CD4+ Effector Memory and Naive T Cells Mediate Graft-Versus-Leukemia Via Direct Recognition of MHCII on Leukemic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 579-579 ◽  
Author(s):  
Hong Zheng ◽  
Catherine C. Matte ◽  
Srividhya Venkatesan ◽  
Britt E. Anderson ◽  
Mark J. Shlomchik ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cd4 T Cells ◽  
Bone Marrow Cells ◽  
Dominant Role ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Effector Memory ◽  
Cd4 Cells ◽  
Graft Versus Leukemia

Abstract One of the major challenges in allogeneic stem cell transplantation (alloSCT) is to separate graft-versus-host-disease (GVHD) from graft-versus-leukemia (GVL). We and others have previously demonstrated, in both major histocompatibility complex (MHC)-compatible/multiple minor histocompatibility antigen-mismatched and MHC-mismatched murine models of alloSCT, that spontaneous effector memory (EM) CD4+T cells depleted of regulatory CD25+ cells (CD4+CD44+CD62L-CD25-) do not cause GVHD. We have also shown that these EM CD4+ T cells can mediate GVL against a model of murine chronic phase of CML (mCP-CML) induced via retroviral transduction of BM cells with the bcr-abl fusion cDNA without causing GVHD (Zheng, et al ASH meeting 2004). In the present study we analyzed the effector mechanisms of these EM CD4+ cells in the B6bm12 → B6 MHCII disparate bone marrow transplantation (BMT) model. First, we demonstrated that the GVL activity of both EM and naïve CD4+ T cells required cognate interactions with CML targets as GVL was ineffective against mCP-CML induced in bone marrow from B6.I-Ab−/− (MHCII−) mice. Recipients of MHCII− mCP-CML died from mCP-CML between day 15-20 post BMT, regardless of whether they received EM or naïve CD4+ cells or no T cells at all. In light of data in the same model that parenchymal MHCII expression is not required for GVHD (Teshima et al, 2002), these data demonstrate distinct mechanisms for the cytotoxicity by CD4+ cells in GVL and GVHD—direct in the former and indirect in the latter. To further investigate the specific mechanisms of T cell killing, we tested the effectiveness of EM CD4+ cells in eradicating mCP-CML induced in bone marrow cells from Fas−/− and TNFR1/R2−/− mice. Both EM and naïve CD4+ cells mediated GVL against these gene deficient leukemias that was similar to that against wild type mCP-CML. In summary, these results suggest that EM and naive CD4+ cells mediate GVL via direct cognate engagement with targets. Their killing, however, does not depend on either FasL or TNF-α which suggests a dominant role for perforin, TRAIL, or both. Interestingly, although the mechanisms of recognition and killing of mCP-CML by either naïve or EM CD4+ T cells are so far indistinguishable, whereas only the naïve cells cause GVHD. Whereas a number of investigators have been able to separate mechanisms of killing in GVHD vs. GVL, this is to our knowledge the first clear demonstration of a difference in the mechanism of recognition between GVHD and GVL.

Sequential Blockade and Engagement of Co-Stimulatory Pathways: A Potential Strategy for Amplifying Graft-Versus-Leukemia Responses without GVHD.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3075-3075
Author(s):  
Ronjon Chakraverty ◽  
Jennifer Buchli ◽  
Guiling Zhao ◽  
Richard Hsu ◽  
Michael Croft ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cd8 T Cells ◽  
Bone Marrow Cells ◽  
Host Cells ◽  
Effector Memory ◽  
Activated T Cells ◽  
Class I Mhc ◽  
Graft Versus Leukemia ◽  
Ctl Activity

Abstract One potential approach for the effective disengagement of graft-versus-leukemia (GVL) effects from graft-versus-host disease (GVHD) following BMT is the use of nonmyeloablative conditioning as a platform for the adoptive transfer of donor T cells. In pre-clinical models, donor CD8+ T cells can induce powerful responses against tumors of host origin, but the effect lacks durability such that a re-challenge with tumor inevitability leads to tumor progression and death. This deficit is associated with the failure of functional CD8+ effector/memory T cells (TE/M) to survive long-term post-DLI. To examine the fate of GVH-reactive CD8+ T cells following DLI, we established mixed hematopoietic chimeras (MC) in a parent →F1 model using a nonmyeloablative protocol that incorporates co-stimulatory molecule blockade. B6D2F1 mice received 3Gy TBI and intra-peritoneal injections of anti-CD154 and anti-CD8 mAb on day 0 followed by infusion of 2 x 107 C57BL/6 bone marrow cells. 10 weeks later, when mAb had cleared from the circulation, MC received DLI that included CD8+ T cells from 2C transgenic mice that bear TCR specific for recipient class I MHC Ld. Using a clonotypic marker to monitor the response, we observed expansion of 2C CD8+ cells, peaking in the spleen on day 7 and then rapidly declining such that 2C CD8+ T cells were <0.1% of splenocytes by day 60. The decline in GVH-reactive T cells was associated with marked apoptosis and a sustained reduction in the expression of IL-7Rα. By day 60, no CTL activity against host cells was detectable. We reasoned that strategies that augment the survival of GVH TE/M might enhance the durability of the GVL response and, in the absence of tissue inflammation induced by conditioning, might not lead to GVHD. Co-stimulation through the tumor necrosis family receptor, OX40, which is expressed on activated T cells, is anti-apoptotic and enhances recruitment of TE/M to the memory pool. Following DLI, OX40 expression on 2C CD8+ T cells peaked on day 7 with somewhat earlier and sustained expression on DLI-derived CD4+ T cells. Since OX40 expression was specific for GVH-reactive T cells, we examined the effect of giving agonistic anti-OX40 antibody on day +5 following DLI. This was associated with rapid and complete conversion to full donor chimerism by day +14, whereas DLI + control antibody recipients had only partially converted by day +28. By day 60 post-DLI, anti-host CTL activity was clearly detectable in anti-OX40 recipients but not in controls. No clinical evidence of GVHD was observed, although histological examination revealed transient mild lymphocytic infiltration of the lamina propria on day +13, which resolved completely by day +18. In further experiments, anti-OX40 administration was associated with marked increases in the numbers of 2C CD8+ T cells in spleen, lymph node and bone marrow following DLI. Furthermore, effector differentiation, as assayed by intracellular expression of interferon-γ by 2C CD8+ T cells, was increased in recipients of anti-OX40 antibody. Of note, we observed a complete inhibition IL-7Rα down-regulation that is normally observed on activated CD8+ T cells following DLI. We conclude that OX40 co-stimulation following delayed DLI to established MC represents a potential means to enhance the magnitude and duration of a GVH reaction without the induction of significant GVHD.

Increased Expression of PD-1 on CD4+ T Cells from Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4154-4154
Author(s):  
Mary M Sartor ◽  
David J Gottlieb
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Effector Memory ◽  
Cd4 Cells ◽  
Normal Peripheral Blood

Abstract Although the predominant finding in patients with chronic lymphocytic leukemia (CLL) is an expansion of monoclonal B lymphocytes, a polyclonal expansion of T cells co-exists in CLL patients. Allogenic stem cell transplants for CLL suggest that a significant graft versus leukaemia effect mediated through recognition of minor MHC or leukaemia specific antigens can be achieved. Since it appears that the immune system and probably T cells recognise CLL cells, it is possible that one or more T cell defects might contribute to the initiation or maintenance of a clone of CLL lymphocytes. PD-1 is a coinhibitory molecule that is expressed on T cells in patients with chronic viral infections. It has been suggested that PD-1 expression might be a marker of cell exhaustion due to antigenic overstimulation. We examined the expression of PD-1 and its naturally occurring ligands PD-L1 and PD-L2 on both B and T cells in patients with CLL and compared this with expression on normal peripheral blood mononuclear cells. We found that PD-1 was expressed on over 10% of CD4+ T cells in 7 of 9 cases of CLL (mean 22±16%) but not on CD4+ T cells in any of 9 normal donors (mean 0±0%), p=0.0009. There was no difference in PD-1 expression on CD8+ or CD14+ PBMCs from CLL patients and normal donors (for CD8+ 24±21% and 19±16% for CLL and normals; for CD14+ 58±16% and 71±31% for CLL and normals). More than 10% of CD5+/19+ CLL cells expressed PD-1 in 7 of 10 cases (mean 18±18%) while more than 10% of normal B cells from 6 of 7 donors also expressed PD-1 (mean 49±30%). We examined the expression of PD-1 on naïve, central memory, effector memory and terminally differentiated subsets of CD4+ cells (CD62L+CD45RA+, CD62L+CD45RA−, CD62L−CD45RA− and CD62L−CD45RA+ respectively) from CLL patients and normal donors. The expression of PD-1 was higher on CD4+ cells from CLL patients in all subsets. The effect was most prominent in the effector memory subset (mean 54±4% for CLL patients versus 26±17% for normal donors, p=0.02). We looked for expression of PD-L1 and PD-L2 on T cells, B cells, monocytes and NK cells from CLL patients and normal donors. PD-L1 was only expressed on monocytes (mean 30±23%) and NK cells (mean 14±19%) from CLL patients and on monocytes from normal donors (mean 35±26%). There was no expression of PD-L2 on any cell type in either CLL patients or normal donors. We conclude that there is increased expression of the co-inhibitory molecule PD-1 on CD4+ T cells in patients with CLL. Ligation of PD-1 by PD-L1 expressed on monocytes or NK cells could inhibit immune responses to tumor and infectious antigens leading to persistence of clonally expanded cells and predisposition to opportunistic pathogens.

scholarly journals Peri-Transplant Steroids Enhance GVL and Attenuate GVHD By Redistributing Alloreactive Donor T Cells from the Gut into the Bone Marrow

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3210-3210
Author(s):  
Takayuki Inouye ◽  
Motoko Koyama ◽  
Ensbey Kathleen ◽  
Nicholas Greene ◽  
Luke Samson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Expansion ◽  
Effector Memory ◽  
Gi Tract ◽  
Donor T Cells ◽  
T Cell Expansion

Leukemia relapse represents a failure of graft-versus-leukemia (GVL) and remains the major limitation of allogeneic stem cell/bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) within the gastrointestinal (GI) tract is the principal determinant of transplant-related mortality and is initiated by a network of alloantigen presentation by professional and non-professional APC that prime donor T cells in the GI tract and related lymphoid structures. Since GVL and lethal GVHD are mediated by donor T cells at spatially distinct sites; bone marrow (BM) and the GI tract respectively, we sought tractable approaches to spatially separate alloreactive responses at these two locations. The administration of high dose steroids in the peri-transplant period is permissive of T cell replete HLA-haploidentical BMT and significant GVL effects (Ogawa H, et al. BBMT. 2006). We utilized murine haploidentical BMT models (B6D2F1 → B6C3F1, B6 → B6D2F1) with recipient background MLL/AF9 primary acute myeloid leukemia (AML), with or without dexamethasone (Dex) administration (5 mg/kg/day i.p., days -1 to +5). Dex-treatment improved transplant survival (from 25% to 68% at day 100, P=0.0012) with significant reductions in GVHD histopathology specifically in the colon (histopathology scores 8.7±1.0 vs 4.6±0.8, P< 0.05), despite excellent leukemia control. To understand this paradox, we analyzed the kinetics of donor T cell expansion after BMT. In the mesenteric lymph node (mLN), Dex treatment significantly suppressed the expansion of both CD4 and CD8 T cells (3.3±0.3 x 105 vs 1.4±0.3 x 105, P< 0.001 and 4.2±0.4 x 105 vs 2.1±0.4 x 105, P< 0.01 respectively) and the activation of CD4 T cells (CD25 MFI: 2021±146 vs 1056±102, P< 0.01). In contrast, donor effector/memory CD44+ CD8 T cells were expanded in the BM of Dex treated recipients (1.9±0.3 x 105 vs 3.1±0.4 x 105, P< 0.05) that demonstrated high per cell cytolytic activity against leukemia (specific lysis: 65±2.4 % vs 62±2.6 % in untreated vs Dex-treated, P> 0.05). Surprisingly, there was no difference in proliferation (cell tracking dye dilution: 63±5.5 % vs 57±5.5 % in untreated vs Dex-treated, P> 0.05) or apoptosis (caspase-3: 6.6±0.4 % vs 6.1±0.6 %, caspase-8: 20±1.6 % vs 17±3.3 % in untreated vs Dex-treated, respectively, P> 0.05) of CD4 T cells in the mLN between the two groups. We undertook experiments with luciferase expressing T cells and noted that Dex-treatment preferentially inhibited T cell accumulation in the GI tract, but not marrow after BMT. Thus, it appeared that Dex treatment preferentially re-distributed donor T cells from the GI tract to the bone marrow. We next determined if Dex exerted effects via direct signaling to the donor T cell. We thus transplanted glucocorticoid receptor (GR)-deficient or intact T cells (GRfl/fl lck-Cre mice). Dex-treatment reduced donor CD4 T cell expansion in the mLN independent of their expression of the GR (untreated vs Dex-treated: 2.8±0.6 x 105 vs 1.2±0.3 x 105, lckCREGRfl/fl and 2.4±0.3 x 105 vs 1.4±0.4 x 105, GRfl/fl littermates, P< 0.05 both groups). Thus steroid effects were mediated indirectly, putatively via effects on recipient alloantigen presentation. There was a marked reduction in recipient dendritic cells (DC) and macrophages expressing the Ea peptide within MHC class II in the GI tract of Dex-treated recipients (terminal Ileum YAe+ DC number 896±93 vs 356±40, P< 0.01, YAe+ macrophage number 1035±136 vs 355±97, P< 0.01). In conjunction with this, expression of the gut homing integrin a4b7 expression was reduced in CD4 T cells from Dex treated recipient mLN (25±1.6 % vs 17±1.7 %, P< 0.01), while the marrow homing integrin VLA-4 (a4b1) was increased (a4: 62±2.2 % vs 75±1.6 %, P< 0.001, b1: 52±2.5 % vs 61±1.6 %, P< 0.05) in donor CD8 T cells from Dex treated recipient BM. Finally, Dex treatment enhanced GVL against a second primary AML (BCR/ABL-NUP98/HOXA9) relative to untreated recipients and those receiving post-transplant cyclophosphamide (PT-Cy) (relapse rate: 0% vs 40% vs 100% at day 35 in Dex vs untreated vs PT-Cy, PT-Cy vs Dex-treated, P< 0.0001; untreated vs Dex-treated, P=0.029). These data suggest a potential therapeutic strategy to modulate antigen presentation in the GI tract and consequent integrin imprinting that minimizes GVHD lethality whilst enhancing GVL within BM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Thymus Transplantation Plus Allogeneic Bone Marrow Transplantation Can Induce Strong Graft-Versus Leukemia Effects with Low Risk of Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5798-5798
Author(s):  
Yuming Zhang ◽  
Xiaoqing Feng ◽  
Cuiling Wu ◽  
Wenling Guo ◽  
Huiping Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Graft Versus Host Disease ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Cd4 Cells ◽  
Graft Versus Host ◽  
Graft Versus Leukemia ◽  
Thymus Transplantation

Abstract Abstract: [Objective] Allogeneic bone marrow transplantation (allo-BMT) has been used for the treatment of refractory leukemia and donor lymphocyte infusion (DLI) was used for the purpose of graft versus leukemia (GVL) effects. However, DLI is associated with a risk of graft versus host disease (GVHD). Thus, new cellular-based methods are desired. In the present study, we performed thymus transplantation (TT) plus allo-BMT to explore it’s anti leukemia effects. [Methods] Recipient B6 mice (H-2b) bearing leukemia (EL-4 cells, H-2b) were irradiated 8 Gy. The next day, bone marrow cells from BALB/c mice (H-2d) were transplanted into the B6 mice. Simultaneously, DLI and thymus transplantation from the same donor were carried out. The survival period of the recipient B6 mice were examined, histological studies were performed in the liver, intestine, and the engrafted thymus from the recipients 4 weeks after the BMT. Surface markers on lymphocytes from the spleen were analyzed by 3-color fluorescence staining using a FACScan system to determine chimerism. [Results]. All mice treated with BMT showed fully donor-derived chimerism(H-2d). The survival rate in mice treated with BMT plus TT was significantly prolonged compared with those treated with BMT alone or BMT plus DLI. Histologically, both the cortex and medullar areas of the engrafted thymus under the renal capsule were clearly shown. Normal T-cell differentiation was also observed in the engrafted thymus. Microscopic founding of small intestine and liver in the BMT plus TT group indicated mild GVHD, whereas those treated with BMT plus DLI showed moderated to serious GVHD. The number of the CD4+ cells was significantly greater in the mice treated with BMT+TT compared with those with BMT alone or those with BMT + DLI. The percentage of FoxP3+ regulatory T cells among CD4+ cells was higher in the mice treated with BMT + TT comparable to those treated with BMT + DLI. The results for CD8+ T cells were similar to those for CD4+ cells. [Conclusion]. Allo-BMT combined with TT induces high thymopoiesis, and can elicit strong GVL effects with mild GVHD reaction. We thus found that donor-derived T cells play an important role in the treatment of leukemia. Also the details of the mechanisms are still unknown, one possibilities may be the continuous supplementation of T cells from the allogeneic thymus. The results of the present study suggest this strategy will become a new way for the treatment of refractory or relapse leukemia in human. Disclosures No relevant conflicts of interest to declare.

scholarly journals Early Homeostatic Expansion of Regulatory T Cells with the Predominant Effector/Memory Phenotype May Stabilize Immune Recovery in the First Month after HSCT

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2501-2501
Author(s):  
Takanori Yoshioka ◽  
Yusuke Meguri ◽  
Takeru Asano ◽  
Taro Masunari ◽  
Kumiko Kagawa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Acute Phase ◽  
Cd4 T Cells ◽  
Chronic Phase ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Ki 67 ◽  
Memory Phenotype ◽  
Post Transplant

Abstract CD4+Foxp3+ regulatory T cells (Treg) play a central role in establishing immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously reported that the long-term severe lymphopenia could result in the collapse of Treg homeostasis leading to the onset of chronic GVHD (Matsuoka et al. JCI 2010). However, Treg homeostasis in the very early phase after HSCT has not been well studied. To address this issue, we here examined the early lymphocytes reconstitution in total 34 patients who received HSCT. Peripheral blood samples were obtained at 2, 4, 8 and 12 weeks after transplant and analyzed the reconstitution of CD4+CD25med-highCD127lowFoxp3+ Treg comparing with CD4+CD25neg-lowCD127highFoxp3- conventional T cell (Tcon) and CD8+ T cells. CD4 T cell subsets are further divided into subpopulations by the expression of CD45RA and CD31. The expressions of Helios, Ki-67, Bcl-2 and C-C chemokine receptor type 4 (CCR4) on these subsets were also examined. These patients were transplanted the grafts from various stem cell sources (7 HLA-matched PBSCT, 12 HLA-matched BMT, 6 HLA-mismatched CBT and 9 HLA-haploidentical PBSCT) and this enables us the opportunity to comparatively evaluate the early lymphocyte reconstitution among the different types of HSCT. After transplant, total lymphocyte counts were significantly lower than the counts before the start of conditioning (median lymphocytes 113/ul at 2 weeks and 223/ul at 4 weeks vs 550/ul before conditioning, P<0.01 and P<0.01, respectively). In the severely lymphopenic condition in the first month after HSCT, all T cell subsets were undergoing aggressive proliferation in this acute phase as compared to proliferation in the chronic phase, however, Treg proliferation was significantly higher than in Tcon at 4 weeks (%Ki-67+ cells; median 56.4%, 23.4%, respectively, P<0.02). %Treg of total CD4 T cells elevated and peaked at 4 weeks post-transplant. At this timepoint, %Treg of CD4 T cells showed the clear inverse correlation with %CD45RA+ of Treg (r2=0.40), suggesting the expansion of Treg in this phase appears to be a result from severe lymphopenia-driven proliferation which involves conversion from naive into memory phenotype. Elevation of %Treg was most evident in the patients who received HLA-haploidentical graft after ATG-containing conditioning (median 8.41% in haplo-HSCT, 5.25% in other groups, P<0.05), again indicating the lymphopenia is critical factor to drive Treg proliferatrion. Expanded Treg showed a predominant Helios+CD45RA-CD31- effector/memory phenotype with the lower level of Bcl-2 expression as compared to CD45RA+ naïve Treg. The elevation of Treg did not sustain and %Treg of CD4 T cells got back to the baseline level by 8 weeks. During the first 3 months after HSCT, CD45RA- Treg exhibited high level of CCR4 and the recovery of this subset was critically delayed in Adult T-cell Leukemia (ATL) patients treated with anti-CCR4 antibody in the peri-transplant period, resulting in the development of acute graft-versus-host diseases. In conclusion, our findings suggest that, not only in the chronic phase but also in the acute phase, the homeostasis of Treg is more susceptible to the post-transplant environment as compared to other lymphocyte subsets. Post-transplant lymphopenia drives aggressive Treg proliferation resulting in the increased percentage of this subset in the very acute phase which may contribute to stabilize the immune recovery. The careful monitoring of Treg from the point of view might provide important information to promote immune tolerance. Disclosures No relevant conflicts of interest to declare.

scholarly journals CTLA-4 on alloreactive CD4 T cells interacts with recipient CD80/86 to promote tolerance

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3475-3484 ◽  
Author(s):  
Josef Kurtz ◽  
Forum Raval ◽  
Casey Vallot ◽  
Jayden Der ◽  
Megan Sykes
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Bone Marrow Cells ◽  
Antigen Presenting Cells ◽  
Cd4 T Cell ◽  
Cell Tolerance ◽  
Antigen Presenting ◽  
Marrow Cells

Abstract Although the inhibitory receptor CTLA-4 (CD152) has been implicated in peripheral CD4 T-cell tolerance, its mechanism of action remains poorly defined. We analyzed mechanisms of CD4 cell tolerance in a model of tolerance induction involving establishment of mixed hematopoietic chimerism in recipients of fully MHC-mismatched allogeneic bone marrow cells with anti-CD154 mAb. Animals lacking CD80 and CD86 failed to achieve chimerism. We detected no T cell–intrinsic requirement for CD28 for chimerism induction. However, a CD4 T cell–intrinsic signal through CTLA-4 was shown to be essential within the first 48 hours of exposure to alloantigen for the establishment of tolerance and mixed chimerism. This signal must be provided by a recipient CD80/86+ non–T-cell population. Donor CD80/86 expression was insufficient to achieve tolerance. Together, our findings demonstrate a surprising role for interactions of CTLA-4 expressed by alloreactive peripheral CD4 T cells with CD80/86 on recipient antigen-presenting cells (APCs) in the induction of early tolerance, suggesting a 3-cell tolerance model involving directly alloreactive CD4 cells, donor antigen-expressing bone marrow cells, and recipient antigen-presenting cells. This tolerance is independent of regulatory T cells and culminates in the deletion of directly alloreactive CD4 T cells.

A Novel Strategy for Expanding Primitive Leukemic Cells from Chronic Phase CML Patients by Forced Overexpression of a NUP98-HOXA10 Homeodomain Fusion Gene.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1078-1078
Author(s):  
Ivan Sloma ◽  
Suzan Imren ◽  
Yun Zhao ◽  
Keith Humphries ◽  
Connie J. Eaves
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fusion Gene ◽  
Low Frequency ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Human Cord Blood ◽  
Hematopoietic Stem

Abstract Analysis of the leukemic stem cell compartment in CML patients with chronic phase disease remains a major challenge. This is due to the usually low frequency of these cells in the bone marrow and blood of most patients regardless of the WBC count and the fact that they are typically outnumbered by normal hematopoietic stem cells from which they cannot be currently separated. Moreover, thus far it has not been possible to identify conditions for their selective expansion in vitro or in vivo. To pursue this goal, we have begun to explore the effects of certain HOX gene-containing constructs on primitive chronic phase CML cells based on previous evidence that these genes markedly enhance the expansion of primitive normal murine and human cord blood cell numbers without inducing leukemia. Lineage-negative peripheral blood or bone marrow cells from 3 chronic phase CML patients (with &gt;93%, &lt;20% and &lt;6% Ph+ LTC-ICs by G-banding karyotyping) were pre-stimulated overnight in a medium containing a serum substitute and 100 ng/ml hSteel Factor (SF), 100 ng/ml hFlt3-ligand and 20 ng/ml each of hIL-3, hIL-6 and hG-CSF. Cells were then exposed to a lenti-PGK-GFP virus with or without an upstream MDUS-NUP98-HOXA10 homeodomain (HD) element for 5 hours in the same medium. After removal of the virus, the cells were maintained in culture under the same conditions for 2 more days to allow full expression of the transduced genes. At this point, both cultures contained the same number of total cells, GFP+ cells and clonogenic progenitors (BFU-E + CFU-GM + CFU-GEMM); i.e., 2.2±0.5 x105 vs 2.2±0.6 x105 total cells, 1.0±0.2 x105 vs 1.3±0.3 x105 GFP+ cells, 3.6±1.7 x104 vs 3.4±1.7 x104 total CFCs and 1.7±0.9 x104 vs 2.4±1.3 x104 GFP+ CFCs per 105 starting lin- cells. However, after the 2-day post-transduction, cells had been maintained for 6 weeks in longterm cultures (LTCs) containing murine stromal cells producing hIL-3, hSF and hG-CSF, we noted a markedly higher (4 to 74-fold) output of CFCs from the NUP98-HOXA10HD-transduced cells. Moreover, whereas the proportion of GFP+ CFCs in the 2-day post-transduction cultures was on average only 31% and 48 % for the control and tested cells respectively, this increased to &gt;98% in the 6-week LTCs initiated with cells that were overexpressing NUP98-HOXA10HD but remained constant at 39% in the control LTCs - suggesting a significant growth advantage conferred by the NUP98A10HD transgene. Importantly, RT-PCR genotyping of the colonies in these assays showed the majority of LTC-IC-derived CFCs from the NUP98-HOXA10HD-transduced cells to be BCR-ABL+, indicative of an even greater output of CFCs by the NUP98-HOXA10HD transduced BCR-ABL+ vs normal cells. These results highlight the potential of NUP98-HOXA10HD to selectively expand primitive CML cells isolated directly from chronic phase patients which will facilitate their further investigation and use to screen and validate new therapeutic agents.

Two Photon Intravital Microscopy Reveals CD4+ T Cells Making Cognate Interactions With Tissue Dendritic Cells In Skin Graft-Versus-Host-Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2004-2004
Author(s):  
Sarah Morin-Zorman ◽  
Christian Wysocki ◽  
Edwina Kisanga ◽  
David Gonzalez ◽  
David Rothstein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Cd4 Cells ◽  
Cd8 Cells ◽  
Graft Versus Host ◽  
Two Photon

Abstract Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs (SLO), we have consistently observed MHCII+ donor-derived tissue APCs (t-APCs), including tissue dendritic cells (t-DCs) in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells. We hypothesize that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease locally. We could not use knockout or APC depletion approaches to study T cell: t-APCs interactions as they impact APCs systemically and might therefore affect T cell stimulation in SLO. We therefore utilized two-photon intravital microscopy to analyze interactions between fluorescent donor CD4+ T cells and t-DCs in skin. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) CD11c-YFP transgenic (Tg) Bone Marrow (BM) with B6 RFP Tg CD4 cells and nonfluorescent B6 CD8 cells. We imaged ear skin in GVHD mice 4 weeks later. In general CD4 cells co-localized with DCs. We observed CD4+ T cells that were highly motile and only in transient contact with DCs and others that made stable contact with DCs. To determine how much TCR: MHCII interactions drive sustained CD4+ T cell: DC interactions and arrest CD4+ T cell motility, mice were imaged and then injected with an MHCII blocking antibody (Ab; Y3P) with continued imaging of the same regions. After injection, T cell mean speed significantly increased and the proportion of T cells in stable contact with DCs decreased, indicating that transient disruption of TCR: MHCII is sufficient to restore motility to some T cells. In a second approach to assess the specificity of CD4+ T cell: t-DC interactions we transplanted 129 mice with B6 RFP+ CD4 cells, nonfluorescent CD8 cells and a mix of CD11c-YFP MHCII-/- and RFP wt BM or a mix of CD11c-YFP wt and RFP Tg MHCII-/- BM. We are currently comparing the motility of CD4 cells that make contact with MHCII+ as compared to MHCII- DCs, with the prediction that contact times will be shorter with the latter. Our data suggest that CD4+ T cells make cognate interactions with t-DCs in skin and we hypothesize that these interactions promote GVHD locally. Because the graft-versus-leukemia effect occurs primarily in bone marrow and secondary lymphoid tissues, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL. Disclosures: No relevant conflicts of interest to declare.

Signaling Profiles of T Cells during Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1262-1262
Author(s):  
Sydney X. Lu ◽  
Vanessa M. Hubbard ◽  
Melanie Y. Chow ◽  
Theo D. Kim ◽  
David Y. Suh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Caspase 3 ◽  
Effector Memory ◽  
Cd4 Cells ◽  
Histone H2b ◽  
Pathway Activity ◽  
Cd8 Cells ◽  
Signaling Activity

Abstract In this study we characterized the signaling profiles of T cells in murine allogeneic bone marrow transplantation (BMT) models. We used a novel high-throughput technique to analyze activation of signaling pathways by flow cytometry. We first studied normal splenic T cells (Table 1) and found that relative to naïve CD4 and CD8 cells, effector and memory cells displayed nearly global increases in activation of signaling pathways including the MAP kinases and STATs. We observed similar increases in signaling activity in effector/memory vs. naive T cells in the mesenteric and peripheral lymph nodes (PLN) and the liver. Signaling in the C57BL/6 spleen CD4 CD8 Naïve (CD44lo CD62Lhi) Effector (CD44hi CD62Llo) Memory (CD44hi CD62Lhi) Naïve (CD44lo) Effector/Memory (CD44hi) Ratios of activation of signaling molecules in effector or memory CD4 or CD8 cells compared with naïve cells. c-Raf (pS259) 1 2.2 1.8 1 1.2 MEK 1/2 (pS217/pS221) 1 2.7 9.7 1 2.2 ERK 1/2 (pT202/pY204) 1 1.6 1.8 1 1.2 JNK (pT183/pY185) 1 1.1 6.5 1 2.3 Pan-p38 (pT180/pY182) 1 2.1 3.0 1 1.8 Pan-Akt (pT308) 1 2.1 3.8 1 1.4 Pan-Akt (pS473) 1 2.6 3.9 1 1.7 Pan-PKC ( β II pS660) 1 2.7 10.1 1 1.4 STAT-1 (pY701) 1 0.9 6.3 1 2.4 STAT-3 (pY705) 1 1.4 1.8 1 1.3 STAT-3 (pS727) 1 1.2 1.3 1 1.3 STAT-4 (pY693) 1 2.0 2.3 1 1.2 STAT-5A (pY694) 1 1.3 1.7 1 1.3 S6 Ribosomal Protein 1 3.0 6.3 1 1.8 (pS235/pS236) Histone H2B (pS14) 1 2.6 4.2 1 1.8 Cleaved Caspase 3 (D175) 1 2.0 3.8 1 1.3 PIP2 1 1.3 0.4 1 0.6 In the thymus, there was a general lack of signaling activity in CD4+CD8+ (DP) cells, and these responded poorly to cytokine stimulation. By contrast, CD4−CD8− (DN) cells of the DN1 and DN2 stage had the greatest signaling activity of all thymocytes. We then studied alloreactive and homeostatically proliferating T cells by infusion of carboxyfluorescein (CFSE) labeled T cells into irradiated hosts. We noted a gradual and global increase in signaling activity with each cell division in the spleen, liver, and PLN. Peak increases relative to the undivided CFSEhi population of up to 2.5-fold (ERK 1/2) in the CD4 cells by the 4th cell division, with similar increases in CD8 cells, were found. Even the undivided CD4 CFSEhi population itself displayed large increases in signal (~4 fold for p38 and ERK 1/2) relative to naïve splenic CD4 cells. We defined slow-cycling cells as homeostatically expanding and fast-cycling cells as alloreactive, and found a distinct signaling pattern in homeostatically expanding T cells, with lower levels of MAP kinase pathway activity, JAK/STAT pathway activity, and pro-apoptotic proteins such as cleaved caspase 3 and phosphorylated histone H2B. However, PKC phosphorylation appeared to be higher in homeostatically proliferating cells than alloreactive T cells. In the setting of T-cell depleted bone marrow transplantation, we found in several models that newly generated, donor-derived splenic T cells had signaling levels generally comparable with that of the naïve populations in a normal spleen, but with a marked deficiency in STAT-3 (pS727) phosphorylation. In the thymus, we observed defects in Akt and STAT-3 (pS727 and pY705) signaling in the DN, DP, CD4, and CD8 subsets relative to signaling levels in the normal thymus. We conclude that the high-throughput analysis of signaling activity in T cells during allogeneic BMT allows for the definition of distinct signaling profiles. These signaling signatures can be used for the development of targeted therapies to enhance or inhibit specific T cell subsets.

Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1261-1261 ◽  
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Masaki Yasukawa ◽  
Toshihiro Miyamoto ◽  
Goichi Yoshimoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Self Renewal ◽  
Human T Cells

Abstract Transplantation of human leukemic cells into severe combined immunodeficiency (SCID) mice has been used to analyze developmental mechanisms of human leukemogenesis. Previous models, however, were limited in efficient or long-term engraftment of leukemia initiating cells. Here we report a new SCID model that supports highly efficient long-term engraftment of primary human acute myelogenous leukemia (AML) cells. We have established a novel immune-compromised mouse by backcrossing a complete null mutation of the common cytokine receptor g chain onto NOD-scid mice (NOD/SCID/IL2rgnull mice), and reported that normal human cord blood-derived hematopoietic stem cells efficiently engrafted in newborn NOD/SCID/IL2rgnull mice as compared to NOD/SCID/b2mnull mice (Ishikawa et al, Blood in press). Injection of 5x106 total bone marrow mononuclear cells from primary AML patients (FAB subtypes: M1, M2, M3, M4 and M7) into sublethally-irradiated newborn NOD/SCID/IL2rgnull mice, however, did not result in efficient engraftment of AML cells, while predominant proliferation of human CD4+ and CD8+ T cells was seen. These human T cells expressed CD45RO, and levels of human IFN-g in sera of the recipients significantly elevated, suggesting that human T cells were activated and inhibited the engraftment of human AML cells in the xenogeneic setting. We thus transplanted AML cells after T cell depletion. Strikingly, transplantation of 4x106 T cell-depleted AML bone marrow cells into neonatal NOD/SCID/IL2rgnull mice resulted in the efficient AML engraftment, whose levels were significantly higher than those in transplantation of the same number of T cell-depleted AML cells into NOD/SCID/b2mnull newborns or NOD/SCID/IL2rgnull adults. We also transplanted 103–104 hCD34+hCD38− bone marrow cells purified from AML patients. These low-doses of hCD34+hCD38− cells also successfully engrafted, progressively giving rise to hCD34+hCD38+ and hCD34− leukemic cells over 16 weeks. hCD34+hCD38− cells purified from the bone marrow of primary NOD/SCID/IL2rgnull recipients again reconstituted AML in secondary recipients, indicating that this system supports self-renewal capacity of AML stem cells within the hCD34+hCD38− fraction. Thus, the NOD/SCID/IL2rgnull newborn system provides a powerful model to study human leukemogenesis as well as the interaction between human T cells and AML cells in vivo.

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