Recipients Of Spleen Cells Research Articles

Activation of ephrin A proteins influences hematopoietic stem cell adhesion and trafficking patterns

To determine if Eph receptors and ephrins can modulate the homing of hematopoietic cells in a murine bone marrow transplantation model. EphA and ephrin A gene expression by mouse hematopoietic stem cells and the progenitor cell line FDCP-1 was determined by real-time reverse transcription polymerase chain reaction and flow cytometry. The effect of ephrin A activation on adhesion of hematopoietic progenitors was determined by in vitro adhesion assays in which cells were exposed to fibronectin or vascular cell adhesion molecule-1 (VCAM-1) and an increasing gradient of immobilized EphA3-Fc. Adhesion to fibronectin and VCAM-1 was further investigated using soluble preclustered EphA3-Fc. We used soluble unclustered EphA3-Fc as an antagonist to block endogenous EphA-ephrin A interactions in vivo. The effect of injecting soluble EphA3-Fc on the mobilization of hematopoietic progenitor cells was examined. We determined the effect on short-term homing by pretreating bone marrow cells with EphA3-Fc or the control IgG before infusion into lethally irradiated mice. Preclustered and immobilized EphA3-Fc increased adhesion of progenitor cells and FDCP-1 to fibronectin and VCAM-1 (1.6- to 2-fold higher adhesion; p < 0.05) relative to control (0 μ/cm(2) EphA3-Fc extracellular molecule alone). Injection of the antagonist soluble EphA3-Fc increased progenitor cell and colony-forming unit-spleen cells in the peripheral blood (42% greater colony-forming unit in culture; p < 0.05, 3.8-fold higher colony-forming unit-spleen) relative to control. Treating bone marrow cells with EphA3-Fc resulted in a reduction by 31% in donor stem cells homing to the bone marrow and accumulation of donor cells in recipient spleens (50% greater than control) and greater recovery of donor stem cells from the peripheral blood.

Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice.

Whereas humanized mouse models have contributed significantly to human immunology research, human T cells developing in mouse thymic environment fail to demonstrate HLA-restricted function. To achieve HLA-restricted human immune response, we created an immune-compromised non-obese diabetic/SCID/IL2rg(null) strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD). Transplantation of purified Lin-CD34+CD38- human hematopoietic stem cells into NSG-HLA-A2/HHD newborns resulted in the development of human CD4+ and CD8+ TCR alphabeta+ T cells and CD4-CD8- and CD8+ TCR gammadelta+ cells in recipient bone marrow and spleen. Human cytotoxic T lymphocytes (CTLs) become functionally mature, as evidenced by the production of granzyme corresponding to phenotypic transition from naïve to effector memory CTLs. In these recipients, human Th17 cells developed along with Th1 and Th2 cells. Epstein-Barr virus (EBV) infection in the humanized NSG-HLA-A2/HHD recipients resulted in the formation of lymphoproliferative lesions consisting mainly of human B cells with scattered human T cells. Human CTLs developing in the recipients recognized EBV-derived peptides in an HLA-restricted manner and exerted HLA-restricted cytotoxicity against EBV-infected human B cells. The HLA-expressing humanized mouse with functional HLA-restricted T cells and consistent representation of rare T-cell subsets overcomes a major constraint in human immunology, and serves as a useful model for investigation of human immune responses against pathogens and for the development of therapeutic strategies against human diseases.

Host CD11c+ Dendritic Cells Are Required for Priming the Lympho-Haematopoietic Graft-Versus-Host Response but Not Graft-Versus-Host Disease.

Abstract 2450Poster Board II-427Following allogeneic bone marrow transplantation (BMT), delayed donor leukocyte infusion (DLI) at a point when conditioning-induced inflammation has resolved, is employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease (GVHD). We have previously shown that host bone marrow (BM)-derived antigen presenting cells (APC) are required to prime GVL responses following delayed donor T cell transfer into MHC-mismatched allogeneic chimeras. The APC population(s) required for induction of graft-versus-host reactivity are not known, although it has been demonstrated by others that certain host-derived dendritic cell (DC) populations (in situ or transferred) are capable of inducing GVHD in models of immediate T cell transfer to freshly conditioned recipients. We hypothesised that host CD11c+ conventional DC would be required for the induction of graft-versus-host reactivity in a model of delayed T cell transfer to established mixed chimeras (MC). To test this hypothesis, we reconstituted lethally irradiated B6 (H2b) mice with a mixture of BALB/c (H2d) and B6.CD11c-DTR T cell depleted bone marrow. In the resulting MC, host CD11chigh cells (predominantly conventional DC) are uniquely sensitive to killing by diphtheria toxin (DT) due to their selective expression of a high affinity DT receptor. 8-10 week old established MC received DLI in form of congenic Thy1.1+ BALB/c splenocytes. MC were injected with DT (or PBS) every 72 hours from day -1 to day 10 following DLI. Depletion of host conventional DC (>90%) abrogated accumulation of donor Thy1.1+ CD4 and CD8 T cells in recipient spleens and reduced in vivo cytotoxicity against host target B cells. These effects associated with a lack of conversion from mixed to full donor chimerism, demonstrating an absolute requirement for host CD11chigh DC in priming the lympho-haematopoietic graft-versus-host response in the steady state.We have shown previously that induction of systemic inflammation by injection of a synthetic TRL7 agonist (R-848) is sufficient to induce systemic GVHD in MC after delayed DLI (Chakraverty et al, JEM, 2006). We therefore considered the requirement for CD11chigh DC in priming graft-versus-host reactivity in the presence of inflammation. We generated BALB/c + B6.CD11c-DTR→ B6 MC as before and then used DT treatment to deplete host conventional DC in the presence of R-848 (given every 72h from day -1 to day 10 following DLI). In MC without depletion of CD11chigh DC, R-848 treatment enhanced accumulation of Thy1.1+ BALB/c CD4 and CD8 donor cells and increased in vivo cytotoxicity against host B cell targets as compared to non-R-848 treated MC. This was associated with histological evidence of GVHD in the skin, gut and liver. In MC with depletion of CD11chigh DC (>90% deletion), R-848 treatment was associated with equivalent donor T cell accumulation and in vivo killing of host targets as observed in non-DC depleted controls. Furthermore, DC-depleted MC treated with R-848 also developed histological GVHD. In vitro experiments using CD11c+ stimulator cells purified from the spleens and lymph nodes of R-848 or PBS-treated mice suggested that priming of the allo-response in the presence of inflammation was not due to a residual population of CD11chigh DC remaining after DT treatment.Taken together, these data demonstrate for the first time that conventional host CD11chigh DC are required to prime lympho-haematopoietic graft-versus-host responses in the steady state following delayed DLI to MC. In contrast, their role is redundant in the context of TLR-agonist induced GVHD. This suggests that other APC populations can prime GVHD in the presence of systemic inflammation. Disclosures:No relevant conflicts of interest to declare.

The dynamic changes of T-bet +/GATA-3 + and RORγt +/FOXP3 + cells in recipient spleens and grafts after rat orthotopic liver transplantation

Background Rejection of transplanted liver occurs when the host generates alloantigen-reactive T cells, and CD4 + T-cell subsets, including Th1, Th2, T17 and iTreg, could be involved in changing the dynamics of graft rejection onset. In the current immunosuppressive strategies, rejection is treated as an undifferentiated process that is uniform, which results in the failure of tolerance induction. Here, we established a rejection model to observe the reciprocal interaction of CD4 + T-cell subsets in the complex networks of the immune system. Methods Orthotopic liver transplantation (OLT) from male inbred Lewis rats ( n = 15) to male inbred Brown Norway (BN) rats was performed by Kamada's two-cuff technique without reconstruction of the hepatic artery. OLT from BN to BN rats ( n = 5) was performed as a control. Recipients were sacrificed on postoperative days 3, 5, 7, 9, 11, 13 and 15. Recipient spleens and grafts were harvested, fixed in 10% neutral formalin, and embedded in paraffin. Meanwhile, hematoxylin and eosin and immunohistochemical staining was done, acute rejection was graded by the Banff scheme, and the number of T-bet +, GATA-3 +, RORγt + and FOXP3 + cells in the spleen and grafts were counted. Results In recipient spleens, the T-bet + and RORγt + cells were increased more significantly in the mild acute rejection (AR) group than in the control group ( P = 0.016, P = 0.009, respectively), while both cell types were decreased in the moderate AR group. Compared with the control group, the RORγt + cells did not differ significantly in the severe AR group, while the T-bet + cells were significantly decreased ( P = 0.465, P = 0.009, respectively). The GATA-3 + cells were significantly decreased in the mild AR group compared with the control group ( P = 0.009). With regard to the FOXP3 + cells, there was no significant difference between the control and mild AR groups ( P = 0.754), while they were significantly decreased in the moderate and severe AR groups ( P = 0.028, P = 0.009, respectively). The ratio of T-bet +/GATA-3 + cells was more associated with AR than was the RORγt +/FOXP3 + cell ratio in the early stage. In the graft, the T-bet + and RORγt + cells were significantly increased in mild, moderate and severe AR groups compared with the control group ( P < 0.009). The expression of GATA-3 + cell was not significantly increased in the mild AR group compared with the control group, while it was significantly increased in the moderate and severe AR groups ( P = 0.028, P = 0.009, respectively). Concerning the FOXP3 + cells, no significant difference was found between the control and mild AR groups ( P = 0.347), while they were significantly increased in the moderate and severe AR groups ( P < 0.009). Conclusions Our study revealed the dynamic changes of T-bet +, GATA-3 +, RORγt + and FOXP3 + cells in the spleen and grafts of recipient rats. It seems that the ratio of T-bet +/GATA-3 + cells was more associated with AR than was RORγt +/FOXP3 + cells in the early stage of rejection, while the ratio of RORγt +/FOXP3 + cells was associated more with the later but more persistent stage of rejection. This study could make a contribution to the optimal selection of immunosuppressive regimens according to the dynamic changes in CD4 + T-cell subsets at different stages of rejection.

B7-H4 transfection prolongs β-cell graft survival

B7-H4, a recently discovered member of B7 family, can negatively regulate T cell responses. However, it is not clear whether B7-H4 negatively function in cell transplantation. In this study we investigated the immunosuppressive effect of B7-H4 on β-cell transplantation. An insulinoma cell line, NIT-1, transfected with B7-H4 (B7-H4-NIT) was established, and transplanted to diabetic C57BL/6 mice by intraperitoneal injection. Proliferation assay of splenocytes in vitro showed that B7-H4-NIT suppressed alloreactive T cell activation. The proportion of IFN-γ-producing cells in recipient spleen was significantly reduced and the number of Treg cells was upregulated in B7-H4-NIT group compared to the control, EGFP-NIT. The expression of mRNA coding IFN-γ was lower but that of IL-4 was higher in B7-H4-NIT transplanted recipients than in the control animals. The results of ELISA also revealed the same trends. Diabetic mice reached normalglycemic quickly and gained weight after transplantation of B7-H4-NIT. More importantly, the survival time for recipients transplanted with B7-H4-NIT cells was significantly longer than that with EGFP-NIT cells. These results indicate that B7-H4 transfection prolongs β-cell graft survival.

The Rapid Ex Vivo Generation of an Immunodominant Antigen-Specific Memory CD8 Population and Its Subsequent Homeostatic Expansion in Ablatively-Conditioned HCT Recipients.

There is currently significant interest in the transplant field to develop adoptive-transfer strategies utilizing T cell populations to provide immediate immune function as well as long-term immune reconstitution following hematopoietic cell transplantation (HCT). Presumably, these pre-selected T cell populations could then be further expanded in the transplant recipient as a consequence of lymphopenia-induced proliferation. However, clinical application of adoptive transfer strategies has been limited by practical (time, expense) and technical (isolation and expansion of antigen-specific T cell populations) difficulties, hence more efficient approaches need to be identified. Recent reports have demonstrated the feasibility for the rapid ex vivo generation of transgenic memory CD8 populations. We investigated the potential of applying this ex vivo approach to generate and expand an immunodominant antigen-specific memory population from primary CD8 T cells. CD8 cells recognizing the mouse H60 epitope were selected as the antigen-specific CD8 population. The H60 glycoprotein is the ligand for NKG2D and the LTFNYRNL peptide is an immunodominant minor transplantation antigen. H60 is expressed by BALB.B (H2b) hematopoietic cells and recognized by C57BL/6 (B6) CD8 cells within the context of the H2Kb molecule. CD8 T cells from normal B6 spleens were positively selected using Miltenyi beads. The purified CD8 cells (97%) were then cultured with bone marrow-derived B6 DC, rmIL-2, and H60 peptide (1μM) for 3 days. Cells were harvested and re-cultured with rmIL-15 for 2–4 days. The resultant CD8 population was enriched 10 fold for tetramer-stained H60+ CD8 T cells (average: 3.0% of CD8 T cells). The H60+ CD8 cells displayed a memory phenotype as characterized by CD44+, Ly6C+, CD62Lintermed, and CD25lo expression. We hypothesized these H60+ CD8 T cells could be further expanded in transplant recipients by lymphopenia-induced proliferation. To determine the expansion and persistence of H60+ TM post-HCT, H60+-enriched CD8 cells were co-transplanted with T cell-depleted B6 bone marrow into 9.0Gy-conditioned syngeneic recipients. The phenotype and number of H60+ cells in recipient spleens and bone marrow were assessed beginning 3 days post-HCT. Notably, the H60+ CD8 cells maintained their memory phenotype and persisted at least 2 months post-transplant. The ex vivo-generated H60+ TM underwent a relative expansion of 1.5–2 fold as assessed in recipient spleens, similar to the post-transplant expansion of H60+ CD8 TM derived in vivo from B6 mice primed to BALB.B cells. Moreover, this post-HCT expansion was also similar to that by an ex vivo-generated, transgenic CD8 TM population. Both (ex vivo and in vivo generated) H60+ TM populations also exhibited expansion (1.5–2 fold) in the bone marrow. In total, an immunodominant antigen-specific CD8 TM population was selectively generated and enriched ex vivo and found to undergo expansion following transplant into ablatively conditioned HCT recipients. The similarities in expansion and persistence between ex vivo generated H60 and in vivo primed H60 populations suggest this approach may have useful applications towards the development of successful adoptive transfer strategies.

The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.

One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.

Role of SPA-1 in Phenotypes of Chronic Myelogenous Leukemia Induced by BCR-ABL–Expressing Hematopoietic Progenitors in a Mouse Model

SPA-1 is a negative regulator of Rap1 signal in hematopoietic cells, and SPA-1-deficient mice develop myeloproliferative disorders (MPD) of long latency. In the present study, we showed that the MPDs in SPA-1(-/-) mice were associated with the increased hematopoietic stem cells expressing LFA-1 in bone marrow and their premature mobilization to spleen with extensive extramedullary hematopoiesis, resembling human chronic myelogenous leukemia (CML). We further showed that human BCR-ABL oncogene caused a partial down-regulation of endogenous SPA-1 gene expression in mouse hematopoietic progenitor cells (HPC) and immature hematopoietic cell lines. Although both BCR-ABL-transduced wild-type (wt) and SPA-1(-/-) HPC rapidly developed CML-like MPD when transferred to severe combined immunodeficient mice, the latter recipients showed significantly increased proportions of BCR-ABL(+) Lin(-) c-Kit(+) cells compared with the former ones. Serial transfer experiments revealed that spleen cells of secondary recipients of BCR-ABL(+) wt HPC failed to transfer MPD to tertiary recipients due to a progressive reduction of BCR-ABL(+) Lin(-) c-Kit(+) cells. In contrast, SPA-1(-/-) BCR-ABL(+) Lin(-) c-Kit(+) cells were sustained at high level in secondary recipients, and their spleen cells could transfer MPD to tertiary recipients, a part of which rapidly developed blast crisis. Present results suggest that endogenous SPA-1 plays a significant role in regulating expansion and/or survival of BCR-ABL(+) leukemic progenitors albeit partial repression by BCR-ABL and that Rap1 signal may represent a new molecular target for controlling leukemic progenitors in CML.

Gene therapy using adenoviral vector encoding 4-1BBIg gene significantly prolonged murine cardiac allograft survival

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, interacts with 4-1BBL expressed on APC and delivers a costimulatory signal for T cell activation and growth. In this study, we investigated the efficacy of an adenoviral vector encoding murine 4-1BB extracellular domain and human IgG1 Fc (Ad4-1BBIg) fusion gene on murine cardiac allograft survival. Abdomen heterotopical heart graft model was performed from Balb/c to C57BL/6 mice. The adenoviral vectors, Ad4-1BBIg or an adenoviral vector containing EGFP gene (AdEGFP), were administered intravenously to recipient animals after cardiac grafting. The cardiac allograft survival was monitored by daily palpation. The serum level of 4-1BBIg and graft histology was assessed. Cytokine profiles in the grafts were detected by RT-PCR. IFN-γ producing cells in recipient spleen were examined by flow cytometry. 4-1BBIg gene expression was achieved highly level at 72 h after vector injection. The proportion of IFN-γ producing cells in recipient spleen was significantly reduced after administration of Ad4-1BBIg, compared to the group given AdEGFP or to the untreated control group. Unlike in controls, cardiac allograft expression of mRNA coding for IL-2 and IFN-γ remained low in the Ad4-1BBIg group. Ad4-1BBIg therapy markedly reduced T cell infiltration into the graft and significantly prolonged recipient survival time (13.5 days), compared to the untreated group (7.5 days) and the AdEGFP-treated group (8.0 days) ( P < 0.05). These results indicate that blockade of 4-1BB/4-1BB ligand interactions by Ad4-1BBIg inhibited alloreactive T-cell activation and attenuated T-cell infiltration into the graft, resulting in significant prolongation of murine cardiac allograft survival. Therefore, Ad4-1BBIg may be useful for preventing allograft rejection.

Both CD4+and CD8+T Cells Can Mediate Vaccine‐Induced Protection againstCoccidioides immitisInfection in Mice

To determine which lymphocytes are required for vaccine-induced immunity to coccidioidomycosis, we used a temperature-sensitive mutant of Coccidioides immitis to immunize mice lacking subsets of lymphocytes or specific cytokines and infected the mice 4 weeks later with virulent C. immitis. After 2 weeks, we determined the number of fungi in their lungs and spleens. Vaccine-induced immunity required alpha beta T lymphocytes. beta -2 microglobulin knockout (KO) mice were protected by immunization, and we transferred protection using CD4+ T cells from immunized mice. However, vaccination also protected CD4+ KO mice, which suggests that CD8+ T cells played a role in vaccine-induced immunity, even though they were not required. We adaptively transferred protection using spleen cells from immunized CD4+ KO mice to nonimmune B6 mice, but CD8+ -depleted spleen cells did not protect against infection. Recipients of spleen cells from immunized CD4+ KO mice had 6 times more tumor necrosis factor (TNF)- alpha mRNA in their lungs than did mice that received nonimmune spleen cells, and TNF receptor-1 KO mice were not fully protected by immunization. These results show that both CD4+ and CD8+ T cells can protect against coccidioidomycosis and that TNF- alpha is a necessary component of the acquired immune response.

Improved survival following induction of GVHD following lipopolysaccharide immunization

Graft-vs-host disease (GVHD) is still the primary limitation to the wider application of allogeneic bone marrow transplantation (BMT). On the one hand, it predisposes transplant recipients to risk of bacterial, fungal, and viral infections, on the other, lipopolysaccharide (LPS), an endotoxin found in the cell walls of gram-negative bacteria, has been shown to play a significant role in the development and severity of GVHD following allogeneic myeloablative BMT. Our study focused on immunization of recipient and donor mice with endotoxin prior to transplantation, in an attempt to reduce mortality caused by gram-negative bacterial infections posttransplantation. In one experiment, recipient mice were immunized with LPS prior to BMT, whereas in another experiment, donor mice were immunized prior to BMT. The mice were evaluated for development of GVHD and for survival. Our results showed that injection of low-dose LPS to mice prior to induction of GVHD with allogeneic spleen cells saved more than 40% of the recipients, whereas all mice in the untreated control group died. The survival of recipients of spleen cells from immunized donors rose to 54% and clinical signs of GVHD were attenuated as compared to control mice inoculated with spleen cells obtained from unimmunized donors. This immunization protocol suggests that immunization of the donor or the recipient against LPS prior to transplantation may be protective against gram-negative bacteria.

Contrasting Effects of Post-Transplant Lymphopenia on Proliferation and Degranulation in Antigen-Specific CD8 Memory T Cells.

Lymphopenia following bone marrow transplant (BMT) can induce expansion of donor T cells which help contribute to reconstitution in recipients. We are interested in elucidating the responses of transplanted CD8 T memory (TM) to lymphopenic signals in the recipient splenic and marrow environments. To compare proliferative responses of marrow and spleen populating antigen-specific CD8 TM to lymphopenic signals, purified (95% pure) CFSE labeled B6 CD8 TM were co-transplanted with syngeneic or allogeneic TCD-BM to ablatively conditioned (9.0 Gy) syngeneic B6 recipients. Antigen-specific CD8 TM were obtianed following priming B6 mice against BALB.B antigens. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to identify immunodominant H60-specific CD8+ cells which expressed a central memory (TM) phenotype (CD44+, Ly6C+). Three days post-transplant of these CD8 TM, recipient spleen and marrow cells were analyzed for CFSE dilution in gated H60+CD8+ T cells. Transferred H60+ CD8+ cells in the marrow compartment consistently showed greater (5 – 6 cell divisions) proliferative activity compared to those populating the spleen (1 – 2 divisions) in recipients of syngeneic (i.e. homeostatic) as well as allogeneic TCD-BM. Thus, the marrow compartment apparently provided greater support for the early homeostatic proliferation of these MiHA-specific CD8 TM. Furthermore, the presence of antigen in B6 recipients of allogeneic BM did not significantly enhance the proliferation of transferred CD8+ TM, indicating that homeostatic signals induced greater overall proliferation than antigen activation in the simultaneous presence of both signals early post-transplant. To examine for non-proliferative functional activity in transplanted antigen-specific CD8 TM, degranulation was assessed by surface expression of LAMP-1 (CD8 107a) which was analyzed 3 days after the transfer of purified splenic CD8 T cells from B6BALB.B into lethally irradiated (9.0 Gy) syngeneic B6 mice. Analysis of recipient spleeen and marrow showed that transferred antigen-specific in these tissues markedly upregulated surface LAMP-1 expression. Interestingly, there was greater frequency (~90%) of CD8+ TM in the spleen expressing LAMP-1 than in the bone marrow (56%). To determine if this degranulation response was limited to transplanted TM, a homogeneous population of naive T cells (TN) from OT-1 RAG−/− TCR tg mice was prepared and transplanted in an identical manner. LAMP-1 expression was marginally detectable in these cells. In total, these results suggest that transplanted CD8 TM and TN respond differently in the homeostatic environment post-transplant. Moreover the observations may suggest that CD8 TM can exhibit effector-like activity in the lymphopenic milieu immediately post-transplant. These findings have implications for the relative contribution of hematopoietic compartments during immune reconstitution after HCT.

Simulataneous Vaccination and Delayed Lymphocyte Infusion for Enhancement of Immune Reactivity Against Acute Lymphoblastic Leukemia or Lymphoma.

BACKGROUND: Delayed lymphocyte infusion (DLI) can be employed as an antileukemia immune therapy when patients with leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT). While effective in patients with chronic myelogenous leukemia, it is generally ineffective with relapse of acute lymphoblastic leukemia (ALL). This ineffectiveness may be multifactorial and possible reasons include rapid leukemia cell growth in ALL, reduced propensity to apoptosis signals, and poor presentation of antigens present on lymphoblasts resulting in failure to generate a significant immune response. If the failure of malignant lymphoblasts in marrow and lymphoid tissues to elicit meaningful T cell responses plays a role in the ineffectiveness of DLI, it is possible that therapeutic vaccines may be able to ameliorate this barrier. We have recently demonstrated that vaccination immediately prior to HSCT enhances antigen specific T cell responses in the post-transplant immune repertoire (Bone Marrow Transplantation, 35:793–801, 2005.)HYPOTHESIS: Vaccination with leukemia related antigens at the time of DLI will increase donor T cell responses to leukemia related antigens without exacerbation of GVHD responses to ubiquitous host minor histocompatibility antigens (mHA).METHODS: We employ a murine MHC matched allogeneic HSCT model in which 6 antigens are molecularly characterized. C3.SW mice are donors and C57BL/6 mice are recipients. In this model the H7 mHA is the immunodominant antigen, while alloresponses to H3 and H13 are also present. By using spontaneously arising lymphoblastic leukemia/lymphoma cells from Ig-cmyc transgenic male C57BL/6 as the model malignancy in a female to female transplant the male HY associated antigens Uty, Dby and Smcy can be used as antigens restricted to the lymphoid malignancy. Four weeks after allogeneic HSCT mice were vaccinated with 107 cells expressing the HY antigens and the C57BL/6 H7, H3 and H13 minor antigens. The cells used for pre-DLI vaccination were either irradiated male C57BL/6 spleen cells or irradiated male malignant lymphoblasts. One day later mice received iv infusion of 107 spleen cells from C3.SW female donor mice previously primed against HY antigens by immunization with male C3.SW cells. Ten days interferon gamma Elispot assays were performed on HSCT recipient spleen cells using the peptide-defined antigens.RESULTS: Vaccination of HSCT recipients with male spleen cells 1 day prior to DLI from HY immune donors significantly increased T cell responses to HY peptide epitopes. Recipients of DLI alone harbored 40 interferon secreting cells per 106, while combination of vaccination and DLI yielded 94 per 106(p < 0.05). In contrast, there was no change in the number of T cells responding to the H7, H3 and H13 mHA. DLI only exhibited 26 per 106 while DLI plus vaccine exhibited 27 per 106 (p < 0.05). Whole cell irradiated lymphoblasts were not as effective as irradiated splenocytes in augmenting the HY responses, although they were equally effective in producing T cell responses in normal, nontransplanted mice.CONCLUSIONS: Simultaneous vaccination and DLI can lead to significant expansion of donor cells potentially reactive with antigens present on malignant lymphoblasts without exacerbation of T cell responses to ubiquitous mHA associated with GVHD. Current work is exploring methods by which indirect presentation of antigens from lymphoblasts can be enhanced, since it is unlikely that vaccines relying on direct antigen presentation by lymphoblasts will be effective.

FK 506 inhibits tolerance induction in mice liver transplantation

Background In the orthotopic mouse liver transplantation model, allografts are accepted without immunosuppression, and donor-specific tolerance is induced upto 40 days. Although FK 506 is a well-known immunosuppressive agent, its influence on tolerance induction is not known. In this study, we examined the influence of FK 506 on tolerance induction in a mouse liver transplant model. Methods Orthotopic liver transplantation was performed from B10.BR (H-2K) to B10.D2 (H-2D mice). In the experimental group, FK 506 (1 mg/kg/d) was given subcutaneously to the recipients from day 0 to day 21, whereas the control group received a placebo (1 mg/kg/d). On day 40, donor skin grafts were transplanted to the recipients to examine the survival times of the recipients and the skin grafts. On day 14, donor-type cells in recipient's blood, spleen, kidney, thymus, and lymph nodes were examined by RT-PCR using specific donor-type MHC class I and II primers. Results All recipients survived for more than 100 days. The mean survival time of skin grafts in the experimental group was significantly reduced compared to that of controls. On day 14, either donor-type MHC class I- or class II-positive cells were detected in the control group, whereas donor-derived MHC class II-positive cells disappeared in the experimental group. Conclusions In the early period after mouse liver transplantation, FK 506 inhibits tolerance induction paradoxically. Some donor-derived MHC class II-positive cells might play an important role in tolerance induction.

Marrow Derived Muscle Colonies Are a Clonal Phenomenon.

We have previously established a model of plasticity of marrow to skeletal muscle that is based on transplantation of GFP positive marrow and cardiotoxin injury to the tibialis muscle. In these experiments, we have observed colonies of GFP positive muscle fibers, defined as 3 or more myocytes. We noted that individual GFP positive fibers showed marked heterogeneity of fluorescence. In sharp contrast, colonies of GFP+ cells, showed homogeneous fluorescence. This was confirmed by using confocal microscopy and by measuring the mean fluorescent intensity of the individual cells in the colonies. When the intensity of the green fluorescence in individual fibers was graded to dull, moderate or bright, in 95% of the colonies counted, showed homogenous intensity of green fluorescence while for single GFP fibers dispersed throughout the specimen the probability of 3 cells being the same color was only 14.12%. Therefore the probability of 95% of colonies to have a minimum of 3 homogenous fibers by random association is less than 0.0001. To show that colonies are not from clumps of cells injected into the muscle, we compared the number of the colonies in two different experimental setting where in both GFP positive marrow cells were injected intravenously after 900cGy of radiation followed by cardiotoxin injury to the muscle. Animals who received two cycle of G-CSF mobilization were compared with those who had lineage negative marrow cells directly injected into their muscle, one day after injury. The incidence of muscle colonies remained the same between the groups. The incidence increased in experiments with multiple cycles of mobilization and injury and also in mdx mouse that has spontaneous ongoing regeneration of its muscles. Cotransplantation of marrow cells from yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) transgenics into C57BL/6 mice followed by cardiotoxin injury showed colonies that were either from YFP or CFP cells. No mixed colonies were found. Transplantation of YFP marrow cells before injury followed by CFP cells after injury and vice versa showed colonies only from cells that were infused at the time of transplantation. Colonies were seen only in samples that received CD45, C-Kit and/or Sca positive marrow cells and not in those negative populations. These cells resulted in a GFP+ CD45 negative population 4 weeks after transplantation and produced Myf5+ and MyoD+ myoblasts three days after injury. These data suggests that marrow derived muscle colonies are a clonal phenomenon similar to CFU-S cells in recipient spleens after transplantation.

Mycophenolate mofetil does not suppress the graft-versus-leukemia effect or the activity of lymphokine-activated killer (LAK) cells in a murine model.

Graft-versus-leukemia (GVL) effect is an essential component in the course of allogeneic stem cell transplantation (SCT). However, both prevention and treatment of established graft-versus-host disease (GVHD), including with drugs such as cyclosporine, can suppress GVL effects. Mycophenolate mofetil (MMF) is becoming a standard of care in SCT recipients for better prevention of GVHD as well as for promoting stem cell engraftment. To evaluate the effect of MMF, an immunosuppressive drug increasingly used for prevention of GVHD, on disease recurrence following SCT in a preclinical animal model. Since GVL effects may be also induced by alloreactive natural killer (NK) cells, the goal was to investigate the effects of MMF on the activity of lymphokine-activated killer (LAK) cells. MMF was administered by daily intraperitoneal injection starting at day 1 post-SCT. Cytotoxic LAK activity was measured by 5-h 35S-release assay, and GVL was tested by the appearance of BCL1 leukemia in a semi-mismatched (C57BL/6 donors to [BALB/c x C57BL/6] F1 recipients) murine model. A dosage regimen of 28-200 mg/kg per day MMF had no negative effect on either cytotoxic LAK activity or GVL (as measured by finding of leukemic cells in recipient spleen by PCR or the appearance of clinical leukemia with adoptive transfer). These results suggest that MMF does not impair GVL effects or reduce LAK cell activity in mice.

Ex vivo fludarabine exposure inhibits graft-versus-host activity of allogeneic T cells while preserving graft-versus-leukemia effects

Allogeneic donor T cells in bone marrow transplantation (BMT) can contribute to beneficial graft-versus-leukemia (GVL) effects but can also cause detrimental graft-versus-host disease (GVHD). A successful method for the ex vivo treatment of donor T cells to limit their GVHD potential while retaining GVL activity would have broad clinical applications for patients undergoing allogeneic hematopoietic cell transplantation for malignant diseases. We hypothesized that donor lymphocyte infusions treated with fludarabine, an immunosuppressive nucleoside analog, would have reduced GVHD potential in a fully major histocompatibility complex-mismatched C57BL/6 → B10.BR mouse BMT model. Recipients of fludarabine-treated donor lymphocyte infusions (F-DLI) had significantly reduced GVHD mortality, reduced histopathologic evidence of GVHD, and lower inflammatory serum cytokine levels than recipients of untreated DLI. Combined comparisons of GVHD incidence and donor-derived hematopoietic chimerism indicated that F-DLI had a therapeutic index superior to that of untreated DLI. Furthermore, adoptive immunotherapy of lymphoblastic lymphoma using F-DLI in the C57BL/6 → B10.BR model demonstrated a broad therapeutic index with markedly reduced GVHD activity and preservation of GVL activity compared with untreated allogeneic T cells. Fludarabine exposure markedly reduced the CD4 +CD44 low-naive donor T-cell population within 48 hours of transplantation and altered the relative representation of cytokine-producing CD4 + T cells, consistent with T-helper type 2 polarization. However, proliferation of fludarabine-treated T cells in allogeneic recipient spleens was equivalent to that of untreated T cells. The results suggest that fludarabine reduces the GVHD potential of donor lymphocytes through effects on a CD4 +CD44 low T-cell population, with less effect on alloreactive T cells and CD4 +CD44 high memory T cells that are able to mediate GVL effects. Thus, F-DLI represents a novel method of immune modulation that may be useful to enhance immune reconstitution among allograft recipients with reduced risk of GVHD while retaining beneficial GVL effects.

Prolonged survival of rat hepatic allografts after pretransplant intraportal administration of recipient blood to the donor

Background: We investigated the effect of the pretransplant administration of recipient's blood to donor on rat hepatic allograft survival. Methods: Serum levels of interferon (IFN)-γ were determined after transplantation. Immunocompetent cells from the donor were identified with anti-donor class II MHC (RT1B a) (OX-76) antibodies. The number of donor-derived CD4 + and CD8 + T cells in hepatic allografts or recipient spleen was determined. Results: LEW rats transplanted with hepatic allografts from ACI rats survived a mean of 11.1±2.1 days. Pretreatment intravenous administration of recipient LEW blood did not prolong the survival of hepatic allografts (10.5±1.7 days). In contrast, pretreatment intraportal administration of recipient LEW blood significantly increased the survival of LEW recipients with ACI livers to 31.7±7.6 days (Table 1). The ratio of ox76 +CD4 + or ox76 +CD8 + T cell to CD4 + or CD8 + cell was significantly greater (25.6% versus 5.7%, 26.6% versus 6.8%) in hepatic allografts pretreated with pretransplant intraportal administration of LEW recipient blood, rather than those cells in recipient spleen (46.2% versus 34.4%, 63.7% versus 71.4%). Conclusions: Pretransplant intraportal administration of recipient LEW blood to ACI donor rats prolonged the survival of hepatic allografts. This may be associated with the regional graft-versus-host reaction.

Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen

BackgroundSynthetic siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally, and RNA interference targeting Fas protects mice from fulminant hepatitis. Fas-mediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx), and blockade of Fas and Fas ligand interaction successfully promotes the repopulation of allogenic liver cells in recipient spleens. In the present study, we further investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens. Materials and methodsHepatocytes were isolated from BALB/c mice and mock transfected or transfected with Fas siRNA or GFP siRNA (n = 8/group). The expression of Fas was examined by RT-PCR and flow cytometric analysis. Forty-eight hours later, the cells were transplanted into spleens of allogenic B6 mice. Spleens were harvested on day 21 after transplantation. Apoptosis was assessed by TUNEL assay, survival of hepatocytes by alanine transaminase (ALT) assay. ResultsFas siRNA transfection reduced Fas expression on hepatocytes at both mRNA and protein levels (P < .05). Upon transplanting into recipient spleens, hepatocytes transfected with Fas siRNA demonstrated a lower percentage of apoptosis detected by TUNEL (6 ± 3% in Fas siRNA group vs 12 ± 5% in GFP siRNA group and 10 ± 3% in mock transfected group; P < .05), and increased survival as determined by ALT assay (38.2 ± 10.6 IU/g in Fas siRNA group vs 21.3 ± 8.4 IU/g in GFP siRNA group and 18.5 ± 5.9 IU/g in mock-transfected animals). ConclusionFas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes, and thus holds promise to inhibit acute rejection after hepatocyte transplantation.

The effect on the proliferation and apoptosis of alloreactive T cells of cell dose in a murine MHC-mismatched hematopoietic cell transplantation model

Activation-induced cell death (AICD) of lymphocytes is an apoptotic pathway involved in the control of T-cell homeostasis. The magnitude of graft-vs.-host disease (GVHD) following allogeneic hematopoietic cell transplantation may be attenuated by the enhancement of AICD. The aim of the present study was to clarify the effect of T cell dose upon the fate (proliferation or apoptosis) of individual activated T cells in a murine GVHD model. To this end, we investigated the kinetics of the proliferation and apoptosis of donor T cells in recipient spleens in the early stage of a fully major histocompatibility complex (MHC)-mismatched murine transplantation model from C57BL/6 (H-2 b) to lethally-irradiated (8.5 Gy) BALB/c (H-2 d) mice. To track the behavior of alloreactive lymphocytes in vivo, we used the fluorescent cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester in combination with flow cytometry. Engraftment of donor T cells to recipient spleens was almost completed within 24 h after transfer. After that, at higher doses of transferred cells, the donor T cells actively divided for up to 72 h resulting in a 30-fold increase in cell number at the maximum cell dose (2.0×10 7). As the transferred cell dose decreased, the proliferation of T cells tended to be suppressed. At cell doses of 0.5×10 7 or less, the proliferation of T cells was profoundly suppressed, ultimately resulting in little proliferation of donor T cells observed from 24 to 72 h at the minimum cell dose (0.1×10 7). The frequency of Annexin-V-positive cells was found to increase gradually as the transferred cell dose decreased. Thus, an increase in apoptotic events appeared to play an important role in the suppression of the proliferation of T cells at lower splenocyte doses. Further analyses revealed that Fas ligand (FasL)-positive T cells were observed exclusively among T cells that divided at least 5 times, and that all of them were positive for Annexin-V, indicating that they were in the process of apoptosis. Together with our finding that the frequency of apoptosis increased with the progression of cell division, these findings strongly suggest that AICD occurred through the Fas/FasL system and that AICD increased as the dose of donor T cells participating in the allogeneic response decreased. When relatively small numbers of T cells are confronted with an excess of antigen, they disappear. This process is called clonal exhaustion-deletion. Our results support the idea that AICD is involved in the process of clonal exhaustion-deletion. Relevant to the clinical aspects of hematopoietic cell transplantation, our findings indicate that AICD may be associated with tolerance induction in T-cell-depleted transplantation from HLA-mismatched donors, in which T cells contaminating marrow grafts do not need to be completely removed for achieving tolerance between donors and recipients. Furthermore, our results indicate that a small change in the quantitative balance between antigens and T cells responding to them leads to a large difference in the fate of T cells activated in response to MHC-incompatible antigens. Thus, the size of the T cell dose is one of the important considerations in tolerance induction, GVHD and rejection.