Cell-depleted Bone Marrow Transplantation Research Articles

Venetoclax imparts distinct cell death sensitivity and adaptivity patterns in T cells

BH3 mimetics are increasingly used as anti-cancer therapeutics either alone or in conjunction with other chemotherapies. However, mounting evidence has also demonstrated that BH3 mimetics modulate varied amounts of apoptotic signaling in healthy immune populations. In order to maximize their clinical potential, it will be essential to understand how BH3 mimetics affect discrete immune populations and to determine how BH3 mimetic pressure causes immune system adaptation. Here we focus on the BCL-2 specific inhibitor venetoclax (ABT-199) and its effects following short-term and long-term BCL-2 blockade on T cell subsets. Seven day “short-term” ex vivo and in vivo BCL-2 inhibition led to divergent cell death sensitivity patterns in CD8+ T cells, CD4+ T cells, and Tregs resulting in shifting of global T cell populations towards a more memory T cell state with increased expression of BCL-2, BCL-XL, and MCL-1. However, twenty-eight day “long-term” BCL-2 blockade following T cell-depleted bone marrow transplantation did not lead to changes in the global T cell landscape. Despite the lack of changes in T cell proportions, animals treated with venetoclax developed CD8+ and CD4+ T cells with high levels of BCL-2 and were more resistant to apoptotic stimuli following expansion post-transplant. Further, we demonstrate through RNA profiling that T cells adapt while under BCL-2 blockade post-transplant and develop a more activated genotype. Taken together, these data emphasize the importance of evaluating how BH3 mimetics affect the immune system in different treatment modalities and disease contexts and suggest that venetoclax should be further explored as an immunomodulatory compound.

Beware of the persistent abscess: Chronic granulomatous disease presenting with suppurative lymphadenitis

Abstract An 18-month old boy presented with axillary suppurative lymphadenitis, which failed to respond to conservative management. His care was transferred to pediatric surgeons for further management. Resolution of the abscess was not achieved despite two incision and drainage procedures. Cultures obtained from the abscess grew Serratia Marcescens , and Staphylococcus aureus . A diagnosis of chronic granulomatous disease (CGD) was made, subsequently identified to be x-linked. Review of a chest X-ray previously reported as normal identified focal consolidation. This was investigated with CT and thought to represent previous granuloma with current Staphylococcus infection. The boy was started on prophylactic septrin and itraconizole, in addition to 2 months of treatment antibiotics. The abscess healed, and a repeat CT demonstrated resolution of the cavitating lung lesion. A gastrostomy was inserted in preparation for a T cell-depleted haploidentical bone marrow transplantation, which to date appears to have been curative. Chronic granulomatous disease is a rare genetic primary immunodeficiency associated with high mortality if diagnosis is delayed. The recent establishment of a UK CGD registry has identified a far higher prevalence than previously thought. Stem cell transplantation now offers a potentially curative treatment, with better outcomes in younger children. It is therefore essential that pediatric surgeons are aware of this diagnosis, and that appropriate investigations are instituted when an abscess fails to resolve, or has cultured unusual organisms.

Apoptotic cell injection-induced tolerance preserved graft versus leukemia effect (P2206)

Abstract Allogeneic hematopoietic cell transplantation (AHCT) is a curative therapy for hematological malignancies unfortunately limited by graft-versus-host-disease (GvHD) occurrence. We have demonstrated that apoptotic cell injection can favor tolerance due to their immunomodulatory properties and thus increase engraftment and limit GvHD, through TGF-β and regulatory T cell dependent mechanisms. However, since apoptotic cell injection favors tolerance we evaluated whether graft-versus-leukemia (GvL) was preserved. In an experimental mouse model of T cell-depleted bone marrow transplantation (TCD BMT), leukemic A20 luciferase+ cells were injected at d0 and allogeneic donor T cell infusion was given on d6. Apoptotic cells were injected either on d0 or d6. Eradication of leukemic cells was observed in all mice receiving T cells, as attested by tumor growth evaluated by luminescence. Mice not receiving T cells died from leukemia within 27 days. Mice demonstrating GvL also developed GvHD according to Ferrara's score, and only 27% survived at d27. In contrast, 50% of mice receiving apoptotic cells on d0 or d6 survived at d27 and demonstrated long-term GvL with the complete absence of leukemic cell growth. Our data clearly demonstrated that GvHD suppression by apoptotic cell injection does not abrogate GvL activity. Thus, apoptotic cell injection should be considered in clinic to favor engraftment, prevent GvHD, conserving high GvL therapeutic activity.

Innate Lymphoid Cell-Derived IL-22 Regulates Epithelial Recovery From Gvhd

Abstract 339There is little understanding of the maintenance and regeneration of epithelial tissues after allogeneic transplant. Most clinical strategies to limit epithelial damage from graft vs. host disease (GVHD) also limit post-transplant immune function. Damage to the gastrointestinal (GI) tract from GVHD is a major cause of morbidity and mortality, and damage to the thymus from pre-transplant conditioning and GVHD can impair immune reconstitution, predispose patients to infection, and increase the risk of relapse. Therefore, understanding of tissue damage and recovery could lead to strategies selectively protecting epithelial tissues, improving intestinal barrier function, and promoting immune reconstitution without worsening post-transplant immunosuppression.We have recently identified that IL-22 from recipient-derived innate lymphoid cells (ILC) is critical for promoting intestinal recovery from GVHD and for promoting thymic recovery from radiation/pre-transplant conditioning. IL-22 deficient mice demonstrated significantly reduced thymopoiesis after total body irradiation (TBI), and IL-22 deficient murine bone marrow transplant (BMT) recipients demonstrated increased GVHD mortality and intestinal histopathology, deficiency of the antimicrobial molecules Reg3γ and Reg3β, and loss of intestinal stem cells needed for epithelial recovery. The source of thymic and intestinal IL-22 was RORγ+CD3−NKp46−IL-7R+CCR6+ lymphoid-tissue-inducer-like cells. Similar to as had been observed in the thymus, intestinal ILC produced IL-22 in response to IL-23, which was upregulated after TBI (p<.05 small intestine, p<.001 large intestine). IL-22 was also upregulated in response to TBI, but not in p40-deficient mice lacking IL-23 (p<.05 small intestine, p<.01 large intestine). ILC were radioresistant, as lethal TBI led to a three-fold increase in the intestinal ILC:CD4 ratio (p<.05). Furthermore, recipient-derived ILC comprised more than 50% of intestinal lamina propria ILC three months after T cell-depleted BMT, well after donor myeloid reconstitution and after donor reconstitution of the intestinal T cell compartment as well (Figure 1).Although intestinal ILC could survive lethal TBI, they were significantly depleted by both MHC mismatched (B6BALB/c) and MHC matched (LPB6) GVHD. Similarly, GVHD led to depletion of thymic IL-22+ ILC and reduction in thymic IL-22 levels (p<.001). Thymic IL-22 was critical for maintaining thymopoiesis during GVHD, as IL-22 deficient BMT recipients demonstrated significantly greater loss of double positive (DP) thymocytes after MHC-mismatched BMT. We previously identified that IL-21 receptor (IL-21R) signaling contributes to the migration of alloreactive donor T cells to the GI tract and that IL-21R-deficent donor T cells mediate significantly reduced GI GVHD. Given the similar homing molecules involved in the migration of donor T cells to the GI tract and thymus in GVHD, we evaluated the role of IL-21 in thymic GVHD. Donor T cell IL-21R deficiency led to increased thymopoiesis and DP thymocytes (p<.001), but not in IL-22-deficient recipients. ILC evaluation indicated that this IL-22 dependency was because IL-21R-deficiencient donor T cells had a reduced capacity to eliminate thymic ILC during GVHD (Figure 2). Therefore, donor T cell IL-21 signaling was critical for the elimination of recipient thymic ILC during GVHD, and preservation of the ILC compartment allowed for the IL-22 mediated regeneration of thymopoiesis. Finally, we also found that administration of rIL-22 post-BMT could reverse the thymic damage caused by GVHD and elimination of ILC, restoring the numbers of DP thymocytes to a level similar to what was observed after T cell-depleted BMT.In summary, IL-22+ ILC are radioresistant and capable of regulating tissue-specific epithelial recovery after allogeneic BMT. However, recipient ILC are extremely sensitive to GVHD, leading to a loss of the IL-22-mediated recovery response. IL-21 blockade can prevent the elimination of recipient thymic ILC by donor T cells in GVHD, and IL-22 administration can restore the thymopoiesis that is lost in GVHD due to ILC elimination. Maintenance of epithelial function post-BMT is thus an active innate immune response requiring cooperation between both recipient stroma and recipient hematopoietic cells. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Post-Transplantation B Cell Function in Different Molecular Types of SCID

Severe combined immunodeficiency (SCID) is a syndrome of diverse genetic cause characterized by profound deficiencies of T, B and sometimes NK cell function. Non-ablative HLA-identical or rigorously T cell-depleted haploidentical parental bone marrow transplantation (BMT) results in thymus-dependent genetically donor T cell development in the recipients, leading to a high rate of long-term survival. However, the development of B cell function has been more problematic. We report here results of analyses of B cell function in 125 SCID recipients prior to and long-term after non-ablative BMT, according to their molecular type. Studies included blood immunoglobulin measurements; antibody titers to standard vaccines, blood group antigens and bacteriophage Φ X 174; flow cytometry to examine for markers of immaturity, memory, switched memory B cells and BAFF receptor expression; B cell chimerism; B cell spectratyping; and B cell proliferation. The results showed that B cell chimerism was not required for normal B cell function in IL7Rα-Def, ADA-Def and CD3-Def SCIDs. In X-linked-SCID, Jak3-Def SCID and those with V-D-J recombination defects, donor B cell chimerism was necessary for B cell function to develop. The most important factor determining whether B cell function develops in SCID T cell chimeras is the underlying molecular defect. In some types, host B cells function normally. In those molecular types where host B cell function did not develop, donor B cell chimerism was necessary to achieve B cell function. 236 words.

The impact of CD4 +Foxp3 + Treg on immunity to murine cytomegalovirus after bone marrow transplantation depends on the peripheral or thymic source of T cell regeneration

Objective The adoptive transfer of regulatory T cells (Treg) in murine models has been shown to ameliorate graft-versus-host disease while it may preserve the graft-versus-leukemia effect. However, the impact of Treg on infectious immunity after bone marrow transplantation (BMT) is still unclear. Immunocompetence against opportunistic viral infections depends on the kinetics of T cell recovery after BMT through two distinctive processes, i.e. lymphopenia-induced proliferation (LIP) of mature T cells and generation of T cells through thymopoiesis. Methods In this study, we set out to assess the effects of adoptively transferred Treg on T cell regeneration in a homeostatic peripheral T cell expansion model and a thymopoiesis-dependent BMT model, and on murine cytomegalovirus (mCMV) clearance and mortality following mCMV challenge. Results Using lymphopenic Rag-2 −/−γc −/− mice that received a limited number of congenic T cells, we demonstrate that adoptively transferred Treg abrogate LIP of T cells. mCMV challenge resulted in a rapid increase of viral load and death in mice that received Treg, but not in controls. In contrast, following syngeneic T cell-depleted BMT in Rag-2 −/−γc −/− mice, adoptively transferred Treg did not delay T cell reconstitution nor suppressed thymic output and had no effect on viral clearance and survival following mCMV-challenge. Conclusion The effect of Treg on T cell-mediated immunocompetence against mCMV early after BMT depends on the relative contribution of peripheral expansion and thymopoiesis to T cell regeneration.

The Role of Missing Killer Cell Immunoglobulin-Like Receptor Ligands in T Cell Replete Peripheral Blood Stem Cell Transplantation from HLA-Identical Siblings

The contribution of natural killer (NK) cells to graft-versus-malignancy (GVM) effects following hematopoietic stem cell transplantation (HSCT) remains uncertain, particularly in the HLA-identical setting. A model considering missing HLA ligands to the donor's inhibitory killer cell immunoglobulin-like receptor (KIR), termed the missing KIR ligand model, has been established in T cell depleted bone marrow transplantation (BMT), but lacks validity in other cohorts with different treatment characteristics. We hypothesized that the impact of missing KIR ligands on relapse-free survival (RFS) and overall survival (OS) in T cell replete peripheral blood SCT (PBSCT) differs from that in the T cell depleted BMT setting, and retrospectively evaluated 100 consecutive, HLA-identical sibling transplantations for hematologic malignancies. In addition to KIR ligand status, we considered the donors' activating KIRs and grafted NK, T, and CD34(+) cell doses. Our findings demonstrate noninferiority for OS (P = .005) and RFS (P = .002) for the heterozygous HLA-C group KIR ligand status (C1/2; n = 47) compared with patients missing either C1 or C2 (n = 53). Similarly, OS (P = .031) and RFS (P = .034) of Bw4-positive patients was noninferior to that of patients missing a Bw4 ligand to KIR3DL1. By multivariate analysis, C1/2 heterozygous patients had a favorable risk ratio (RR) for relapse (RR = 0.28; P = .003), RFS (RR = 0.56; P = .046), and acute graft-versus-host disease grade II-IV (RR = 0.36; P = .05). Following reduced-intensity conditioning (RIC), but not standard-intensity conditioning, myeloablative (MA) transplantation, a grafted NK cell dose above the median (3.4 x 10(7)/kg) was associated with a lower risk of relapse (RR = 0.57; P = .003) and improved survival (RR = 0.78; P = .03). Overall, our findings support a role for NK alloreactivity in HLA-identical HSCT, but argue against a favorable impact of missing KIR ligands in the given setting. We conclude that the mechanism favoring the missing KIR ligand constellation in T cell depleted BMT may not operate in T cell replete PBSCT. The reasons for this differential effect remain unresolved.

Protection of the Intestinal Epithelium from Conditioning with R-spondin1 Ameliorates Graft-Versus-Host Disease

Damage to the gastrointestinal (GI) tract by pretransplant conditioning regimen plays a critical role in amplifying graft-versus-host disease (GVHD). Thus protection of the GI tract from conditioning may represent a novel approach to prevent GVHD. R-Spondin1 (R-Spo1) is a novel class of soluble activator for Wnt/□-catenin signaling, and has potent and specific proliferative effects on the intestinal crypt cells; injection of R-Spo1 protects mice from chemotherapy-induced intestinal mucositis. We therefore hypothesized that administration of R-Spo1 could modulate GVHD by reducing the GI tract damage and improve outcome of allogeneic bone marrow transplantation (BMT). Lethally irradiated B6D2F1 (H-2b/d) mice were injected with 5 × 106 BM and 2 × 106 T cells from MHC-mismatched B6 (H-2b) donors on day 0. Mice were intravenously injected with 200 μg of R-Spo1 or diluent from days −3 to −1 and +1 to +3 after BMT. In vivo labeling assay of mitotic cells with BrdU demonstrated that the proliferative index, as determined by the percentages of BrdU-positive cells among crypt epithelial cells, was significantly greater in the small intestine of R-Spo1 treated mice than controls 4 days after BMT (57% ± 3% vs 48% ± 1%, P<0.05). Analysis of the mesenteric lymph nodes and spleens on day +7 demonstrated significantly reduced expansion of donor T cells in R-Spo1 treated recipients in association with reduced serum levels of IFN-□ and TNF-□ on day +7 when compared to controls (Table). GVHD was severe in allogeneic controls, with 12.5% survival by day +40, whereas 62.5% of R-Spo1-treated animals survived this period (Table). Histopathologic examination of the small and large bowel and liver showed significantly reduced GVHD pathology in R-Spo1 treated animals than in controls (Table). A flowcytometric analysis of the spleen and thymus after BMT showed that administration of R-Spo1 did not impair donor cell engraftment and T and B cell immune reconstitution. We next evaluated the impact of R-Spo1 on graft-versus-leukemia (GVL) effects. BMT was performed similarly as above with the addition of 5 × 104 host-type P815 leukemia cells (H-2d). All recipients of T cell-depleted BM died from leukemia by day +20 after BMT, while no leukemia death was observed in R-Spo1 treated allogeneic animals. Overall, R-Spo1 treatment improved outcome of allogeneic BMT by reducing GVHD, while maintaining immune reconstitution and GVL effects. Thus, administration of R-Spo1 reduces the GI tract damage and suppresses donor T cell activation and systemic GVHD, supporting a hypothesis that the GI tract plays a major role in the amplification of systemic GVHD. Brief treatment with R-Spo1 may serve as an effective adjunct to clinical regimens of GVHD prophylaxis.Pathology ScoresGroupSurvivals on day+40 (%)Small bowelLarge bowelLiverINF □ (ng/ml)TNF □ (pg/ml)TCD diluent1002.4± 0.93.5± 1.00.3± 0.3NDND+T diluent12.58.3± 2.77.3± 1.92.0± 0.86.0 ± 2.4103.7 ± 9.9+T R-Spo162.5*3.4±1.9**3.9± 0.3**0.8± 0.7**2.3 ± 1.5**55.4 ± 6.6**TCD: T cell-depleted BMT, +T: T cell-repleted BMT, ND: not detectedData are expressed as mean ± SD. *P<0.01 vs control, **P<0.05 vs control

Disruption of Gamma Interferon Signaling through the STAT1 Pathway Enhances Alloreactivity While Abrogating GvHD

BACKGROUND: Strategies that allow for sufficient alloreactivity to mediate graft-versus-tumor (GVT) effects after allogeneic bone marrow transplant (BMT) while minimizing graft-versus-host-disease (GVHD) remain elusive. Historically, gamma interferon (IFNγ) has been implicated as a critical mediator of both GVT and GVHD pathophysiology, and high levels of IFNγ have been implicated in immunosuppression. Given that immunosuppression is also a hallmark of GVHD, we hypothesized that modulation of IFNγ signaling could have important effects on both GVHD and GVT following BMT. We therefore compared immunocompetence to tumor challenge and tumor vaccination, in the presence or absence of IFNγ receptor (R1) and STAT1 signaling (a primary downstream mediator of IFNγ signaling) on donor bone marrow.METHODS: We utilized a clinically relevant, minor histocompatibility antigen-mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) into thymectomized mice to ensure that T cell recovery occurred exclusively from the DLI, as occurs in most clinical settings during the first 6 months post-BMT. Lethally irradiated female C57BL/6 (B6) × C3H.SW recipients were engrafted with normal B6, B6 IFNγR1-deficient, normal B6129, or B6129 STAT1-deficient bone marrow on day 0 and reconstituted with donor T cells on days 14 and 28 at a dose that induced sublethal GVHD, evidenced by weight loss, histology, and abrogated quantitative immune responses to an HY-directed tumor vaccine. Recipients were then challenged on day 42 with an HY-expressing tumor and followed for survival. Immune reconstitution was assessed by flow cytometry both prior to and after DLI administration.RESULTS: Mild GVHD prevents vaccine-mediated immune responses and HY-expressing tumor protection in thymectomized recipients. Both IFNγR1 and STAT1-deficient marrow abrogate GVHD. STAT1-deficient marrow increased absolute numbers of splenic plasmacytoid dendritic cells (CD11cint/Class II+/Gr-1+/B220+) prior to DLI administration. In addition, an increase in the percentage and absolute number of splenic regulatory T cells was observed at the time of tumor challenge. Surprisingly, despite the immunosuppressive milieu, recipients of IFNγR1-deficient marrow had enhanced vaccine-mediated tumor protection as measured by overall survival, similar to syngeneic BMT controls.CONCLUSION: IFNγ signaling is a critical mediator of GVHD pathophysiology, including the immunosuppression associated with this condition. Disrupting IFNγ signaling through the JAK1/STAT1 pathway is a novel strategy to selectively modulate alloreactivity, and results in enhanced anti-tumor immune responses following allogeneic BMT without exacerbating GVHD.

Early Immune Response Against Retrovirally Transduced Herpes Simplex Virus Thymidine Kinase-Expressing Gene-Modified T Cells Coinfused with a T Cell-Depleted Marrow Graft: An Altered Immune Response?

Administration of herpes simplex thymidine kinase (HSV-tk)-expressing, gene-modified T cells (GMCs) with T cell-depleted bone marrow transplantation (TCD-BMT) can allow modulation of posttransplantation alloreactivity. Twelve patients received 2 x 10(5) to 2 x 10(6) CD3+ donor GMCs per kilogram with HLA-identical sibling TCD-BMT. Despite extensive T cell depletion of bone marrow, an intensive conditioning regimen, and immunosuppressive graft-versus-host disease (GvHD) prophylaxis, infusion at the time of TCD-BMT of this low number of GMCs sufficed to induce a rapid GMC-specific immune response, as detected by interferon- enzyme- linked immunospot assay in six of eight patients, preferentially targeting HSV-tk. Maximal responses were reached early (median time, 49 [35-68] days post-BMT), with a subsequent rapid and significant decrease in five of six evaluable patients. Immune responses were negatively correlated with the maximal circulating GMC counts. However, such immune response did not result in the elimination of circulating GMCs and was not associated with measurable ex vivo cytotoxic activity against GMCs. Furthermore, alloreactive GMCs still could induce GCV-sensitive GvHD in one patient despite an ongoing immune response. Overall, infusion of HSV-tk-expressing GMCs at the time of BMT results in an early immune response. Such immune response may be altered and may not prevent persistent GCV-sensitive alloreactivity.

Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT).

MRL-lpr/lpr mice spontaneously develop a severe autoimmune syndrome, characterized by massive generalized lymphadenopathy, arthritis, arteritis, dermatitis and immune complex-mediated glomerulonephritis. Bone marrow transplantation (BMT) from MHC-matched systemic lupus erythematosus (SLE)-resistant donors to susceptible recipients has proved effective in correcting autoimmune manifestations in autoimmune-prone mice. We investigated the effect of syngeneic BMT from MRL/lpr (donor) to immunocompromised MRL/lpr (recipient), after purging the bone marrow inoculum with MoAbs against mature T cells (anti-Thy 1.2). All the untreated mice developed lymphadenopathy and by the age of 36 weeks five of the eight were dead; in contrast, all the mice which underwent syngeneic BMT following acute immunosuppression with total body irradiation (900 cGy) (TBI) remained disease-free. In an additional experiment, it was found that conditioning with cyclophosphamide (CY) before BMT was more effective than TBI in inhibiting delayed-onset autoimmune manifestations (mean survival 350 days in the CY group and 305 days in the TBI group, versus 197 days in untreated controls). Under both immunosuppressive regimens T cell-depleted bone marrow grafts produced far better results than did unmanipulated BMT. Following syngeneic BMT the incidence of proteinuria and the level of serum anti-DNA (dd) antibodies were significantly reduced, compared with that of the age-matched untreated controls. CY was more effective than TBI in reducing the anti-DNA titres. Likewise, T depletion of bone marrow inocula before BMT induced a more drastic drop in autoantibodies, following both CY and TBI conditioning protocols. After syngeneic BMT (either CY or TBI) no signs of lymphadenopathy were observed even at an advanced age. Upon histopathological examination, the BMT-treated mice displayed normal glomeruli with occasional minimal signs of glomerulonephritis. Syngeneic T cell-depleted BMT following acute cytoreduction of anti-self immune lymphocytes may represent a new therapeutic approach for drug-resistant autoimmune diseases.

Expression of p75 chain of IL-2 receptor in the early immunological reconstitution after allogeneic bone marrow transplantation

Expression of p55 and p75 chains of IL-2 receptor (IL-2R) on the surface of both T and natural killer (NK) circulating lymphocytes was investigated in 14 paediatric patients given allogeneic bone marrow transplantation (BMT) from HLA-identical sibling or partially matched family donors. IL-2-induced proliferative and cytotoxic responses were also studied and all analysis was performed within 45 days from transplant. We found that, early after transplant, the percentage of p55+ and of p75+ peripheral blood lymphocytes (PBL) was not significantly different in patients who had received HLA-identical BMT (p55+ 8.04 +/- 4.87%; p75+ 28.27 +/- 18.85%) compared with healthy controls (p55+ 7.26 +/- 6.17%; p75+ 19.42 +/- 10.49%), while recipients of T cell-depleted marrow included a remarkably high percentage (76-90%) of p75+ PBL, which were mostly CD3- and co-expressed CD56 molecule. Comparable values of p55+ lymphocytes were observed in all patients and controls. However, in contrast to the other two groups, in recipients of T cell-depleted BMT the majority of these cells co-expressed p75 chain and CD56 antigen. IL-2-induced proliferation and lymphokine-activated killer (LAK) activity were detectable in all patients, and their values did not correlate with expression of p55 or p75 chains. Our data suggest that expansion of NK subsets expressing IL-2R chains after T cell-depleted BMT may be related to early reconstitution of natural immunity in the presence of allogeneic stimuli.

726. Effect of Vaccine on Growth Suppression of Cancers and Hematopoietic Neoplasms by Total Body Irradiation and T Cell Depleted Bone Marrow Transplantation

726. Effect of Vaccine on Growth Suppression of Cancers and Hematopoietic Neoplasms by Total Body Irradiation and T Cell Depleted Bone Marrow Transplantation

Loss of IFNγR1 Signaling on Donor Bone Marrow Abrogates GVHD but Maintains Immunocompetence.

BACKGROUND: Acute graft versus host disease (GVHD) remains as the major complication after allogeneic bone marrow transplant (BMT) resulting in organ toxicity and immune dysfunction. Indeed, we have previously demonstrated that GVHD impairs responses to dendritic cell vaccines. The pathophysiology of GVHD involves preparative regimen-induced inflammation of target organs, release of inflammatory mediators such as gamma interferon (IFNg), and subsequent activation of alloreactive T cells. Given that IFNg can both contribute to GVHD and provide beneficial immune responses, we explored the potential role of IFNg on GVHD and post-transplant immunocompetence.METHODS: We utilized a minor histocompatibility antigen mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) as a means of controlling the induction of acute GVHD and to provide a source of immunocompetence in a thymectomized mouse. To study the role of IFNg on GVHD, we chose either IFNg receptor 1 (IFNgR1) −/− marrow or DLI to permit the normal production of IFNg in GVHD while influencing which cells that can respond to the cytokine. Normal C57BL/6 (B6) or IFNgR1 −/− B6 mice were used as bone marrow donors on day 0 into lethally irradiated, thymectomized B6 × C3H.SW (F1) mice. Normal B6 or IFNgR1 −/− DLIs given with or without a dendritic cell vaccine were introduced at days 14 and 28 post-BMT both to control the induction of GVHD and to provide a population of vaccine-responding cells. F1 recipients were observed for signs of GVHD. ELISPOT of the number of antigen-reactive IFNg-producing splenocytes were also performed to measure functional response to vaccine.RESULTS: The absence of IFNgR1 in the DLI abrogates GVHD as shown by % change in weight (B6 DLI = −6.3 +/− 4.7 vs. IFNgR1−/− DLI = 6.6 +/− 6.1, p=0.001) and allows for greater doses of DLI to be tolerated by the host, however, there is also decreased vaccine responses by ELISPOT (B6 DLI = 1631 vs. IFNgR1−/− DLI = 72, p=0.03). Surprisingly, using IFNgR1−/− bone marrow also abrogates GVHD as shown by % change in weight (B6 marrow = −6.3 +/− 4.7 vs. IFNgR1−/− marrow = 4.4 +/− 4.2, p less than 0.05) and splenocyte count (B6 marrow = 31.48 +/− 12.54 vs. IFNgR1−/− marrow = 63.54 +/− 15.92, p=0.008), but vaccine responses by ELISPOT can be restored to levels that are equivalent of syngeneic control mice, even in the presence of a normal B6 DLI (B6 marrow = 1631 vs. IFNgR1−/− marrow = 9283, p=0.0002). The abrogation of GVHD by IFNgR1−/− marrow does not appear to be a dominant effect since mixtures of IFNgR1 −/− and normal B6 bone marrow still cause GVHD.CONCLUSIONS: Recipients of allogeneic bone marrow and T cells developed GVHD and had decreased vaccine responses by ELISPOT. Loss of IFNgR1 on allogeneic donor lymphocytes abrogates their ability to cause GVHD, but also diminished their ability to respond to vaccine. Surprisingly, loss of IFNgR1 on a donor bone marrow-derived, non T cell results in equivalent abrogation of GVHD, while restoring immunocompetence through favorable responses to a vaccine. Further studies will attempt to identify the phenotype of the responsible bone marrow-derived cell. These results demonstrate a strategy of providing higher doses of DLIs to enhance anti-tumor activity without exacerbating GVHD, and thus, have implications for immune modulation post-allogeneic BMT.

Improvement of Canine XSCID by In Vivo Gene Therapy Using RD114/TR-Pseudotyped SIV Lentiviral Vectors.

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency (dysfunctional B cells; absence of T and NK cells) and early mortality, caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins (IL)-2,-4,-7,-9,-15 and -21. The standard therapy for XSCID in infants is a T cell depleted bone marrow transplant with either none or very modest conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution that may be limited to only the T cell lineage. Ex vivo autologous stem cell gene therapy has emerged as an alternate treatment capable of achieving substantial to complete immune reconstitution in infants without a sibling donor. However, some children have developed lymphocytic leukemia, which appears in part to be related to vector insertional mutagenesis. The gammaretroviruses currently in use have potent enhancers in the LTR and have a predilection to insert at the 5′ end of genes. Self-inactivating (SIN) lentivirus vectors may be advantageous because they do not show this property and can be constructed with internal promotors that have less enhancer activity together with insertion of insulators. We have constructed SIN simian immunodeficiency viral vectors (SIVmac) encoding human γc (hγc), with or without a double copy chicken insulator core element in the 3′ LTR. In addition we have pseudotyped the vector with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicity of the VSV-G envelope traditionally used with lentivectors. We have previously shown that it is possible to correct a dog model of XSCID using a RD114-pseudotyped gammaretroviral vector encoding the dog γc for in vivo gene therapy. Unlike the mouse model of XSCID which lacks B cells, the dog model more closely resembles the phenotype in humans. We decided to use our in vivo dog model for preclinical testing of safety and efficacy of our SIV hγc vectors. Viral particles were produced by transient transfection of 293T cells with a 4-plasmid system and concentrated by high-speed centrifugation. 30 mls (average 2.4–3×107 viral particles in total) was injected IV into 2–5 day-old pups. Transgene marking in blood lymphocytes was detected as early as 2 wks after treatment, increased within the first 6–8 wks and became relatively stable thereafter. The absolute lymphocyte count was normalized in one dog (injected on the second day after birth) by wk 6 (4000, 76% hγc+), and improved in two other dogs (422, 29% hγc+ at 18 wks; 498, 27% hγc+, at 16 wks) which had received the same amount of virus over 2 days. Furthermore, up to 5% of the myeloid lineage showed gene marking at 10–18 wks after viral delivery, indicating that early committed progenitors or HSCs had been transduced. It is noteworthy that our finding that the hγc can improve the disease phenotype in XSCID dogs makes it an excellent large animal model for preclinical evaluation of vectors. In summary, we have demonstrated that in vivo delivery of SIV lentiviral vectors expressing hγc efficiently reconstitute the T-lymphoid compartment in the XSCID canine model. We plan to monitor these dogs closely for potential adverse events.

Injury to the Thymic Niche for DN2/DN3 Developing T Cells Impairs Reconstitution.

Despite recovery of most hematopoietic functions, prolonged defects in generating functional T lymphocytes is a common occurrence after T cell depleted bone marrow transplant. While the mechanisms of these defects have not all been elucidated, contributing factors include age, T cell depletion of the graft, therapy with radiation and cytotoxic agents, and graft versus host disease (GvHD). We have designed an in vivo functional assessment of the ability of thymic stroma to support de novo T lymphocyte development. Mice deficient for the alpha chain of the IL-7 receptor (IL7Rα−/−) support robust thymic reconstitution after transplant of limited numbers of congenic precursors. We demonstrated that this capacity for reconstitution of immunodeficient strains depends on the paucity of specific endogenous precursors (DN3) cells in the IL7Rα−/− thymus and reflects the presence of functionally normal but empty stromal niches in this immunodeficient strain. We have proceeded to demonstrate that a variety of chemotherapeutic agents as well as aging, impair the ability of IL7Rα−/− thymic stroma to support de novo T cell development. Some agents allow donor chimerism in the thymus, but not rescue of the hypocellularity (eg cyclophosphamide) while others do not affect reconstitution (eg fludarabine). Multi-agent regimens have been administered and in some instances demonstrate an additive detrimental impact on thymic reconstitution. For several agents, damage has been localized to specific stromal niches by isolating changes in lymphoid subsets and stromal keratin expression. Decreased availability of the stromal niche for DN3 progenitors is associated with a decreased frequency of DN3s and an absolute block in recipient IL7Rα−/− T cell development. In addition, RNA from treated and untreated thymic stromal cells has been used to evaluate the gene expression pattern in thymic stroma of IL7Rα−/− mice treated with cytotoxic agents. In one example to be presented we find decreased thymic reconstitution in mice treated with Busulfan with sustained changes in stromal elements. These findings are consistent with sustained damage to thymic stromal cells. Among other changes, busulfan leads to decreased expression of laminin 5 by cortical thymic epithelial cells. The integrin heterodimerα6β4 is a binding partner for laminin 5 and is expressed uniformly by DN2 thymocytes. The significance of laminin 5/α6β4 signaling during T cell development was assessed using mice with a targeted mutation in the integrin β4 signaling domain. In these experiments mutant fetal liver was used to create hematopoietic chimeras and evaluate T cell development. Our studies provide insight into the nature of damage to thymic stroma by cytotoxic regimens and an understanding of the effect of this damage on subsequent immune reconstitution.

The Effect of Unrelated Donor Marrow Transplantation on Health-Related Quality of Life: A Report of the Unrelated Donor Marrow Transplantation Trial (T-Cell Depletion Trial)

The primary objective of this study was to compare health-related quality of life (HRQL) in adult patients undergoing either ex vivo T cell–depleted bone marrow transplantation or conventional marrow transplantation. Data on patients’ HRQL were gathered as part of a multicenter randomized trial comparing the effect of ex vivo T-cell depletion versus methotrexate and cyclosporine immunosuppression on disease-free survival. HRQL assessments were conducted at baseline, day +100, 6 months, 1 year, and 3 years. There were no treatment arm differences 1 year after transplantation on the Functional Assessment of Cancer Therapy, Bone Marrow Transplantation, the Medical Outcomes Study Short-Form 36, and the Centers for Epidemiological Studies of Depression. The lack of treatment differences was robust across types of data analyses that took baseline functioning into account and that recognized the sensitivity of outcome measures to assumptions concerning missing data. The trajectory of recovery revealed an initial decrease in function and then a recovery to pretreatment levels that were similar for both treatment arms. Furthermore, the patients in both treatment groups returned to a functional level that approximated general US population norms. Even though the incidence of acute graft-versus-host disease was slightly higher in the conventional treatment arm, T-cell depletion did not differentially affect HRQL at 1 year after transplantation.

A comparison of postengraftment infectious morbidity and mortality after allogeneic partially T cell–depleted peripheral blood progenitor cell transplantation versus T cell–depleted bone marrow transplantation

Postengraftment infections are a major cause of transplant-related morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-SCT). Allogeneic peripheral blood progenitor cell transplantation (PBPCT) is associated with faster hematopoietic recovery compared to bone marrow transplantation (BMT) and unmanipulated PBPCT may be associated with fewer postengraftment infections. We set out to evaluate and compare the incidence, cause, and outcome of postengraftment infections following HLA-identical sibling T cell-depleted PBPCT vs T cell-depleted BMT between days 30 and 365 posttransplant. Forty recipients of peripheral blood progenitor cells (PBPC) and 47 recipients of bone marrow (BM) were included. The two groups of patients were comparable with respect to their baseline characteristics. PBPC grafts contained significantly more CD34+ cells and PBPCT was associated with significantly faster neutrophil and lymphocyte recovery as compared to BMT. PBPC recipients experienced more chronic graft-vs-host disease (GVHD; 55% vs 34%; p=0.02). The number of definite and clinical infections per 100 patient days was comparable between recipients of PBPC and BM with similar contribution of causative microorganisms. At one year post SCT, 68% of PBPC recipients had experienced at least one CTC grade 3-4 infection vs 65% of BM recipients. Treatment-related mortality at one year from transplantation was 34% after PBPCT vs 30% after BMT, and no difference in infection-related mortality was observed. Postengraftment infectious morbidity and mortality were comparable between recipients of PBPC and BM despite a higher CD34+ cell content of PBPC grafts and faster lymphocyte recovery after PBPCT, which may in part be explained by the higher incidence of chronic GVHD.

Anti-NK cell treatment induces stable mixed chimerism in MHC-mismatched, T cell-depleted, nonmyeloablative bone marrow transplantation

To clarify natural killer (NK) cell-mediated resistance under cytoreductive conditioning and T cell-depleted bone marrow transplantation, we investigated the effects of host NK cell depletion on engraftment and induction of stable mixed chimerism. BALB/c mice (H-2kd) were injected intraperitoneally with anti-asialoGM1 antibody (anti-NK Ab) on day -1. On day 0, they received total body irradiation (TBI) at a dose of 500 cGy, followed by intravenous infusion of 2 x 10(7) T cell-depleted (TCD) bone marrow cells from C57BL/6 mice (H-2kb). Early engraftment and chimerism were determined by the relative ratio of peripheral blood (PB) lymphocytes expressing either H-2kd or H-2kb on day +21. Long-term engraftment and chimerism were evaluated on PB and spleen by multicolor flow cytometry. Although no recipients treated with TBI alone showed engraftment, all the recipients conditioned with anti-NK Ab and TBI showed successful engraftment as well as a donor-dominant pattern of mixed chimerism in both PB and spleen. Spleen cells from recipients with mixed chimerism showed specific tolerance to both host and donor strains, but not to a third party (C3H/He). None of the reconstituted mice showed signs of graft vs host disease, and all survived up to day +330. These observations indicate that host NK cell depletion may be used to reduce the intensity of conditioning regimens for engraftment of TCD grafts, and can contribute to establishment of stable mixed chimerism in major histocompatibility complex-mismatched nonmyeloablative transplantation.

Cord blood transplantation for children with acute leukaemia: a Eurocord registry analysis

Objectives Report results of unrelated cord blood transplants collected by Eurocord Registry in children with acute leukemia. Results Children with AML: 95 children were analyzed. The two year leukemia free survival was 42% in patients transplanted in first remission, 50% in second remission and 21% in children not in remission. Children with poor prognostic cytogenetic features had the same survival compared to other patients. Children with ALL: 195 patients with ALL were analyzed. The two year leukemia free survival was 36% in patients transplanted in remission and 15% in patients transplanted in relapse. Results of unrelated cord blood transplants compared to unrelated bone marrow transplants in children with acute leukemia: 416 children with acute leukemia received a HLA matched unrelated bone marrow transplant and were compared to 99 children transplanted with an unrelated HLA mismatched cord blood. The long term outcome between these groups were comparable with delayed engraftment in cord blood transplant, more relapse in T cell depleted bone marrow transplant and more GVH in the unmanipulated bone marrow transplant resulting in similar 5 years leukemia free survival. Conclusion These results show that use of unrelated cord blood transplant is an option in patients lacking an HLA identical sibling donor.