G-CSF Activated Granulocytes Inhibit Experimental Graft-versus-Host Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4989-4989
Author(s):  
Zilton F.M. Vasconcelos ◽  
Julia Farache ◽  
Bruna M. Santos Grad ◽  
Tereza S. Palmeira Grad ◽  
Luis Fernando Bouzas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Activity ◽  
Control Group ◽  
Low Density ◽  
Graft Versus Host ◽  
The One

Abstract Acute Graft versus host diseas (aGVHD) is a major complication of stem cell transplantation. The disease is mediated by T cells and a higher incidence/severity would be expected when higher numbers of T cells are inoculated. However, the incidence of aGVHD in PBST, which carries about 10 times more T cells then BMT, is not higher than the one found in later. This finding indicates a modulatory role for G-CSF over T cell activity. We had previously shown that T cells from G-CSF treated PBSC donors do not produce g-IFN nor IL-4 and that this inhibition was mediated by low density, G-CSF activated, granulocytes. In order to test if in fact G-CSF activated granulocytes could inhibit disease, we first checked if G-CSF could generate low density granulocytes, in vivo and in vitro. Indeed, either in vivo(21mg /day - 5 days) or in vitro (150 ng -12hs) with G-CSF generates low density granulocytes which co-purify with the mononuclear cells in the ficoll® gradient. Moreover, as we had shown in humans, these low density cells, inhibit the production of g-IFN by anti-CD3 activated T cells on flow cytometry studies (17%-T cells alone versus 3% T cells with granulocytes 1:1). Radiation quimaeras were set with (B6 X BALB/c)F1 as hosts reconstituted with T cell depleted C57Bl6 bone marrow, in the presence or absence of nylon wool selected spleen cells (NWSC), as T cell source, from normal or G-CSF treated mice. As previously shown by others, NWSC from G-CSF treated mice diminishes the incidence of acute disease on day 20 post-transplant, from 75 to 25%. In order to investigate if this inhibition was dependent on the activated granulocytes present in the NWSC from G-CSF treated mice, granulocytes were depleted with anti-GR1 and complement. In this case, the incidence of disease is the same or even higher (75% experiment#1 and 100% in experiment #2) than the one observed on the control group (NWSC from control mice). These results strongly suggest that activated granulocytes could indeed inhibit aGVHD. We then generated activated granulocytes in vitro, by treating spleen derived high density granulocytes with 150ng of G-CSF for 12 hs. After the incubation period, a new ficoll® gradient was performed and the low density cells were obtained. T cell contamination on the second gradient was eliminated by anti-CD4 and CD8 complement lysis. These activated granulocytes were inoculated together with NWSC from control mice in the radiation quimaeras at a 1:1 ratio. In this case 100% disease inhibition was observed when compared to the positive control group, where 75% of the animals got sick. Our data indicate that activated granulocytes are the major mediators of the G-CSF immunossupressive effects and that these cells can be used as a novel immune modulator in clinical transplantation to prevent acute GVHD.

Prevention of Transfusion-Associated Graft-Versus-Host Disease by Photochemical Treatment

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3140-3147 ◽  
Author(s):  
Joshua A. Grass ◽  
Tamim Wafa ◽  
Aaron Reames ◽  
David Wages ◽  
Laurence Corash ◽  
...  
Keyword(s):  
T Cells ◽  
Gamma Radiation ◽  
Graft Versus Host Disease ◽  
Clinical Signs ◽  
Peripheral Blood Cell ◽  
Control Group ◽  
Host Disease ◽  
Graft Versus Host

Abstract Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 108 splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 μmol/L S-59 and 2.1 J/cm2UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 μmol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 105.1 murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.

Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3045-3045
Author(s):  
Yoshinobu Maeda ◽  
Pavan Reddy ◽  
Chen Liu ◽  
D. Keith Bishop ◽  
James L.M. Ferrara
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Graft Versus Host Disease ◽  
Serum Levels ◽  
Host Disease ◽  
Graft Versus Host ◽  
Full Intensity

Abstract Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P<0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P<0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P<0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P<0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.

Roscovitine Prevents TCR-Mediated T Cell Clonal Expansion In-Vitro and Protects Against GvHD In-Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 318-318 ◽  
Author(s):  
Lequn Li ◽  
Hui Wang ◽  
Vassiliki A. Boussiotis
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
T Cell Responses ◽  
Clonal Expansion ◽  
Control Group ◽  
Cell Responses

Abstract Cell cycle re-entry of quiescent T lymphocytes is required for generation of productive T cell responses. Cyclin-dependent kinases (cdk), particularly cdk2, have an essential role in cell cycle re-entry. Cdk2 promotes phosphorylation of Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Besides Rb, cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits cell cycle progression from G1 to S phase, and impaired phosphorylation on the cdk-mediated sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. Recently, we determined that induction of T cell tolerance resulted in impaired cdk2 activity, leading to reduced levels of Smad3 phosphorylation on cdk-specific sites and increased Smad3 antiproliferative function due to upregulation of p15. We hypothesized that pharmacologic inhibition of cdk2 during antigen-mediated T cell stimulation might provide an effective strategy to control T cell expansion and induce tolerance. (R)-roscovitine (CYC202) is a potent inhibitor of cdk2-cyclin E, which in higher concentrations also inhibits other cdk-cyclin complexes including cdk7, cdk9 and cdk5. It is currently in clinical trials as anticancer drug and recently was shown to induce long-lasting arrest of murine polycystic kidney disease. We examined the effect of roscovitine on T cell responses in vitro and in vivo. We stimulated C57BL/6 T cells with anti-CD3-plus-anti-CD28 mAbs, DO11.10 TCR-transgenic T cells with OVA peptide or C57BL/6 T cells with MHC disparate Balb/c splenocytes. Addition of roscovitine in these cultures resulted in blockade of cell proliferation without induction of apoptosis. Biochemical analysis revealed that roscovitine prevented phosphorylation of cdk2, downregulation of p27, phosphorylation of Rb and synthesis of cyclin A, suggesting an effective G1/S cell cycle block. To determine whether roscovitine could also inhibit clonal expansion of activated T cells in vivo, we employed a mouse model of GvHD. Recipient (C57BL/6 x DBA/2) F1 mice were lethally irradiated and were subsequently infused with bone marrow cells and splenocytes, as source of allogeneic T cells, from parental C57BL/6 donors. Roscovitine or vehicle-control was given at the time of allogeneic BMT and on a trice-weekly basis thereafter for a total of three weeks. Administration of roscovitine protected against acute GvHD resulting in a median survival of 49 days in the roscovitine-treated group compared to 24 days in the control group (p=0.005), and significantly less weight loss. Importantly, roscovitine treatment had no adverse effects on engraftment, resulting in full donor chimerism in the treated mice. To examine whether tolerance had been induced by in vivo treatment with roscovitine, we examined in vitro rechallenge responses. While control C57BL/6 T cells exhibited robust responses when stimulated with (C57BL/6 x DBA/2) F1 splenocytes, responses of T cells isolated from roscovitine-treated recipients against (C57BL/6 x DBA/2) F1 splenocytes were abrogated. These results indicate that roscovitine has direct effects on preventing TCR-mediated clonal expansion in vitro and in vivo and may provide a novel therapeutic approach for control of GvHD.

Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1972-1972
Author(s):  
Gerald P. Morris ◽  
Geoffrey L Uy ◽  
David L Donermeyer ◽  
Paul M Allen ◽  
John F. DiPersio
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Peripheral Blood ◽  
Thymic Selection ◽  
Cell Repertoire ◽  
Host Disease ◽  
Graft Versus Host

Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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 ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4484-4484 ◽  
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.

scholarly journals Antigen-specific suppressor T-cell activity in genetically restricted immune spleen cells.

1978 ◽  
Vol 148 (5) ◽  
pp. 1271-1281 ◽  
Author(s):  
C W Pierce ◽  
J A Kapp
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Antibody Responses ◽  
Spleen Cells ◽  
Suppressor T Cells ◽  
Specific Suppressor T Cells ◽  
Immune Spleen Cells

Virgin spleen cells develop comparable primary antibody responses in vitro to syngeneic or allogeneic macrophages (Mphi) bearing the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT), whereas immune spleen cells primed with syngeneic or allogeneic GAT-Mphi develop secondary responses preferentially when stimulated with GAT-Mphi syngeneic to the GAT-Mphi used for priming in vivo. These restrictions are mediated by products of the I-A subregion of the H-2 complex and are operative at the level of the GAT-Mphi-immune helper T-cell interactions. To investigate why these immune spleen cells fail to develop a significant antibody response to GAT-Mphi other than those used for in vivo immunization and determine the mechanism by which the restriction is maintained, spleen cells from virgin and syngeneic or allogeneic GAT-Mphi-primed mice were co-cultured in the presence of GAT-Mphi of various haplotypes. Antibody responses to GAT developed only in the presence of GAT-Mphi syngeneic to the Mphi used for in vivo priming; responses in cultures with GAT-Mphi allogeneic to the priming Mphi, whether these Mphi were syngeneic or allogeneic with respect to the responding spleen cells, were suppressed. The suppression was mediated by GAT-specific radiosensitive T cells. Thus, development of GAT-specific suppressor T cells appears to be a natural consequence of the immune response to GAT in responder as well as nonresponder mice. The implications of stimulation of genetically restricted immune helper T cells, and antigen-specific, but unrestricted, suppressor T cells after immunization with GAT-Mphi in vivo are discussed in the context of regulatory mechanisms in antibody responses.

Blocking the regulatory T cell molecule LAG-3 augments in vivo anti-tumor immunity in an autochthonous model of prostate cancer

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2573-2573
Author(s):  
C. G. Drake ◽  
C. Kelleher ◽  
T. Bruno ◽  
T. Harris ◽  
D. Flies ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cell ◽  
Specific Antigen ◽  
Cell Activity ◽  
Cd8 T Cell ◽  
Regulatory Function

2573 Background: LAG-3 is a CD4 homolog expressed on activated T cells, NK cells, tumor infiltrating lymphocytes (TIL), and plasmacytoid dendritic cells. Recently, we showed that LAG-3 was relatively overexpressed in specific T cells rendered unresponsive in vivo by the presence of cognate self-antigen. These anergic T cells display regulatory function both in vitro and in vivo, and blockade of LAG-3 with a non-depleting monoclonal antibody significantly mitigates their regulatory T cell activity. Methods: Using a novel model of prostate cancer in which a tumor-specific antigen is expressed in autochthonous tumors, we tested whether treatment with a non-depleting anti-LAG-3 antibody affected trafficking and function of tumor-specific T cells. Results: LAG-3 blockade significantly augments specific CD8 T cell trafficking to antigen-expressing tumors, but not to normal tissue. Most significantly, LAG-3 blockade functionally reversed CD8 T cell tolerance as assayed by an in vivo cytotoxic T lymphocyte (CTL) assay. Combining LAG-3 blockade with specific anti-tumor vaccination results in a dramatic increase in activated CD8 T cells in the tumor parenchyma. Conclusions: Taken together, these data support the concept that treatment with a LAG-3 blocking antibody may significantly delay disease progression in patients with cancer. We have recently generated a panel of monoclonal antibodies directed against human LAG-3; several of these antibodies significantly augment human T cell responses in vitro. No significant financial relationships to disclose.

Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1311-1311
Author(s):  
Corinna Leng ◽  
Cuiling Li ◽  
Judy Ziegler ◽  
Anna Lokshin ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Histone Deacetylase ◽  
Graft Versus Host Disease ◽  
Host Disease ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ

Abstract Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.

Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3728-3728
Author(s):  
Kenrick Semple ◽  
Antony Nguyen ◽  
Yu Yu ◽  
Claudio Anasetti ◽  
Xue-Zhong Yu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Graft Versus Host ◽  
Cell Immunity ◽  
Cd28 Costimulation ◽  
Insight Into

Abstract Abstract 3728 CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

2020 ◽  
Vol 8 (2) ◽  
pp. e001419
Author(s):  
Samanta Romina Zanetti ◽  
Paola Alejandra Romecin ◽  
Meritxell Vinyoles ◽  
Manel Juan ◽  
José Luis Fuster ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Cell Responses ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Severe Colitis

BackgroundAlthough adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied.MethodsWe performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model.ResultsWhile B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC.ConclusionsCollectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.

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