Number Of Donor CD4 Research Articles

Impaired bone marrow B-cell development in mice with a bronchiolitis obliterans model of cGVHD

AbstractChronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients after allogeneic bone marrow (BM) or stem cell transplantation (allo-SCT). Recent work has indicated that both T and B lymphocytes play an important role in the pathophysiology of cGVHD. Previously, our group showed a critical role for the germinal center response in the function of B cells using a bronchiolitis obliterans (BO) model of cGVHD. Here, we demonstrated for the first time that cGVHD is associated with severe defects in the generation of BM B lymphoid and uncommitted common lymphoid progenitor cells. We found an increase in the number of donor CD4+ T cells in the BM of mice with cGVHD that was negatively correlated with B-cell development and the frequency of osteoblasts and Prrx-1–expressing perivascular stromal cells, which are present in the B-cell niche. Use of anti-DR3 monoclonal antibodies to enhance the number of donor regulatory T cells (Tregs) in the donor T-cell inoculum ameliorated the pathology associated with BO in this model. This correlated with an increased number of endosteal osteoblastic cells and significantly improved the generation of B-cell precursors in the BM after allo-SCT. Our work indicates that donor Tregs play a critical role in preserving the generation of B-cell precursors in the BM after allo-SCT. Approaches to enhance the number and/or function of donor Tregs that do not enhance conventional T-cell activity may be important to decrease the incidence and severity of cGVHD in part through normal B-cell lymphopoiesis.

Expression Of PD-L1 Is Required To Control Acute GvHD In Allo-HSCT Recipients

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with both malignant and non-malignant hematologic disorders. But life-threatening graft-vs-host diseases (GvHD) caused by alloreactive donor T cells limits its clinical use. Alloreactive T cells are also required for graft-vs-leukemia (GvL) and to fight opportunistic infections. Hence, a method that modulates donor T cells activity to reduce GvHD but to retain GvL effect is highly desirable. The inhibitory receptor programed cell death-1 (PD-1) reduces T cell activation through binding with its ligand PD-L1 or PD-L2. Interaction between PD-1 and PD-L1 induces cardiac allograft tolerance and expression of PD-L1 is upregulated in presence of inflammatory stimuli. Here, we studied the role of PD-L1 expression on hematologic and non-hematologic tissues and PD-1 - PD-L1 binding in the development of GvHD. MethodsWild type C57BL/6 (WT B6), PD-L1 knock out B6 (KO) and PD-L2 KO B6 mice were transplanted with 2 x106 splenic T cells and bone marrow (BM) cells from H-2K B10.BR donors. The average acute GvHD scores were determined by combining the GvHD scores obtained from the histological tissue sections of small intestine, large intestine and liver, and weight-loss, posture, activity, fur texture and skin integrity data following standard published procedures. The activation status of splenic T cells was analyzed by flow cytometry. Serum cytokines were determined by using 26 plex Luminex assay. The requirement of hematopoietic and or non-hematopoietic tissues expressing PD-L1 to reduce GvHD was investigated by generating radiation chimeras using WT B6 mice engrafted with PD-L1 KO BM and vice versa. Two months later radiation chimeras were transplanted again with 2 x106 splenic T cells along with 2 x106 BM cells from congenic na•ve H-2K donors. The role of PD-L1 expressing donor hematopoietic cells on the development of acute GvHD was tested by transplanting B10.BR mice with donor PD-L1 KO B6 and WT B6 splenocytes. ResultsPD-L1 KO B6 recipients had significantly increased acute GvHD (scores 1.68 ± 0.07) compared with WT B6 GvHD (0.78 ± 0.024, p<0.0005) and B6 PD-L2 KO B6 (0.86 ± 0.14, p<0.0005) within day 8 after transplant. All PD-L1 KO B6 recipients had severe GvHD with >25% weight loss on day 8 after transplant and were sacrificed. The WT B6 and PD-L2 KO B6 recipients survived 75% and 80%, respectively until 34 days of transplantation with similar levels of chronic GvHD. To test whether excessive activation of donor T cells caused severe acute GvHD in PD-L1 KO B6 recipients, we determined the activation status of donor T cells in the spleen. The numbers of donor CD4+ and CD8+ T cells expressing ICOS-1 and PD-1 in the spleen were significantly higher (p<0.005) in PD-L1 KO B6 recipients compared with the WT B6 and PD-L2 KO B6 recipients. Additionally, significantly increased levels of serum inflammatory cytokines (IFN-g and TNF-a) were also detected in the PD-L1 KO B6 recipients compared with the WT B6 recipients on day 8 post transplant (Figure 1). Using WT B6 or PD-L1KO hematopoietic cell radiation chimeras as allo-HSCT recipients, we further confirmed that both allo-HSCT radiation chimeras having PD-L1 expressing hematopoietic (10% survival, open square) and non-hematopoietic cells (10% survival, closed triangle) were required to protect from GvHD (Figure 2). We next investigate whether PD-L1 KO donor cells cause increased GvHD. The B10.BR recipients transplanted with donor PD-L1 KO B6 splenocytes had 70% survival while the same recipients transplanted with WT B6 donor splenocytes had only 20% survival until 100 days post transplant. These data suggest that only PD-L1 expressed by host tissues is required to inhibit the development of GvHD. [Display omitted] [Display omitted] In summary, our data suggest that PD-L1 expressed by host tissues are required to control GvHD and method(s) that enhance expression of PD-L1 in allo-HSCT recipients may represent a novel strategy to control GvHD. Disclosures:No relevant conflicts of interest to declare.

Attenuation by Targeting the B- and T-Cell Attenuator

BTLA is a member of the immunoglobulin family thought to serve as a negative costimulatory molecule on activated T cells. Several recent studies, including the one published in this issue by del Rio and colleagues, have used BTLA-deficient mice and/or anti-BTLA antibodies to investigate the role of BTLA in regulating graft versus host reaction (GvHR) (1–3). Curiously, targeting BTLA either via genetic deletion, or blocking antibody, or agonistic antibody, all resulted in amelioration of GvHR. This suggests that BTLA may have several functions. Indeed, if BTLA’s function were solely inhibitory, agonistic antibody cross-linking should inhibit T cell function, reducing GvHR, whereas genetic deletion or antibody blockade should enhance T cell function, exacerbating GvHR. BTLA is expressed on activated T cells, B cells, dendritic cells, macrophages and some NK cells. Supporting its inhibitory effects, BTLA-deficient T cells display increased proliferation upon mitogenic stimulation and BTLA-KO mice are more susceptible to some autoimmune diseases. BTLA binds the TNFR family member herpes virus entry mediator (HVEM), which can also bind the coinhibitory receptor CD160 and the coactivating ligand LIGHT. The similar phenotype observed in HVEM-deficient and BTLA-deficient mice suggests that BTLA/HVEM is the predominant interaction on T cells (4). To interpret experiments using antibodies that target a surface molecule, it is essential to know whether a given antibody can drive depletion of the targeted cells, block the epitope for the endogenous ligand, or trigger signaling by the targeted molecule. To study the role of BTLA in GvHR, the Murphy group used BTLA-deficient mice as well as the non-depleting and presumably blocking anti-BTLA antibody 6A6 in a hematopoietic stem cell transfer model in which irradiated F1 recipients received parental bone marrow and/or splenocytes (2). Their experiments demonstrated reduced recovery of donor T cells with blocked or genetically absent BTLA, with the protective effect of 6A6 requiring direct engagement of BTLA on donor T cells. Sakoda et al., in a model of GvHR induced by transfer of donor splenocytes into either non-irradiated or lethally irradiated F1 recipients, used their own agonistic, non HVEM-blocking anti-BTLA mAb, BYK-1, and also BTLA-deficient mice (3). Similar to the Murphy model, reduced GvHR observed after transfer of BTLA-KO T cells was associated with diminished recovery of donor T cells whereas BYK-1 did not prevent T cell engraftment. Moreover, these authors demonstrated that reconstitution of BTLA-deficient T cells with a signaling-deficient BTLA molecule restored T cell survival. These 2 studies suggest that T cell-BTLA can serve as a ligand to the receptor HVEM that then delivers a pro-survival signal to T cells. Sakoda also showed that agonistic targeting of BTLA resulted in reduced cytotoxicity and cytokine production against allogenic stimuli (3). In the current study, del Rio and colleagues used their own anti-BTLA antibody, 4G12b, in non-irradiated F1 recipients of parental splenocytes, and confirmed the prevention of GvHR with no reduction in numbers of donor CD4+ and CD8+ T cells (1). Rather, they reported markedly reduced in vivo lysis of host cells and reduced production of cytokines by donor CD8+ T cells in response to alloantigen, suggesting that this agonistic antibody is preventing an alloreactive and cytotoxic pathway independently of any effects on T cell survival. Interestingly, the 6A6 used by the Murphy group and the 4G12b antibody used by del Rio recognize an overlapping epitope on BTLA, but binding of 4G12b to BTLA does not prevent BTLA from engaging HVEM (5), therefore presumably allowing BTLA to drive HVEM-mediated pro-survival effects. Moreover, their distinct Fc portions (hamster IgG for 6A6 versus rat IgG for 4G12b and BYK-1) may trigger different FcR- and complement-mediated signals which may contribute to their distinct consequences on T cell survival in vivo. Together, these 3 studies suggest that targeting BTLA is capable of reducing GvHR by alternative mechanisms depending on whether the endogenous HVEM-BTLA interaction is preserved or not (Figure 1). Figure 1 Schematic model of the dual effects of BTLA engagement Although these studies clarify the dual role of BTLA on T cells, a few issues remain unresolved. For instance, donor B cells express BTLA and may produce alloantibodies in this model. The agonistic anti-BTLA treatment may inhibit B cells either directly or indirectly by reducing T cell help such that reduced production of alloantibodies could contribute to the diminished in vivo cytotoxicity in anti-BTLA-treated animals. In addition, the contribution of interactions by remaining receptor/ligand pairs in the HVEM pathway needs to be considered. This may be challenging given the evidence of bi-directional signaling downstream of the BTLA/HVEM interaction as well as the potential contributions of HVEM binding to CD160 and LIGHT.

Kinetics of CD4 + and CD8α + T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii

We have previously demonstrated the presence of graft-versus-host reaction (GVHR) in fish employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. To elucidate the role of CD8α + T cells in the induction of GVHR, we investigate the kinetics of CD4 + and CD8 + T-cell subsets in GVHR along with the pathological changes associated with GVH disease (GVHD) in ginbuna. GVHR was not induced with a leukocyte fraction lacking CD8α + T cells separated by magnetic cell sorting. Ploidy and immunofluorescence analysis revealed that CD4 + and CD8α + T cells from sensitized donors greatly increased in the host trunk kidney, constituting more than 80% of total cells 1–2 weeks after donor cell injection, while those from non-sensitized donors constituted less than 50% of cells present. The increase of CD4 + T cells was greater and more rapid than that of CD8α + T cells. The number of donor CD4 + and CD8α + T cells was highest in trunk kidney followed by spleen. Increases in donor CD4 + and CD8α + T cells were also found in liver and PBL, although the percentages were not as high. Pathologic changes similar to those in human and murine acute GVHD were observed in the lymphoid organs as well as target organs such as skin, liver and intestine, including the destruction of cells and tissues and massive leukocyte infiltration. The pathologic changes became more severe with the increase of CD8α + T cells. These results suggest that donor-derived CD8α + T cells play essential roles for the induction of acute GVHR/D in teleosts as in mammals.

Enhanced T Cell Reconstitution by Cord Blood Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1

Previously, we demonstrated that activation of Notch receptors by culture of CD34+CD38− cord blood (CB) hematopoietic progenitors with the Notch ligand Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of CB cells cultured with Delta1ext-IgG compared to recipients of control IgG-cultured or non-cultured CB cells. Furthermore, data suggest that addition of Delta1ext-IgG-cultured CB cells to a non-cultured CB graft facilitated engraftment of the non-cultured CB cells. CD34+CD38− CB cells cultured on immobilized Delta1 in serum free media with Il3, Il6, Flt3l, SCF and TPO for 16 days generated a107-fold increase in total number of cells as well as a 105-fold increase in the number of T-lymphoid biased (CD34+CD7+CD45RA+) cells. Sub-lethally irradiated (275cGy) NOD/SCIDγc−/− mice received 103 non-cultured CB cells or the progeny of 103 Delta1ext-IgG-cultured or IgG-cultured cells. At 6 and 8 wks after HCT, blood samples from recipients of Delta1ext-IgG-cultured cells contained 9- and 3-fold higher numbers of CD3+ cells compared to recipients of non-cultured CB cells (p<0.03 and p<0.04). At 4 wks after HCT, thymuses from recipients of Delta1ext-IgG-cultured cells showed a 9-fold increase in the number of donor CD4+ and/or CD8+ cells compared to recipients of non-cultured cells (p<0.05). These data suggest that Delta1ext-IgG-cultured CB cells have the potential for enhancing early T cell recovery after HCT compared to the noncultured CB cells. Next, we transplanted equal numbers of cells by injecting 106 Delta1ext-IgG-cultured or 106 IgG-cultured CB cells into sub-lethally irradiated NOD/SCIDγc−/− mice. Human engraftment was not observed in mice up to 20 wks after HCT in recipients of IgG-cultured CB cells. Recipients of Delta1ext-IgG-cultured CB cells had detectable CD3+ cells 4 wks after HCT. Despite injecting the equivalent number of cells, the IgG-cultured CB cells did not engraft while the Delta1ext-IgG-cultured cells were able to provide early T cell reconstitution. Lastly, we determined the effect of T cell reconstitution by augmenting a non-cultured CB graft with Delta1ext-IgG-cultured CB. The relative contribution of T cells by the two different CB cells was determined using two HLA disparate CB units. Sub-lethally irradiated NOD/SCIDγc−/− mice received 106 Delta1ext-IgG-cultured cells along with 103 non-cultured CB cells or 106 IgG-cultured CB cells along with 103 non-cultured CB cells or 103 non-cultured CB cells alone. At 6 and 8 wks after HCT, blood samples from the recipient of the non-cultured CB cells augmented with Delta1ext-IgG-cultured had 5-fold higher number of CD3+ cells compared to the other two control groups (p<0.04). Furthermore, CD3+ cells derived from the non-cultured CB cells was 2-fold higher in the mice that received the addition of Delta1ext-IgG-cultured cells compared to mice that received non-cultured cells alone (p<0.05), suggesting a facilitating effect of the Delta1ext-IgG-cultured cells. Augmentation of engraftment was not observed in mice that received IgG-cultured cells. In summary, although both non-cultured and Delta1ext-IgG-cultured CB cells were able to reconstitute the T cell lineage, the Delta1ext-IgG-cultured CB cells had the potential to enhance immune reconstitution early after HCT. We also found that the addition of Delta1ext-IgG-cutlured cells to non-cultured CB cells facilitated the lymphoid engraftment of non-cultured CB. This report describes a novel and clinically feasible exvivo culture system using Delta1 for the generation of CB cell progenitors as a means to accelerate initial T-cell recovery after HCT.

A Key Role for CC Chemokine Receptor 1 in T-Cell-Mediated Respiratory Inflammation

CC chemokine receptor 1 (CCR1) is found on a variety of cells in the immune system and has been shown to play an important role in the host response to pathogens. These studies used a murine model of virus-induced exacerbation of allergic airway disease to examine the role of CCR1 on T cells associated with immune responses taking place in the lung. Lungs of virally exacerbated allergic animals contained elevated levels of interferon-gamma and interleukin-13 and increased levels of CCR1 ligands CCL3 and CCL5. CCR1 expression on T cells was increased in virally exacerbated allergic animals over the level observed in mice sensitized to allergen or exposed to viral infection alone. Using mice deficient for CCR1, we observed decreased airway hyperreactivity and Th2 cytokine production from CD4(+) T cells when this receptor was absent. Transfer studies demonstrated that neither CD4(+) nor CD8(+) T cells from CCR1(-/-) mice migrated to the lymph node as efficiently as wild-type T cells. Intracellular cytokine staining in wild-type mice revealed that CCR1(+) CD4(+) and CD8(+) T cells are associated with interleukin-13 production. Thus, these studies identify CCR1 as a potential target for alleviating T-cell accumulation during exacerbation of asthmatic disease.

The paradoxical effect of glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) activation on alloreactive CD4 and CD8 T cells and the development of GVHD

GITR is a member of the tumor necrosis factor receptor family and is involved in regulatory T cell function and TCR activation. GITR is expressed at low levels on resting T cells, B cells and macrophages. Upon activation, CD4+ and CD8+ T cells upregulate GITR expression while CD4+CD25+ regulatory T cells (Treg) constitutively express GITR. Treg stimulation through GITR inhibits their suppressor function and in vivo administration of GITR agonistic antibodies induce autoimmune disease. Here we show that GITR is a relevant molecule for immune responses in graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo BMT). We used a MHC class I/II disparate model for murine allo BMT (C57BL/6 (B6) into BALB/c) to determine the effects of a GITR activating antibody (GITR Ab) on alloreactive T cells during the development of GVHD. We found that addition of GITR Ab to a mixed lymphocyte reaction (MLR) with irradiated BALB/c splenic stimulators, CD4+CD25− B6 effectors and B6 Tregs induced the blockade of Treg suppressor function, as previously described. Interestingly, GITR Ab also had a direct effect on CD4+CD25− and CD8+CD25− T cell populations. Addition of GITR Ab in an MLR showed a 2-fold decrease in CD4+CD25− proliferation while CD8+CD25− proliferation increased by 3.5-fold when compared to the control. Fas deficient CD4+CD25− T cells had an intact alloreactive proliferative response, suggesting that GITR can induce Fas-mediated apoptosis of CD4+CD25− alloreactive T cells. We used the adoptive transfer of CFSE-labeled donor CD3+ T cells into irradiated allogeneic recipients to assess the expression of GITR in vivo and found increased levels of GITR on both CD4+ and CD8+ dividing alloreactive T cells. When we administered the GITR Ab to allogeneic recipients of CD4+CD25− or CD8+CD25− CFSE-labeled T cells, we observed a 45% reduction in the number of donor CD4+CD25− CFSE-labeled T cells recovered at day 3 after infusion. In contrast, there was a 54% increase in the recovery of CFSE-labeled CD8+CD25− T cells. More importantly, mouse recipients of an allo BMT accompanied by CD8+CD25− donor T cells had increased morbidity and mortality in the presence of GITR Ab. Conversely, mice treated with GITR antibody that received allogeneic CD4+CD25− T cells in the allograft showed a significant decrease in GVHD. Our findings identify a novel and paradoxical effect of GITR stimulation on alloreactive T cells during the development of GVHD.

T-lymphocyte production of macrophage inflammatory protein-1α is critical to the recruitment of CD8+ T cells to the liver, lung, and spleen during graft-versus-host disease

AbstractTo investigate the mechanism by which macrophage inflammatory protein-1α (MIP-1α) affects graft-versus-host disease (GVHD), the expression and function of MIP-1α in 2 murine models of GVHD were evaluated. In irradiated class I and class II disparate recipients, the expression of messenger RNA (mRNA) and protein for MIP-1α was significantly increased in GVHD target organs after transfer of allogeneic lymphocytes compared to syngeneic lymphocytes. When lymphocytes unable to make MIP-1α were transferred, there was a decrease in the production of MIP-1α in the liver, lung, and spleen of bm1 (B6.C-H2bm1/By) and bm12 (B6.C-H2bm12/KhEg) recipients compared to the transfer of wild-type splenocytes. At day 6 there was a 4-fold decrease in the number of transferred CD8+ T cells in the lung and approximately a 2-fold decrease in the number of CD8+ T cells in the liver and spleen in bm1 recipients after transfer of MIP-1α–deficient (MIP-1α−/−) splenocytes compared to wild-type (MIP-1α+/+) splenocytes. These differences persisted for 13 days after splenocyte transfer. In contrast, the number of donor CD4+ T cells found in the liver and lung was significantly increased after the transfer of MIP-1α−/− compared to wild-type splenocytes in bm12 recipients from day 6 through day 10. Thus, the transfer of allogeneic T cells was associated with the enhanced expression of MIP-1α in both a class I and class II mismatch setting. However, the increased expression only led to enhanced recruitment of CD8+, but not CD4+, donor T cells. Production of MIP-1α by donor T cells is important in the occurrence of GVHD and functions in a tissue-dependent fashion.

T-lymphocyte production of macrophage inflammatory protein-1α is critical to the recruitment of CD8+ T cells to the liver, lung, and spleen during graft-versus-host disease

To investigate the mechanism by which macrophage inflammatory protein-1alpha (MIP-1alpha) affects graft-versus-host disease (GVHD), the expression and function of MIP-1alpha in 2 murine models of GVHD were evaluated. In irradiated class I and class II disparate recipients, the expression of messenger RNA (mRNA) and protein for MIP-1alpha was significantly increased in GVHD target organs after transfer of allogeneic lymphocytes compared to syngeneic lymphocytes. When lymphocytes unable to make MIP-1alpha were transferred, there was a decrease in the production of MIP-1alpha in the liver, lung, and spleen of bm1 (B6.C-H2(bm1)/By) and bm12 (B6.C-H2(bm12)/KhEg) recipients compared to the transfer of wild-type splenocytes. At day 6 there was a 4-fold decrease in the number of transferred CD8(+) T cells in the lung and approximately a 2-fold decrease in the number of CD8(+) T cells in the liver and spleen in bm1 recipients after transfer of MIP-1alpha-deficient (MIP-1alpha(-/-)) splenocytes compared to wild-type (MIP-1alpha(+/+)) splenocytes. These differences persisted for 13 days after splenocyte transfer. In contrast, the number of donor CD4(+) T cells found in the liver and lung was significantly increased after the transfer of MIP-1alpha(-/-) compared to wild-type splenocytes in bm12 recipients from day 6 through day 10. Thus, the transfer of allogeneic T cells was associated with the enhanced expression of MIP-1alpha in both a class I and class II mismatch setting. However, the increased expression only led to enhanced recruitment of CD8(+), but not CD4(+), donor T cells. Production of MIP-1alpha by donor T cells is important in the occurrence of GVHD and functions in a tissue-dependent fashion.