Induction Of Graft-vs-host Disease Research Articles

Tumor-Specific Donor Lymphocyte Infusion for Tumor Relapse after MHC-Haploidentical Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for refractory hematologic malignancies such as leukemia and myelodysplastic syndrome. Tumor relapse, graft-vs host disease (GVHD) and infections are, however, major obstacles to successful allo-HSCT. Donor lymphocyte infusion (DLI) is carried out in some cases of tumor relapse and infections after allo-HSCT. DLI potentially induces or aggravates GVHD due to allo-reactivity of the lymphocytes used for infusion and the efficacy of DLI for tumor relapse is actually limited. Therefore, development of the method of DLI with enhanced anti-tumor effects without induction or aggravation of GVHD is expected to improve the results of DLI in the context of tumor relapse. Tumor antigen-specific T cell receptor (TCR)-expressing cell infusion is one of the potentially effective immunotherapies for refractory tumors. We previously reported that tumor antigen-specific TCR-gene-transduced human lymphocytes engineered with a novel retrovirus vector silencing endogenous TCRs showed reduced allo-reactivity (ASH annual meeting 2014). Utilizing this technology, we may be able to enhance anti-tumor effects without induction or aggravation of GVHD. In this study, we conducted a mouse pre-clinical model that mimics tumor-specific TCR-engineered DLI for tumor relapse after MHC-haploidentical HSCT and explored the effect of DLI on tumor growth and GVHD.We performed experimental study utilizing the following model: MHC-haploidentical BALB/c(H-2d)→CB6F1(H-2b/d) HSCT, BALB/c-derived sarcoma CMS5a and T cells from TCR-transgenic mouse DUC18. CMS5a has a unique mutated form of a mitogen-activated kinase ERK2, and a nonamer peptide, called 9m, incorporating the resulting amino acid substitution is presented on H-2Kd and recognized by the CD8+ CTL clone C18. DUC18 is a BALB/c-background TCR transgenic line that expresses the rearranged Vα10.1 and Vβ8.3 genes of the C18. CD8+ T cells of DUC18 mice can suppress CMS5a tumor growth in the syngeneic BALB/c hosts. Allo-reactivity of DUC18 T cells was reduced as compared to that of wild type BALB/c T cells. Based on the results of preliminary experiments, we set standard experimental conditions as follows: X-ray irradiation (10 Gy) for pre-conditioning, 5x106 whole bone marrow and 0.25x106 T cell transplant (BMT) for mild GVHD induction, 1x106 CMS5a inoculation for progressive tumor growth in BMT recipients, and 4x106 CD8+ T cells for observation of the effect of DLI on tumor growth and GVHD. In some experiment, CD4+ T cells were co-infused with CD8+ T cells. DLI was carried out 3 days after CMS5a inoculation at 2, 4 or 8 weeks after BMT.We first used CD8+ T cells from naïve BALB/c or DUC18 mice for DLI at 8 weeks after BMT. No GVHD aggravation was observed in either BALB/c or DUC18 CD8+ T cell recipients and, as expected, suppression of tumor growth was observed only in the DUC18 CD8+ T cell recipients. Next, DLIs using CD8+ T cells only, or both CD8+ and CD4+ (CD8+/CD4+) T cells from naïve mice were carried out at 4 or 2 weeks after BMT. No apparent effect of CD4+ T cell co-infusion on tumor growth and GVHD was observed in both types of donor T cell recipients when DLI was carried out at 4 weeks after BMT. However, GVHD was aggravated in CD8+/CD4+ T cell recipients from both types of donors when DLI was carried out at 2 weeks after BMT, and no CD4-driven additional anti-tumor effect was observed in DUC18 T cell recipients. We then performed DLI using in vitro activated/expanded T cells including both CD8+ and CD4+ as a more clinically relevant model. BALB/c and DUC18 splenocytes were activated and expanded in vitro and infused (adjusted CD8+ T cell number to 4x106/mouse) into recipients at 2 or 8 weeks after BMT. Aggravation of GVHD was observed in either BALB/c or DUC18 activated/expanded T cell recipients when DLI was carried out at 2 weeks after BMT. On the other hand, it was not observed when DLI was carried out at 8 weeks after BMT. Anti-tumor effect of DUC18 T cells was observed regardless of timings of DLI.Taken together, infusion of tumor-specific donor lymphocytes with reduced allo-reactivity for tumor relapse after MHC-haploidentical HSCT will be promising strategy. Timing of DLI and condition of recipients such as inflammatory status may affect GVHD. Multiple model studies are required for development of more effective DLI strategies without aggravation of GVHD. DisclosuresTawara:Astellas: Honoraria. Katayama:Nippon Shinyaku: Honoraria; Chugai: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Dainippon Sumitomo Pharma: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Shionogi: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Taisho Toyama Pharma: Honoraria; Shire: Honoraria; Astellas: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Eisai: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb Japan: Honoraria. Ikeda:Ono Pharmaceutical Co., Ltd: Honoraria; Daiichi Sankyo Co., Ltd: Honoraria.

Pharmacologic inhibition of PKCα and PKCθ prevents GVHD while preserving GVL activity in mice

AbstractAllogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell–mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα−/−/θ−/− donor T cells to induce GVHD was further reduced compared with PKCθ−/− T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.

The Liver Can Host Donor Hematopoiesis and Maintains a Strong Population of Innate Immune Cells That Contributes to Host Protection After Transplantation of Purified Hematopoietic Stem Cells.

Abstract 3545Poster Board III-482Standing in the line of first defense, the liver is a critical immunocompetent organ. It is armed with lymphocytes, including T cells (TC), natural killer (NK) cells, NK T cells, and a variety of antigen-presenting cells (APC), such as dendritic cells and resident macrophages (Mph), called Kupffer cells. Because it is exposed to large amounts of toxins and antigens, both destructive and harmless, liver immunity must provide immunogenic and tolerogenic mechanisms. Moreover, as the organ of fetal blood production the liver can, if required, resume its hematopoietic function. Here, we studied the role of the liver as a hematopoietic and lymphatic organ after hematopoietic cell transplantation (HCT). Lethally irradiated BALB.K and BALB.B mice were given MHC-matched, FACS purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin-) from AKR/J and C57BL/6 donors, respectively, alone or supplemented with 10∧7 splenocytes (SP) for GVHD induction. Mononuclear cells (MNC) were Ficoll-separated from flushed livers 1 to 6 weeks (w) post transplant (pTX) and FACS analyzed. In recipients of TC-containing grafts, the liver was a major target organ of acute graft-vs-host disease (GVHD) with prominent donor lymphocyte expansion causing destruction of the hepatic portal morphology. Rare HSC-derived cells were observed in the livers. In contrast, mice given purified HSC showed no clinical or histological signs of GVHD, yet early pTX a high proportion of donor HSC-derived MNC was observed within the livers, comprising ∼75% of the MNC at 2w. Phenotype analysis revealed that these HSC-derived MNC were primarily NK cells (DX5+CD122+) or Mph (Mac1+F4/80+). In fact, amongst all nucleated cells, NK cells represented >10% and were mixed donor/host type. Interestingly, the Mph were all donor derived. This observation of over-representation by cells of innate immunity (including NK cells and Mph) in livers of recipients of HSC alone led us to hypothesize that these cells might exert protective functions against increased amounts of pathogens and toxins entering the circulation from irradiation-damaged intestines. Thus, to suppress donor Mph reconstitution pTX, silica was injected intraperitoneally on d-1, and every 3d thereafter. All recipients of HSC alone recovered rapidly after irradiation (d5-7), while at this time point recipients of HSC plus silica showed severe weight loss, hunched posture, ruffled fur, diarrhea, with <50% (7/15) survival. These survivors clinically stabilized around d12, suggesting that the intestines recovered from injury. To test if the presence of the HSC derived NK cells and APC could contribute to host protection from GVHD, a lethal dose of SP (10∧7) was injected simultaneously with HSC, or with a delay of 7d or 9d. All mice given SP on d0 died within 9d and 3/5 of those receiving SP on d7 died by d12. However, all mice given SP on d9 recovered fully and showed no signs of GVHD, despite the lymphopenic host environment that usually promotes homeostatic expansion of mature donor TC. In conclusion, the role of the liver as an immunologically active organ after ‘conventional' HCT is often masked by donor TC expansion with subsequent GVHD. Here, we provide evidence that if grafts are devoid of mature lymphoid cells, innate immunity recovers rapidly, and in fact exceeds unmanipulated controls. Donor Mph may protect the host from pathogens and endotoxemia. Moreover, they may neutralize activated donor TC and thereby mediate tolerance between donor and host. Likewise, the elevated proportion of donor and host NK cells, which is lacking in GVHD affected mice, suggest another beneficial mechanism of protection, as NK cells have been reported to be capable of reducing GVHD. Immunohistochemical studies for a better quantitative assessment of resident immune cells in the liver pTX are underway. Disclosures:No relevant conflicts of interest to declare.

The Immunological Impact of Genetic Drift in the B10.BR Congenic Inbred Mouse Strain

The MHC-matched, minor histocompatibility Ag (miHA)-mismatched B10.BR-->CBA strain combination has been used to elucidate the immunobiology of graft-vs-host disease (GVHD) following allogeneic bone marrow transplantation. Studies conducted in the 1980s had established that B10.BR CD8+ T cells were capable of mediating GVHD in the absence of CD4+ T cells, and that CD4+ T cells were unable to induce lethal disease. In more recent studies with this GVHD model, we detected etiological discrepancies with the previously published results, which suggested that genetic drift might have occurred within the B10.BR strain. In particular, there was increased allorecognition of CBA miHA by B10.BR CD4+ T cells, as determined by both TCR Vbeta spectratype analysis and the induction of lethal GVHD in CBA recipients. Additionally, alloreactivity was observed between the genetically drifted mice (B10.BR/Jdrif) and mice rederived from frozen embryos of the original strain (B10.BR/Jrep) using Vbeta spectratype analysis and IFN-gamma ELISPOT assays, suggesting that new miHA differences had arisen between the mice. Furthermore, T cell-depleted B10.BR/Jdrif bone marrow cells were unable to provide long-term survival following either allogeneic or syngeneic bone marrow transplantation. Gene expression analysis revealed several genes involved in hematopoiesis that were overexpressed in the lineage-negative fraction of B10.BR/Jdrif bone marrow, as compared with B10.BR/Jrep mice. Taken together, these results suggest that genetic drift in the B10.BR strain has significantly impacted the immune alloreactive response in the GVHD model by causing altered expression of miHA and diminished capacity for survival following transplantation into lethally irradiated recipients.

Fas Expression on Antigen-Specific T Cells Has Costimulatory, Helper, and Down-Regulatory Functions In Vivo for Cytotoxic T Cell Responses but Not for T Cell-Dependent B Cell Responses

Fas-mediated apoptosis is an important contributor to contraction of Ag-driven T cell responses acting only on activated Ag-specific T cells. The effects of targeted Fas deletion on selected cell populations are well described however little is known regarding the consequences of Fas deletion on only activated Ag-specific T cells. We addressed this question using the parent-into-F(1) (P-->F(1)) model of acute or chronic (lupus-like) graft-vs-host disease (GVHD) as a model of either a CTL-mediated or T-dependent B cell-mediated response, respectively. By transferring Fas-deficient lpr donor T cells into Fas-intact F(1) hosts, the in vivo role of Ag-specific T cell Fas can be determined. Our results demonstrate a novel dichotomy of Ag-specific T cell Fas function in that: 1) Fas expression on Ag-activated T cells has costimulatory, helper, and down-regulatory roles in vivo and 2) these roles were observed only in a CTL response (acute GVHD) and not in a T-dependent B cell response (chronic GVHD). Specifically, CD4 T cell Fas expression is important for optimal CD4 initial expansion and absolutely required for help for CD8 effector CTL. Donor CD8 T cell Fas expression played an important but not exclusive role in apoptosis and down-regulation. By contrast, CD4 Fas expression played no detectable role in modulating chronic GVHD induction or disease expression. These results demonstrate a novel role for Ag-specific T cell Fas expression in in vivo CTL responses and support a review of the paradigm by which Fas deficiency accelerates lupus in MRL/lpr lupus-prone mice.

CTL-promoting effects of CD40 stimulation outweigh B cell-stimulatory effects resulting in B cell elimination and disease improvement in a murine model of lupus.

CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA-->F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.

Inter-Strain Tissue-Infiltrating T Cell Responses to Minor Histocompatibility Antigens Involved in Graft-Versus-Host Disease as Determined by Vβ Spectratype Analysis

Lethal graft-vs-host disease (GVHD) can be induced between MHC-matched murine strains expressing multiple minor histocompatibility Ag differences. In the B6->BALB.B model, both CD4(+) and CD8(+) donor T cells can mediate lethal GVHD, whereas in the B6->CXB-2 model, only CD8(+) T cells are lethal. TCR Vbeta CDR3-size spectratyping was previously used to analyze CD8(+) and CD4(+) T cell responses in lethally irradiated BALB.B and CXB-2 recipients, which showed significant overlap in the reacting repertoires. However, CD4(+) T cells exhibited unique skewing of the Vbeta2 and 11 families in only BALB.B recipients. These Vbeta family reactivities were confirmed by immunohistochemical staining of lingual epithelial infiltrates, and by positive and negative selection Vbeta family transfer experiments for GVHD induction in BALB.B recipients. We have now extended these studies to examine the T cell repertoire responses involved in target tissue damage. Infiltrating B6 host-presensitized CD8(+) and CD4(+) T cells were isolated 8-10 days post-transplant from the spleens, intestines and livers of CXB-2 and BALB.B transplant recipients. For both T cell subsets, the results indicated overlapping tissue skewings between the recipients, also between the tissues sampled within the respective recipients as well as tissue specific responses unique to both the BALB.B and CXB-2 infiltrates. Most notably, the CD4(+) Vbeta 11(+) family was skewed in the intestines of BALB.B but not CXB-2 recipients. Taken together, these data suggest that there are likely to be target tissue-related anti-multiple minor histocompatibility Ag-specific responses in each of the strain recipients, which may also differ from those found in peripheral lymphoid organs.

NKT Cells Are Potent Regulators of GVHD Following Adoptive Transfer in Allogeneic BMT.

NKT cells, which are CD1d reactive and express an invariant TCR, are thought to play an immunoregulatory role in suppressing dysfunctional immune reactions including graft vs. host disease (GVHD). Whether non-manipulated donor-type NKT can suppress GVHD following adoptive transfer has not been addressed, nor has the trafficking pattern of NKT in a hematopoetic transplantation (HCT) setting. To determine how effectively NKT proliferate and traffic in a HCT setting, 5.5x105 highly purified (>95%) NKT (DX5+TCRβ+CD4+) from luciferase positive (luc+) C57BL/6 (H-2b) mice were transferred into lethally irradiated Balb/c (H-2d) recipients along with 5x106 T-cell depleted bone marrow (TCD-BM) from wild-type C57BL/6 mice. Proliferation and migration of luc+NKT was monitored by bioluminescence imaging (BLI). By day 4 after transfer, a prominent signal was observed in the spleen and lymph node (LN) sites. Between days 7 and 10, the NKT migrated to skin, while still remaining present in high numbers in LNs, but decreasing to low levels in spleen. The total photons emitted per mouse reached a peak at approximately 25 days after transplantation, followed by a steady decline. The NKT expansion was more vigorous than that of luc+CD4+CD25+ regulatory T cells (Tregs), which also peak around day 25, but do not show extensive migration to skin. Expansion of NKT was far less than conventional (CD4+ and CD8+) T cells (Tcon), with approximately 10 times more photons/mouse being emitted from 5x105 luc+Tcon as from 5.5x105 NKT. The NKT did not cause GVHD where Tcon rapidly resulted in acute GVHD and animal mortality. To assess the impact of donor-type NKT on GVHD induction by Tcon, we co-transferred various doses of highly purified wt NKT at day 0 with 5x106 TCD-BM, followed by 5x105 luc+Tcon at day 2. Weight and survival of groups were monitored, as well as the proliferation of Tcon by BLI. In groups receiving only Tcon, 50% of the mice died within 2 weeks, and 90% died by day 80. Remarkably, if 2.5x104 sorted NKT were transferred, 100% of the mice survived past day 100. To achieve the same effect with Tregs, 2.5x105 Tregs were required. Mice treated with 2.5x104 NKT lost more weight at early time points than those receiving 2.5x105 Tregs, but both groups recovered from this weight loss and did not exhibit other signs of GVHD (hair loss, hunched back, diarrhea, etc). Interestingly, 2.5x104 NKT caused only a slight reduction in Tcon proliferation, whereas 2.5x105 Treg strongly reduced Tcon proliferation. Surprisingly, when the dose of NKT was increased to 5x104, survival was only 62%, and when increased to 1x105 cells, only 50% of mice survived past day 100. To determine how NKT reduce GVHD, we examined intracellular levels of various cytokines in Tcon with or without 2.5x104 NKT, following HCT. At 8 days after HCT, mice receiving NKT had reductions in the number of IL-17-positive cells (CD4: 2.6% to 0.84%; CD8: 2.5% to 0.66%), and TNFα+ cells (CD4: 45% to 27%; CD8 36% to 24%) in cells from LNs. By day 11, IL-17-positive cells had declined to undetectable levels and TNF levels between groups were equivalent. IFNg levels, which were high in both NKT treated and untreated groups at day 8 (85%–95%), decreased significantly in NKT treated mice by day 11 (CD4: 40%; CD8: 43%), but were still abundant in Tcon only mice (CD4: 78%; CD8: 80%). Together these data indicate that NKT cells persist in vivo upon adoptive transfer and suppress GVHD by decreasing the percentage of Tcon secreting pro-inflammatory cytokines.

Improved Immune Function with Donor B-cell Infusion after Semi-Allogeneic Bone Marrow Transplantation in Mice

Regeneration of the immune system after bone marrow transplantation (BMT) is a slow process, often prolonged by the development and treatment of graft vs. host disease (GVHD). Donor lymphocyte infusion using allogeneic T-cells is widely applied for the induction of GVHD, which is associated with the desired graft vs. leukemia effect. Due to the slow immune recovery, our objective was to accelerate the immune recovery post-BMT by B-cell injections. T-cell-depleted stem cells obtained from female C57BL/6 (B6) mice were transplanted into lethally irradiated (Balb/c x C57BL/6) F-1 female mice. Seven days post-transplantation, murine B-cells of male C57BL/6 origin were infused into the T-cell-depleted chimeras. Thirty and 60 days post-transplantation, PCR analysis of the Y-chromosome was carried out to detect male B-cells in the transplant recipients. In order to evaluate the specific antibody response, the donors were immunized by specific T-cell-dependent and -independent antigens. None of the T-cell-depleted transplanted mice developed GVHD during a follow-up period of 650 days, whereas all non-T-cell-depleted recipients died. At 60 days post-transplantation, significantly higher levels of immunoglobulins (IgA, IgG1, IgG3 isotypes) were seen in chimeras supplemented with male B-cells than in chimeras reconstituted with T-cell-depleted stem cells alone. Our data document the feasibility of administering B-cell therapy post-allogeneic BMT to improve recovery of the humeral arm of the immune system while avoiding GVHD. Furthermore, post-transplant B-cell administration may have an important impact as an alternative to IV immunoglobulin infusions.

Experimental Study on Prevention of GVHD by Selective Elimination of Alloreactive Donor Lymphocytes Prior to Stem Cell Transplantation.

Objective: To investigate a new concept aiming for induction of graft-vs-leukemia (GVL) effect prior to stem cell transplantation (SCT). Mismatched lymphocytes given pre-SCT will be followed by selective elimination of alloreactive donor lymphocytes, thus avoiding lethal graft-vs-host disease (GVHD).Methods: Female (BALB/c×C57BL/6)F1 mice (H-2d/b) as recipients received sublethal total body irradiation (TBI) of 4 Gy (60Coγ-ray) on day 0 followed by being inoculated with 0.5×107 P388D1 leukemia cell line on day 1, injection of 1.5×107 allogeneic splenocytes supplied by C57BL/6 male mice(H-2b)for induction of GVHD, intraperitoneally injection of cyclophosphamide (Cy) (200 mg/kg) or TBI (9 Gy) were given on day 7, one day later, treated mice were rescued with 3×107 syngeneic bone marrow cells supplied by (BALB/c×C57BL/6)F1 male mice(H-2d/b). Recipients were observed clinical manifestation, phenotype, re-establishment of haematogenesis, histopathologic changes of internal organs suffered from GVHD and investigated donor chimerism by the semi-quantitate analyses of polymerase chain reaction (PCR). Data was analyzed by SPSS 10.0 software and expressed as mean ± SD.Results: Recipients had no occurrence of leukemia and GVHD by selective elimination of alloreactive donor lymphocytes by Cy and TBI, survived more than 210 days, to become complete-donor chimerism on day +21. The ratio of chimerism descended subsequently, but still displayed mixed-chimerism on day +90. Control mice died of evident GVHD, leukemia or other death-related-transplantation within 20 to 36 days(p<0.01). Attempting to induce GVL effects by mismatched lymphocytes given before stem cell transplantation followed by selective elimination of alloreactive donor lymphocytes, thus avoiding graft-vs-host disease (GVHD) was feasible.

Donor CD4+ T and B cells in transplants induce chronic graft-versus-host disease with autoimmune manifestations

AbstractChronic graft-vs-host disease (GVHD) is a major cause of morbidity and mortality of long-term survivors of allogeneic hemato-poietic cell transplantation (HCT). Chronic GVHD can have features of an autoimmune collagen vascular disease with clinical manifestations similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T and B cells are generated in chronic GVHD recipients. We have recently developed a new chronic GVHD model by transplantation of donor DBA/2 (H-2d) spleen cells into major histocompatibility complex (MHC)-matched but minor antigen-mismatched sublethally irradiated BALB/c (H-2d) recipients as well as athymic BALB/cnu/nu and adult-thymectomized BALB/c recipients. Both euthymic and athymic BALB/c recipients developed high levels of serum IgG autoantibodies, sclerodermatous skin damage, and glomerulonephritis. Disease induction required both donor CD25-CD4+ T and B cells in transplants. In contrast, donor CD25+CD4+ T regulatory (Treg) cells prevented the disease induction. These results indicate that host thymus is not required for induction of chronic GVHD and that quiescent autoreactive T and B cells in transplants from nonautoimmune donors may be activated and expanded to cause chronic GVHD with autoimmune manifestations in allogeneic recipients, and donor Treg cells can suppress this process.

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.

The Influence of Migration, Alloreactive Repertoire and Memory Subset on the Differential Ability of Naive and Memory T Cells To Induce GVHD.

Allogeneic stem cell transplantation (alloSCT) can cure many hematologic malignancies and hematopoietic stem cell disorders but is frequently complicated by graft vs. host disease (GVHD). We and others have published that memory phenotype (CD62LloCD44hi) T cells do not cause GVHD but can engraft and mount immune responses, including graft-vs.-tumor (GVT) effects. Importantly, these findings apply to GVHD induced by CD4 or CD8 T cells, and across major MHC differences. Thus, the inability to induce GVHD seems a fundamental property of memory phenotype (M) cells. We are investigating several hypotheses to explain why naïve cells (N) cause potent GVHD but M cells do not, including: 1. M cells lack CD62L and fail to traffic to LN and PP, two sites that may be essential for initial priming to allogeneic antigens and 2. M cells have a restricted alloreactive repertoire. There are at least two types of memory cells: central memory (CM) and effector memory (EM). EM cells (CD62LloCCR7neg) quickly express effector functions upon restimulation, preferentially migrate to tissues and spleen (bypassing LNs) and have relatively lower proliferative potential. CM cells (CD62LhiCCR7pos) have hybrid properties of both N and effector cells. Like N cells, their adhesion and chemokine receptors promote migration to LNs; however, their effector functions are more vigorous than N cells, yet slower than EM cells. It is of clinical interest for the design of M cell transfusion to determine the contributions of CM vs. EM cells in GVHD, but prior studies have not clearly investigated CM cells. We therefore compared GVHD initiated by N, EM and CM CD4 cells in the B6 (H-2b) ->BALB/c (H-2d) model. CM cells caused severe GVHD, similar to that induced by N cells. This suggests that LN homing enables M cells to initiate GVHD and/or that CM cells have unique functional properties required to initiate GVHD which are lacking in EM cells. We next asked whether LN entry was required for donor cells to initiate GVHD. N cells induced GVHD, albeit less severe than in WT recipients, in recipients lacking all secondary lymphoid organs (LN, PP and spleen), suggesting that priming in tissues is sufficient. Both of these findings support those of Beilhack, et al. ( Blood 2005; 106:1113) that migration affects T cell initiation of GVHD. However, we also found that N cells from CD62L-deficient donors caused GVHD. Thus, because CD62L function and even secondary lymphoid tissue are dispensable for GVHD induction, we conclude that homing differences alone do not control GVHD and that critical functional properties other than homing must limit EM cells' ability to induce GVHD. To address the role of repertoire, we increased the alloreactive precursor frequency of M cells. We isolated Thy1.1 (B6.C) (H-2d) effector T cells from mice with active GVHD (BALB/c recipients), and therefore with expanded anti-BALB/c repertoire, and parked them in syngeneic RAG1-/- (B6.C) recipients to allow them to differentiate into M cells. After 4 weeks we re-isolated the alloreactive M cells and transplanted them into new BALB/c recipients. The alloreactive M cells engrafted and caused transient skin GVHD without gut GVHD, unlike the more severe chronic GVHD induced by fresh N cells. This muted GVHD argues that although M cells can cause limited GVHD if their repertoire is artificially increased, they are still lacking in other functional properties required for optimal GVHD induction.

Donor CD4+ T and B Cells in Transplants Induce Autoimmune-Like Chronic Graft Versus Host Disease.

Chronic graft-vs-host disease (GVHD) is a major morbidity and mortality of long-term survivors of allogeneic hematopoietic cell transplantation (HCT). Chronic GVHD can have features of an autoimmune collagen-vascular disease with clinical manifestations similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T and B cells are generated in chronic GVHD recipients. We have recently developed a new autoimmune-like chronic GVHD model by transplantation of donor DBA/2 (H-2d) spleen cells into MHC-matched but minor antigen-mismatched sublethally irradiated BALB/c (H-2d) recipients, as well as athymic BALB/c nu/nu and adult thymectomized BALB/c recipients. Both euthymic and athymic BALB/c recipients developed high-levels of serum IgG autoantibodies, sclerodermatous skin damage and glomerulonephritis. Disease induction required both donor CD25−CD4+ T and B cells in transplants. In contrast, donor CD25+CD4+ T regulatory (Treg) cells prevented the disease induction. These results indicate that host thymus is not required for induction of autoimmune-like chronic GVHD, and that quiescent autoreactive T and B cells in transplants from non-autoimmune donors may be activated and expanded to cause autoimmune-like chronic GVHD in allogeneic recipients, and donor Treg cells can suppress this process.

288. A System for GMP Expansion and Transduction of Human T Cells with High Functionality Proven by Consistent Induction of GvHD in a Mouse Xenotransplant Model

Top of pageAbstract Here we demonstrate the GMP scale-up process for a human gene therapy clinical trial utilizing retroviral vector transduced allogeneic T cells eliciting a Graft vs. Leukemia effect in a Donor Lymphocyte Infusion (DLI) setting. After elimination of leukemic cells by DLI, a fusion CD34-TK suicide gene conferring sensitivity to ganciclovir will allow killing of transduced T cells and abrogation of Graft vs. Host Disease (GvHD), a serious side effect of DLI. The truncated CD34 receptor, normally not present on T cells, allows for purification of transduced cells to >95% in a magnetic cell sorter (CliniMacs, Miltenyi, Auburn, CA). PBMCs from 8 donors were stimulated in a closed bag system using the new GMP grade Stemline serumfree T cell medium (Sigma, St. Louis, MO). This medium allowed for elimination of donor variability, which we experienced in T cell expansions using other serumfree media. T cell stimulation was done using clinical grade magnetic beads coated with CD3 and CD28 antibodies (Xcyte Therapies, Inc., Seattle, WA). At least a 2 fold T cell expansion is needed, since transduction and expression of the transgene using a Moloney leukemia virus based retroviral vector depends on cell division and activation. Maintenance and transduction of CD4 and CD8 cells in a physiological ratio is imperative for normal function of infused T cells. Requirements stipulated by regulatory agencies demanded not to introduce more than 1-2 vector copies per cell to keep the risk for insertional mutagenesis to a minimum. A transduction frequency of 20-30% obtained at an MOI of 1-2 was required to generate this copy number (Rettig et al., 2003). In the past we showed that high concentrations of IL-2 (500 U/ml, as previously used in clinical T cell expansions) impair the in vivo functionality of T cells, using our NOD SCID/B2 M deficient mouse model of T cell expansion. We therefore lowered the IL-2 concetration to 50 U/ml. After 48 hours of pre-stimulation, T cells were transduced twice, medium was replaced by bag spinning, removing 2/3 of the supernatant and replacing it with fresh vector containing medium. Cells were harvested on day 4 by disrupting bead/cell clumps and removing the magnetic beads by application of a strong magnetic field. Although a low IL-2 concentration was used, a 3 fold expansion of T cells with 30% transduction efficiency, equally well distributed in the CD4 and CD8 compartment, was observed. The CD4 and CD8 ratios were maintained at input ratio, cell viability was greater than 95%. The most remarkable result was the outstanding activation and in vivo functionality of the expanded T cells. All NOD SCID/B2 M deficient mice (n=12) injected at a cell dose of 107 developed lethal GvHD at day 15 post injection. Activated cells clearly outperformed the GvHD potential of na|[iuml]|ve T cells (Nervi et al., 2005). For the first time, these results demonstrate consistent GvHD elicited in a mouse xenotransplant model by 107 expanded and activated human T cells produced in a closed system, serumfree GMP manufacturing process.

CD30/CD30 ligand (CD153) interaction regulates CD4+ T cell-mediated graft-versus-host disease.

CD30, a TNFR family member, is expressed on activated CD4(+) and CD8(+) T cells and B cells and is a marker of Hodgkin's lymphoma; its ligand, CD30L (CD153) is expressed by activated CD4(+) and CD8(+) T cells, B cells, and macrophages. Signaling via CD30 can lead to proliferation or cell death. CD30-deficient (-/-) mice have impaired thymic negative selection and increased autoreactivity. Although human alloreactive T cells preferentially reside within the CD30(+) T cell subset, implicating CD30 as a regulator of T cell immune responses, the role of CD30/CD153 in regulating graft-vs-host disease (GVHD) has not been reported. We used a neutralizing anti-CD153 mAb, CD30(-/-) donor mice, and generated CD153(-/-) recipient mice to analyze the effect of CD30/CD153 interaction on GVHD induction. Our data indicate that the CD30/CD153 pathway is a potent regulator of CD4(+), but not CD8(+), T cell-mediated GVHD. Although blocking CD30/CD153 interactions in vivo did not affect alloreactive CD4(+) T cell proliferation or apoptosis, a substantial reduction in donor CD4(+) T cell migration into the gastrointestinal tract was readily observed with lesser effects in other GVHD target organs. Blockade of the CD30/CD153 pathway represents a new approach for preventing CD4(+) T cell-mediated GVHD.

A novel approach for prevention of lethal GVHD by selective elimination of alloreactive donor lymphocytes prior to stem cell transplantation

Objective Experiments were designed to investigate a new concept aiming for induction of graft-vs-malignancy (GVM) effect prior to stem cell transplantation (SCT). Mismatched lymphocytes given pre-SCT will be followed by a selective elimination of alloreactive donor lymphocytes, thus avoiding lethal graft-vs-host disease (GVHD). Methods Recipient mice treated with sublethal total-body irradiation (TBI) received a single injection of allogeneic splenocytes, either naïve or rIL-2 activated (ADL), for induction of GVHD. To prevent lethal GVHD, cyclophosphamide (Cy) (200 mg/kg) or TBI (9 Gy) were given 4 days after cell inoculation. One day later, treated mice were rescued with syngeneic bone marrow cells. Results Both Cy and TBI significantly ( p < 0.001) prevented GVHD in all recipients inoculated with either naïve cells or ADL and all recipients survived more than 250 days. Control mice not rescued with CY or TBI died of GVHD within 20 to 25 days. A significant proportion of recipients inoculated with 4T1 tumor cells, treated with ADL followed by TBI 9 Gy, survived more than 250 days disease-free in comparison with 22 days in untreated control mice ( p <0.001). Conclusions Attempting to induce short, yet effective, GVM effects before rather than after SCT, thus avoiding lethal GVHD, may represent an innovative approach for more effective yet safer use of SCT for tumor immunotherapy.

Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease.

Interleukin-7 has been shown to enhance T cell reconstitution after allogeneic bone marrow transplantation, in part, by expansion of mature donor T cells, but whether IL-7 also exacerbates graft-vs-host disease (GVHD) remains unresolved. To address this issue, we examined the effect of IL-7 on GVHD induction using a well-defined murine GVHD model (B6-->B6AF1/J). Administration of IL-7 to nonirradiated B6AF1/J recipients of B6 T cells resulted in expansion of splenic donor CD4(+) and CD8(+) T cells and increased GVHD mortality. In contrast, administration of IL-7 on the same schedule failed to increase GVHD mortality in either sublethally or lethally irradiated animals that received graded doses of T cells designed to induce varying degrees of GVHD severity. Moreover, IL-7 failed to increase the number of alloreactive T cells when examined in a murine model (B6-->BALB.B) that allowed for direct quantitation of graft-vs-host-reactive T cells. The combination of irradiation and transplantation of alloreactive donor T cells resulted in significantly increased levels of endogenous splenic IL-7 mRNA when compared with nonirradiated transplanted animals, providing a potential explanation for why exogenous IL-7 did not increase GVHD severity in these mice. We conclude that host conditioning modulates the ability of exogenous IL-7 to exacerbate GVHD and that this occurs through induction of endogenous IL-7 production.

Minor histocompatibility antigens in human stem cell transplantation.

Minor histocompatibility antigens (mHags) play a major role in graft rejection, the induction of detrimental graft-vs-host disease (GVHD), and the development of the beneficial graft-vs-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT). mHags can be defined as amino acid polymorphisms in cellular proteins that can lead to differential presentation of antigenic peptides in HLA molecules and therefore to differential recognition by T cells. The tissue distribution of the mHags and the HLA molecules by which they can be presented play a significant role in the clinical outcome of T-cell responses against these antigens. In part, differential recognition by T cells of mHags specifically expressed in hematopoietic cells, including the malignant cells from the recipient may result in GVL reactivity without concurrent GVHD. Furthermore, T-cell responses against proteins solely expressed in hematopoietic cell lineages from which the malignancy is derived may be appropriate mediators of GVL reactivity without GVHD induction. Characterization of clinical immune responses in patients treated for relapsed leukemia after allogeneic SCT with donor lymphocyte infusion in the absence of GVHD may lead to the characterization of new mHags that can be exploited to generate tumor-specific immune responses. By in vitro generation of T-cell responses against defined mHags, the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic transplantation may be improved.

IL-6 deficiency allows for enhanced therapeutic value after bone marrow transplantation across a minor histocompatibility barrier in the twitcher (globoid cell leukodystrophy) mouse.

Bone marrow transplantation (BMT) has therapeutic value for twitcher (globoid cell leukodystrophy) mice, which suffer from a genetic deficiency of the lysosomal enzyme galactosylceramidase that leads to progressive demyelination and early death. Preliminary investigations indicated that a semiallogeneic BMT resulted in graft vs. host disease (GVHD) in twitcher mice but not normal mice. Increased production of the cytokine IL-6 has been demonstrated in twitcher mice, and it has been linked with induction of GVHD. We investigated the effects of BMT in twitcher/IL-6 deficient mice and compared these findings with those from transplanted twitcher and control mice. After a semiallogeneic BMT, 11.4% of controls died within few weeks while the rest survived >100 days without GVHD. In contrast, 85% of the transplanted twitcher mice died by 70 days and 65% developed clinical signs of GVHD, e.g., alopecia and weight loss. In transplanted twitcher/IL-6 deficient mice, only 21% died by Day 70, none had alopecia, and 23% had weight loss. There was no difference in the onset day and severity of twitching between twitcher and twitcher/IL-6 deficient mice after BMT. In transplanted twitcher/IL-6 deficient mice, there was improvement of BBB integrity and a decrease in globoid cell number compared with nontransplanted twitcher/IL-6 deficient mice. In summary, these results demonstrate that an underlying pathology like globoid cell leukodystrophy leads to activation of GVHD responses in a donor-host combination that would not normally induce GVHD. Furthermore, IL-6 seems to play a key role because a deficiency of IL-6 results in a better prognosis.