cells are regulated by AzaC-induced Tregs. Conclusions: In vivo administration of AzaC after donor T cell infusion mitigates GvHD while preserving GvL via peripheral conversion of alloreactive donor T cells to FOXP3+ Tregs that preferentially inhibit alloreactive T cells while sparing anti-tumor T cells. These data provides the foundation for future clinical trials using epigenetic therapy aimed at mitigating GvHD without abrogating GvL and overcoming HLA barriers. Disclosures: No relevant conflicts of interest to declare.

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Microrna-17-92 Cluster: Novel Target for Controlling Gvhd While Preserving GVL Effect

Blood ◽

10.1182/blood.v124.21.845.845 ◽

2014 ◽

Vol 124 (21) ◽

pp. 845-845

Author(s):

Yongxia Wu ◽

David Bastian ◽

Jessica Lauren Heinrichs ◽

Jianing Fu ◽

Hung Nguyen ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Cell Death ◽

T Cell ◽

Cd8 T Cells ◽

Locked Nucleic Acid ◽

T Cell Proliferation ◽

Allogeneic Bone

Abstract Graft-versus-host disease (GVHD) remains a life threatening complication after allogeneic hematopoietic stem cell transplantation (HCT). Donor T cells are the key pathogenic effectors in the induction of GVHD. MicroRNAs (miRs) have been shown to play an important role in orchestrating immune response, among which miR-17-92 cluster is one of the best characterized miR clusters that encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1 and 92-1. Although regulatory functions of miR-17-92 cluster have been elaborated in a variety of immune responses including anti-infection, anti-tumor, and autoimmunity, the role of this miR cluster in the modulation of T-cell response to alloantigens and the development of GVHD has not been explored previously. Based on the previous report that miR-17-92 promotes Th1 responses and inhibits induced regulatory T-cell (iTreg) differentiation in vitro, we hypothesized that blockade of miR-17-92 would constrain T-cell alloresponse and attenuate GVHD. To evaluate the function of miR-17-92 on T-cell alloresponse, we utilized the mice with miR-17-92 conditional knock-out (KO) on T cells as donors, and compared the alloresponse of WT and KO T cells after allogeneic bone marrow transplantation (allo-BMT). We observed that KO T cells had substantially reduced ability to proliferate and produce IFNγ as compared to WT counterparts 4 days after cell transfer. Interestingly, CD4 but not CD8 KO T cells had increased cell death in the population of fast-dividing T cells. Thus, miR-17-92 cluster promotes activation and expansion of both CD4 and CD8 T cells, and inhibits activation-induced cell death of CD4 but not CD8 T cells at the early stage of alloresponse in vivo. We further evaluated the role of miR-17-92 on T cells in the development of acute GVHD in a fully MHC-mismatched BMT model. In sharp contrast to WT T cells that caused severe GVHD and resulted in 100% mortality of the recipients, KO T cells were impaired in causing severe GVHD reflected by mild clinical manifestations and no mortality. These observations were extended to MHC-matched but minor antigen-mismatched as well as haploidentical BMT models that are more clinically relevant. We next addressed the critical question whether T cells deficient for miR-17-92 are still capable of mediating graft-versus-leukemia (GVL) effect. Using A20 lymphoma and P815 mastocytoma cell lines, we demonstrated that the KO T cells essentially retained the GVL activity in MHC-mismatched and haploidentical BMT model, respectively. Mechanistic studies revealed that miR-17-92 promoted CD4 T-cell proliferation, survival, migration to target organs, and Th1-differentiation, but reduced Th2-differentiation and iTreg generation. However, miR-17-92 had less impact on CD8 T-cell proliferation, survival, IFNγ production, and cytolytic activity reflected by granzyme B and CD107a expression. Moreover, miR-17-92 negatively regulated TNFα production by both CD4 and CD8 T cells. We therefore conclude that miR-17-92 cluster is required for T cells to induce severe GVHD, but it is dispensable for T cells to mediate the GVL effect. To increase translational potential of our findings, we designed the locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, which have been reported to be the key members in this cluster. We observed that the treatment with anti-miR-17 significantly inhibited T-cell expansion and IFNγ production in response to alloantigen in vivo, and anti-miR-19 was more effective. Furthermore, our ongoing experiment showed the treatment with anti-miR-17 or anti-miR-19 was able to considerably attenuate the severity of GVHD as compared to scrambled antagomir in a MHC-mismatched BMT model. Taken together, the current work reveals that miR-17-92 cluster is essential for T-cell alloresponse and GVHD development, and validates miR-17-92 cluster as promising therapeutic target for the control of GVHD while preserving GVL activity in allogeneic HCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon γ production

Blood ◽

10.1182/blood-2002-02-0445 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2899-2907 ◽

Cited By ~ 59

Author(s):

Duncan Howie ◽

Susumo Okamoto ◽

Svend Rietdijk ◽

Kareem Clarke ◽

Ninghai Wang ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cell Surface ◽

Dna Synthesis ◽

Interferon Γ ◽

T Cell Proliferation ◽

Wild Type ◽

Ifn Γ

CD150 (signaling lymphocyte activation molecule [SLAM]) is a self-ligand cell surface glycoprotein expressed on T cells, B cells, macrophages, and dendritic cells. To further explore the role of CD150 signaling in costimulation and TH1 priming we have generated a panel of rat antimouse CD150 monoclonal antibodies. CD150 cell surface expression is up-regulated with rapid kinetics in activated T cells and lipopolysaccharide/interferon γ (IFN-γ)–activated macrophages. Anti-CD150 triggering induces strong costimulation of T cells triggered through CD3. DNA synthesis of murine T cells induced by anti-CD150 is not dependent on SLAM-associated protein (SAP, SH2D1A), because anti-CD150 induces similar levels of DNA synthesis in SAP−/− T cells. Antibodies to CD150 also enhance IFN-γ production both in wild-type and SAP−/− T cells during primary stimulation. The level of IFN-γ production is higher in SAP−/− T cells than in wild-type T cells. Anti-CD150 antibodies also synergize with interleukin 12 (IL-12) treatment in up-regulation of IL-12 receptor β2 mRNA during TH1 priming, and inhibit primary TH2 polarization in an IFN-γ–dependent fashion. Cross-linking CD150 on CD4 T cells induces rapid serine phosphorylation of Akt/PKB. We speculate that this is an important pathway contributing to CD150-mediated T-cell proliferation.

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Role of Interleukin 12 and Costimulators in T Cell Anergy In Vivo

Journal of Experimental Medicine ◽

10.1084/jem.186.7.1119 ◽

1997 ◽

Vol 186 (7) ◽

pp. 1119-1128 ◽

Cited By ~ 65

Author(s):

Luk Van Parijs ◽

Victor L. Perez ◽

Andre Biuckians ◽

Robert G. Maki ◽

Cheryl A. London ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Proliferation ◽

Interleukin 12 ◽

Potent Inducer ◽

T Cell Anergy ◽

Cell Anergy

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti–CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti–CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one.

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T Cell Activation By Plasmacytoid Dendritic Cells Is Augmented By Inhibition of Vasoactive Intestinal Polypeptide Signaling: Implications for Cellular Immunotherapy

Blood ◽

10.1182/blood.v126.23.3438.3438 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3438-3438

Author(s):

Jingxia Li ◽

Reema Panjwani ◽

Jian-Ming Li ◽

Cynthia R. Giver ◽

Maryellen Malone ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Murine Models ◽

Donor T Cells ◽

Cells Cultured

Abstract Introduction: Data from clinical allogeneic bone marrow transplant (allo-BMT) and pre-clinical murine models of allo-BMT have shown that donor plasmacytoid dendritic cells (pDC) have important roles in regulating graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activities of donor T cells. Using murine models of allo-BMT we have previously shown that 1) donor pDCs induce Th1 polarization of donor T cells and augment the GvL activity of T cells; and 2) the addition of pDC to grafts composed of purified T cells and HSC limited the subsequent development of GvHD. VIP is an immunosuppressive neuropeptide that regulates adaptive immune responses. We reasoned that VIP signaling may regulate activation of allo-specific T-cells, and the VIP pathway may be target for regulating GvHD and GvL in allo-BMT. Methods: To explore the mechanisms by which pDC and VIP signaling regulate T cell activation we used: 1) transgenic mice expressing GFP under the control of the VIP promoter to measure VIP expression in vivo in allo-BMT recipients; 2) one-way mixed lymphocyte reaction (MLR) to measure the proliferative response of transgenic luciferase positive T cells in response to allo-antigens via bioluminescence imaging (BLI); and 3) a model system of indirect presentation of allo-peptides derived from a H2-Ab MHC class II molecule by pDC to transgenic T cells expressing the TEa TCR. The effect of blocking vasoactive intestinal polypeptide (VIP) signaling during activation of allo-reactive T cells was assessed by using VIP-KO cells and by the addition of VIP peptide or a peptide antagonist of VIP (VIPhyb) to the one-way MLR. T cell proliferation and activation was measured by flow cytometry. Results: Analysis of VIP expression in donor pDC in murine models of allo-BMT showed >100-fold induction of VIP promoter activity in donor pDC and donor T cells during the first two weeks post-transplant, indicating that VIP expression in donor pDC may regulate T cell activation.Addition of endogenous native VIP suppressed T cell proliferation in one-way MLR but was reversed by addition of a 10x concentration of the VIP antagonist peptide (Figure 1B). Furthermore, adding 3 uM of the VIP antagonist to the MLR cultured significantly enhanced T cell proliferation. TEa peptide-primed T cells cultures with peptide-primed pDC from VIP knock-out mice had increased proliferation and expressed more of the activation markers CD69 and CD71 compared with T cells cultured with VIP-WT pDCs. Comparing pDC purified from marrow versus spleen, we found significantly more proliferation in T cells cultured with bone marrow VIP-KO pDCs than splenic VIP-KO pDCs, indicating that the less mature marrow pDC have greater antigen presenting ability than the more mature splenic pDC. Conclusion: These data suggest that 1) VIP is produced by donor pDC early after allo-BMT; 2) VIP inhibits T cell allo-proliferation in one-way MLR's; and 3) blocking VIP signaling using donor cells that cannot produce VIP or through the use of pharmacological inhibitors of VIP can augment activation and proliferation of T cells in response to indirect antigen presentation.The present findings support studies of VIP antagonist in allo-BMT to augment the GvL activity of T cells through indirect antigen presentation. Future studies include using the BLI analysis of MLR to determine the effect of novel drugs on T cell proliferation. **Jingxia Li and Reema Panjwani were equal contributors to this abstract. Disclosures No relevant conflicts of interest to declare.

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Low-Dose Decitabine Augments the Activation and Anti-Tumor Immune Response of IFN-γ+ CD4+ T Cells Through Enhancing IκBα Degradation and NF-κB Activation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.647713 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xiang Li ◽

Liang Dong ◽

Jiejie Liu ◽

Chunmeng Wang ◽

Yan Zhang ◽

...

Keyword(s):

Immune Response ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Low Dose ◽

T Cell Proliferation ◽

Ifn Γ ◽

Iκbα Degradation

BackgroundCD4+ T cells play multiple roles in controlling tumor growth and increasing IFN-γ+ T-helper 1 cell population could promote cell-mediated anti-tumor immune response. We have previously showed that low-dose DNA demethylating agent decitabine therapy promotes CD3+ T-cell proliferation and cytotoxicity; however, direct regulation of purified CD4+ T cells and the underlying mechanisms remain unclear.MethodsThe effects of low-dose decitabine on sorted CD4+ T cells were detected both in vitro and in vivo. The activation, proliferation, intracellular cytokine production and cytolysis activity of CD4+ T cells were analyzed by FACS and DELFIA time-resolved fluorescence assays. In vivo ubiquitination assay was performed to assess protein degradation. Moreover, phosphor-p65 and IκBα levels were detected in sorted CD4+ T cells from solid tumor patients with decitabine-based therapy.ResultsLow-dose decitabine treatment promoted the proliferation and activation of sorted CD4+ T cells, with increased frequency of IFN-γ+ Th1 subset and enhanced cytolytic activity in vitro and in vivo. NF-κB inhibitor, BAY 11-7082, suppressed decitabine-induced CD4+ T cell proliferation and IFN-γ production. In terms of mechanism, low-dose decitabine augmented the expression of E3 ligase β-TrCP, promoted the ubiquitination and degradation of IκBα and resulted in NF-κB activation. Notably, we observed that in vitro low-dose decitabine treatment induced NF-κB activation in CD4+ T cells from patients with a response to decitabine-primed chemotherapy rather than those without a response.ConclusionThese data suggest that low-dose decitabine potentiates CD4+ T cell anti-tumor immunity through enhancing IκBα degradation and therefore NF-κB activation and IFN-γ production.

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Donor Plasmacytoid Dendritic Cells Regulate GvHD in a VIP Dependent Manner in Allogeneic BMT Recipients

Blood ◽

10.1182/blood-2021-152945 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1687-1687

Author(s):

Jingru Zhu ◽

Pankoj Kumar Das ◽

Yitong Wang ◽

Jingxia Li ◽

Tamas Nagy ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Gene Expression Analysis ◽

T Cell Proliferation ◽

Post Transplant ◽

Donor T Cells ◽

Th1 Polarization

Abstract Introduction: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide known to induce differentiation of regulatory dendritic cells and regulatory T cells. Using allogeneic hematopoietic stem cell transplantation (allo-HSCT) models, we have shown that donor bone marrow (BM) plasmacytoid dendritic cells (pDCs) facilitate HSC engraftment and attenuate pathogenesis of graft vs. host disease (GvHD) through regulation of recipient T cells. However, the mechanism by which pDCs mitigate the GvHD activity of recipient T cells is not clearly understood. Here, we report that donor pDCs limit pathogenic T cell inflammation by VIP production. Methods: To study VIP production by pDCs, FACS-sorted pDCs from B6 mouse BM were cultured with or without PMA/ionomycin in-vitro. After activation and cytospin slide preparation, pDCs were labeled with anti-PDCA1 (pDC marker) and anti-VIP antibodies for confocal fluorescence microscopy. To investigate the effects of VIP production on T cell proliferation, an in-vitro co-culture assay was performed using R848 and CpG-activated WT or VIP-KO pDCs with anti-CD3-activated, CFSE-labeled syngeneic T cells. For GvHD experiments, irradiated B10.BR (H-2K k) mice received 5x10 3 HSCs, 5x10 4 pDCs and 1x10 6 T cells from WT B6 (H-2K b) or VIP-KO B6 (H-2K b) mice. H&E histology of intestine and colon was performed for GvHD scoring 7 days post-transplant. Graft vs. leukemia (GvL) effects were tested by inoculating recipient mice with 5x10 5 LBRM 33-5A4 cells in the same model. Recipient mice were monitored twice weekly using a 10-point GvHD scoring system. Gene expression analysis of FACS-sorted donor T-cells from recipient spleens was performed using the Nanostring Myeloid Innate Immunity Panel at days 8 and 15 post-transplant. Results: Confocal microscopic images of PMA/ionomycin stimulated or unstimulated sorted pDCs show that VIP is synthesized by pDCs (anti-VIP, green; anti-PCDA-1, red; DAPI counterstain, blue) (Fig 1). After in-vitro culture, VIP expression and frequencies of VIP + pDCs were similar in PMA/ionomycin treated or untreated cells (not shown). VIP-KO mice have significantly higher percentages of pDCs in BM compared to WT (Fig 2a). T cells co-cultured with VIP-KO pDCs showed higher proliferation than T cells co-cultured with WT pDCs, demonstrating that VIP secreted by pDCs reduces T cell proliferation (Fig 2b). Moreover, VIP-KO pDCs induce significantly greater proliferation of IFN-gamma + CD8 T cells compared to WT, indicating that pDCs lacking VIP promote Th1 polarization in-vitro (Fig 2c). The data are consistent with results from GvHD experiments showing increased frequencies of Th1 polarized T cells and fewer regulatory T cells in recipients of VIP-KO pDCs compared with recipients of WT pDCs. Intestinal GvHD scores and crypt apoptosis in the colon were higher in recipient groups transplanted without pDCs or with VIP-KO pDCs compared with recipients of WT pDCs (Fig 3a, b, c). These results indicate that VIP secreted from pDCs limits GvHD in the gut. In the GvL model, administration of pDCs lacking VIP did not alter the anti-tumor effect of donor T cells. Nanostring analysis of T cells recovered from VIP-KO pDC recipients had increased expression of the pro-inflammatory transcription factor Bhlhe40 during the first two weeks post-transplant, and higher transcription levels of the inflammatory mediator Cyclophilin A at day 15 post-transplant than T cells from recipients of WT pDCs. Conclusion: Data from in vitro and in vivo experiments suggest that VIP secreted by pDCs limits pathogenic T cell proliferation. In murine allo-BMT, increased gut GvHD scores and crypt apoptosis in recipients transplanted without pDCs or with VIP-KO pDCs indicates that VIP secreted by pDCs consolidates gut integrity without altering GvL. Gene expression analysis also supports a mechanism by which VIP-secreting donor pDCs reduce T cell inflammation through negative regulation of Bhlhe40. Our findings suggest paracrine VIP signaling is a novel immune checkpoint pathway by which donor pDCs limit T cell activation, Th1 polarization, and inflammation, and improve outcomes of allo-BMT by reducing GvHD activity. Figure 1 Figure 1. Disclosures Waller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

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