Delayed Donor Leukocyte Infusion Research Articles

Distinct graft-versus-leukemic stem cell effects of early or delayed donor leukocyte infusions in a mouse chronic myeloid leukemia model

AbstractAmong hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen–mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1–transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5− splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.

The role of regulatory T cells during the attenuation of graft-versus-leukemia activity following donor leukocyte infusion in mice

We investigated how the graft-versus-leukemia (GVL) effect is attenuated in the tumor microenvironment using a murine model of non-myeloablative allo-HSCT (NM-HSCT) plus delayed donor leukocyte infusion (DLI) in a haploidentical B6 → F1 strain combination. In-line with aggravated leukemia growth, the proportions of effector T cells expressing IFN-γ (Teffs) in spleen were reduced and attenuated GVL activity was found to be accompanied by a rebound in CD4 +Foxp3 + regulatory T cells (Tregs) in tumor-draining lymph nodes and tumor tissues. DLI-derived Tregs and Teffs may be potential indicators of presence of leukemic progression after DLI in this GVL model.

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.

Conversion to Full Donor Chimerism without Gvhd Using High-Dose DLI in Minimally Conditioned Miniature Swine Recipients of Haploidentical HCT.

Non-myeloablative hematopoietic cell transplantation (HCT) followed by delayed donor leukocyte infusion (DLI) is a promising immunotherapeutic approach to the treatment of hematologic malignancies. Major limitations of this approach, however, include the risks of graft failure, graft-versus-host disease (GVHD), and infection. MHC-defined, MGH miniature swine provide a pre-clinical model for studies of HCT. Similar to humans, pigs that receive haploidentical HCT following standard myeloablative conditioning develop severe GVHD or hematopoietic failure. We previously developed a successful haploidentical HCT protocol in miniature swine using a minimally myelosuppressive regimen consisting of a very low-dose of total body irradiation (100cGy TBI) in combination with transient recipient T-cell depletion and a short course of cyclosporine A (CyA), referred to as “ITC”. Strikingly, GVHD is not observed in engrafted animals despite infusion of an enormous number of donor T cells (>5 x 109 T cells/kg) with the initial HCT. Preliminary data demonstrate that regulatory mechanisms play an important role in preventing GVHD and may facilitate engraftment and tolerance in this model. Our data also demonstrate that subsequent DLIs often fails to convert ITC conditioned mixed chimeric swine to full (donor) chimerism. Despite administration of very high numbers of T-cells (5 x 107 T-cells/kg), neither GVHD nor an increase of donor chimerism levels in the peripheral blood post-DLI were observed. We hypothesize that very strong regulatory mechanisms persist in ITC conditioned HCT recipients that not only prevent GVHD but also interfere with DLI-mediated increases in donor chimerism. In the current study, we have investigated 2 approaches to overcome this strong immune regulation and facilitate DLI mediated conversion to full donor chimerism. In the first approach, 2 chimeric animals underwent leuko-depletion in an attempt to reduce putative regulatory cell populations in chimeric recipients prior to DLI. Animals receiving leuko-depletion prior to DLI demonstrated a modest increase (~12%) in thymic chimerism at 4 weeks, suggesting some transient effect. However, a sustained increase in peripheral blood chimerism levels was not achieved. As a second approach, we infused a higher DLI dose (>15 x 107 T-cell equivalents/kg). This dose was calculated based on the dose of DLI given to mixed chimeric mice that results in consistent conversion of low-level chimeras to full donor chimerism without GVHD. Following this high-dose DLI, we observed successful conversion to full donor chimerism in all hematopoietic cell lineages (>95%) within 1 month (Figure 1) without the induction of clinically significant GVHD. The recipient of the DLI underwent a gradual period of conversion between 3 and 4 weeks, with the percent chimerism increasing without a corresponding increase in absolute number of donor cells. A transient leukopenia was observed, suggesting that donor cells were killing recipient lymphohematopoietic cells without inducing tissue damage in the classical GVHD target organs. In summary, these experiments demonstrate for the first time in this large animal model conversion from low to full donor chimerism without GVHD using high-dose DLI in minimally conditioned recipients of haploidentical HCT. [Display omitted]

Peripheral Alloantigen Drives Early Dysfunction and Eventual Exhaustion of CTL Following Delayed Donor Leukocyte Infusions.

T cell immunotherapies for cancer should ideally generate high levels of anti-tumor activity, with minimal host injury and permit the prolonged survival of functional memory/effector cells to prevent tumor recurrence. Following allogeneic stem cell transplantation, delayed donor leukocyte infusion (DLI) is one strategy employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease. However, patients remain at significant risk of relapse following DLI and murine models of delayed DLI indicate that this results from the eventual loss of functional, alloreactive cytotoxic T lymphocytes (CTL) [Mapara et al. Transplantation 2003]. We hypothesised that the loss of functional CTL is driven by persistent stimulation of donor CD8 cells by alloantigen expressed by peripheral tissues. In order to follow and characterise an alloreactive CD8 response under conditions in which alloantigen was present or absent in peripheral tissues, we employed a model in which either parental B6 (H2b) or B6 x DBA-2 F1 (BDF1, H2dxb) mice were lethally irradiated and reconstituted with a mixture of B6 and BDF1 T cell depleted bone marrow. 8-10 weeks later congenic CD45.1 B6 splenocytes were transferred into the established mixed chimeras. This allowed us to test the importance of peripheral antigen in the loss of alloreactive CTL responses, since alloantigen was either restricted to the hematopoietic system (B6 +BDF1 → B6) or was ubiquitously expressed (B6 +BDF1 → BDF1). Following transfer of CD45.1 B6 splenocytes, the ensuing alloantigenspecific T cell response in both groups led to the elimination of alloantigen-positive (BDF1-derived) hematopoietic elements. Thereafter, alloreactive CD8 cells resided in an environment in which peripheral alloantigen was present (PA+) or absent (PA-). We observed similar kinetics of initial CD45.1+ CD8 cell proliferation and expansion and similar acquisition of a CD44highCD62Llow phenotype. However, by day 60, there were striking differences in the phenotype and function of transferred CD8 cells. In PA- hosts, CD45.1+ CD8 cells killed allogeneic target cells effectively both in vitro and in vivo, underwent rapid proliferation in a mixed leukocyte reaction and produced the effector cytokine, IFN-γ. In contrast CD45.1+ CD8 cells from PA+ hosts had little or no cytotoxic activity, did not proliferate to alloantigen and were IFN-γlow. Moreover, CD45.1+ CD8 cells from PA+ hosts displayed high levels of the co-inhibitory receptor PD-1, low levels of the IL-7Rα chain and responded poorly to IL-7 and IL-15 in vitro, a phenotype typical of the ‘exhaustion’ signature observed in CTL following chronic antigen exposure. In comparison, CD45.1+ CD8 cells from PA- hosts expressed significantly lower levels of PD-1, higher levels of IL-7Rα and demonstrated better responsiveness to IL-7 and IL-15 in vitro. In vitro PD-1 or PD-L1 blockade restored IFN-γ generation to CD45.1+ CD8 cells from PA+ hosts, suggesting that the PD-1 pathway may play a functional role in driving exhaustion of these cells. Importantly we observed no loss of long-term alloreactive CD4 responses in either PA+ or PA- hosts. This finding is consistent with a model in which peripheral alloantigen drives exhaustion since the majority of cells expressing Class II alloantigens in PA+ and PA- hosts would be restricted to the hematopoietic system and thus, would have been cleared in the initial alloresponse. The full exhausted phenotype of alloreactive CD8 cells described above was not seen until at least 30 days after transfer to PA+ hosts. However, as early as day 14, CTL primed in PA+ hosts produced less IFN-γ in comparison to those primed in PA-hosts, even though they were still equivalent in terms of their cytotoxicity. Furthermore, when CD8 cells primed in PA+ hosts were transferred to secondary antigen-free hosts, they still displayed reduced ‘fitness’ compared to CTL originally primed in PA- hosts. These data show that peripheral alloantigen qualitatively affects donor CTL function during priming and drives their eventual exhaustion. Additionally they suggest that blockade of co-inhibitory signals may have potential in restoring function to such cells as has been demonstrated in models of chronic infection.

Non-Myeloablative T-Cell Depleted (TCD) Haploidentical Hematopoietic Cell Transplantation (HCT) Followed by Donor Leukocyte Infusion(s) for Hematologic Malignancies: The MGH Experience.

We developed a haploidentical HCT protocol based on MHC-mismatched murine models, in which mixed chimerism (MC) is induced as a platform for adoptive immunotherapy via delayed donor leukocyte infusions (DLI). While the scientific concept has remained the same, this haplo-HCT protocol has undergone multiple revisions since its inception, such as substitution of MEDI-507 (a CD2 monoclonal antibody) for ATG, addition of ex vivo TCD of donor graft (via CD34+ cell selection), changes in MEDI-507 doses/schedules and finally addition of fludarabine-all based on evolving clinical experience and new discoveries from murine models. A total of 14 patients with advanced, mostly therapy-refractory disease (NHL, n=7; HD, n=3; t-AML, n=2; ALL, n=1; MDS, n=1), received our most recent nonmyeloablative strategy consisting of cyclophosphamide, fludarabine, MEDI-507, thymic irradiation on day -1 and cyclosporine for GVHD prophylaxis (with taper and discontinuation by day 35 for patients with MC and no evidence of GVHD) followed by GCSF-mobilized ex vivo TCD haplo-HCT (HLA 1/6 mismatched, n=7; HLA 2/6 mismatched, n=7). Median number/kg (range) of infused CD34+ and CD3+ cells were 6.84 x106 (3.19 x 106 – 14.7 x 106) and 4.73 x 104 (0.06 x 104 – 11.8 x 104), respectively. One patient died shortly after the transplantation due to intracranial hemorrhage. Of 13 evaluable patients, all initially achieved MC without GVHD, but 3 lost detectable donor graft despite multiple DLIs. Split lineage MC occurred in 10 patients, with a predominance of early donor granulocyte chimerism (mean 91%, 87%, 96%) and a lower percentage of early donor T-cell chimerism (57%, 60%, 97%) at 30, 90 and 150 days post-HCT. Ten out of 10 patients ultimately achieved full donor chimerism (FDC) in myeloid lineage and 9 achieved FDC in T-cell lineage (spontaneously, n=8; following DLI, n=2). Natural killer cells recovered relatively early, despite the presence of circulating MEDI-507. Eight patients developed acute GVHD: grade I, n=3; grade II, n=4; grade IV, n=1. One died from GVHD-related complications, 1 died of late idiopathic pneumonitis and 5 died from progressive disease. Median overall survival (OS) of all 14 patients is 195 days (range, 31–1028) post-HCT; at 1 year the OS is 39.2% for all 14 patients. Six patients are alive at a median of 604 days (range, 180–1028) post-HCT, including 3 in complete remission of whom 2 had therapy-related MDS/AML. HLA mismatches in the GVH or HVG direction did not influence graft outcome. We also assessed inhibitory killer immunoglobulin-like receptor (KIR)-HLA epitope mismatches (missing ligand) in the GVH and HVG (host versus graft) directions based on HLA and KIR genotyping of patients and their donors. There was a trend in the association between spontaneous FDC and fewer missing donor HLA ligands for host KIR receptor (<2 v. ≥ 2 missing donor ligands, p = 0.075). In conclusion, using our current haploidentical HCT protocol, we reliably achieved initial MC that converted to FDC, either spontaneously or following DLI with manageable GVHD and with the achievement of sustained anti-tumor responses in some patients with therapy-refractory hematologic malignancies.

Lymphopenia Promotes the Development of Graft-Versus-Host Disease upon Donor Leukocyte Infusion.

In murine models of hematopoeitic cell transplantation (HCT), a graft-versus-leukemia (GVL) effect can be effectively separated from graft-versus-host disease (GVHD) by giving delayed donor-leukocyte infusions (DLI) to established mixed hematopoietic chimeras (MC) (Mapara, Transplantation 2003). In these models, the absence of conditioning-induced inflammation at the time of DLI prevents trafficking to GVHD target tissues, limiting the GVH response to the lymphohematopoeitic system (Chakraverty, J Exp Med 2006). While clinical trials have shown that the same outcome is possible in humans, some patients develop severe GVHD following delayed DLI (Dey, Blood Biol Marrow Transplant 2003). In contrast to mice, who recover T cell numbers quickly after HCT through de novo thymopoiesis, humans remain lymphopenic for many months. The current study investigates the role of recipient lymphopenia in the development of GVHD following delayed DLI to MC. Allogeneic (B10.A) or syngeneic (B6) lymphocytes (30 × 106 whole splenocytes) and bone marrow cells (10 × 106) were administered to unconditioned RAG–1 knockout (KO) B6 recipients or to established MC, and animals were followed for development of GVHD. By day 7 after injection, the RAG KO recipients of allogeneic lymphocytes (allogeneic group) had lost significantly more weight (p<0.001) and had significantly higher clinical GVHD scores (p<0.0001) than RAG KO recipients of syngeneic lymphocytes (syngeneic group) or MC recipients of allogeneic lymphocytes (MC group). Histologic evaluation on day 7 showed significantly increased GVHD in liver and colon of the allogeneic group. Chimerism analysis on day 7 showed the presence of an ongoing lymphohematopoietic GVH response (LGVHR) in both the allogeneic and MC groups, though the response was slower in the MC group. When followed over time, clinical GVHD in the allogeneic group resolved by day 56, weight differences disappeared by day 65, but mild histologic GVHD persisted as late as day 239. Importantly, the same dose of allogeneic T cells given to freshly conditioned animals results in rapid, lethal GVHD, whereas these unconditioned, lymphopenic animals developed only a relatively mild, largely self-limited GVHD. These results demonstrate that in the absence of conditioning-induced inflammation, lymphopenic recipients of allogeneic T cells do develop mild GVHD, consistent with the possibility that recipient lymphopenia contributes to the GVHD seen in human MC recipients of DLI. The mechanisms by which lymphopenia promotes GVHD in non-conditioned MC recipients of DLI require further investigation, but may include the absence of regulatory T cells, the potential for lymphopenia-induced proliferation of T cells, and the presence of infection-induced inflammation.

Host MHC class II+ antigen-presenting cells and CD4 cells are required for CD8-mediated graft-versus-leukemia responses following delayed donor leukocyte infusions

AbstractFollowing bone marrow transplantation, delayed donor leukocyte infusions (DLIs) can induce graft-versus-leukemia (GVL) effects without graft-versus-host disease (GVHD). These antitumor responses are maximized by the presence of host hematopoietic antigen-presenting cells (APCs) at the time of DLI. Using a tumor-protection model, we demonstrate here that GVL activity following administration of DLIs to established mixed chimeras is dependent primarily on reactivity to allogeneic MHC antigens rather than minor histocompatibility or tumor-associated antigens. CD8+ T-cell–dependent GVL responses against an MHC class II–negative tumor following delayed DLI require CD4+ T-cell help and are reduced significantly when host APCs lack MHC class II expression. CD4+ T cells primed by host APCs were required for maximal expansion of graft-versus-host reactive CD8+ T cells but not their synthesis of IFN-γ. In contrast, the GVL requirement for CD4+ T-cell help was bypassed almost completely when DLI was administered to freshly irradiated recipients, indicating that the host environment is a major factor influencing the cellular mechanisms of GVL.

Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

Graft-versus-leukemia effect in minor antigen mismatched chimeras given delayed donor leucocyte infusion: immunoregulatory aspects and role of donor T and ASGM1-positive cells.

Previous studies have demonstrated that delayed donor leukocyte infusion (DLI) can increase graft-versus-leukemia (GVL) without increasing graft-versus-host-disease (GVHD) in MHC mismatched bone marrow (BM) chimeras. In our report, the immune status of minor antigen mismatched BM chimeras given DLI was studied. Particularly the role of donor ASGM1 positive or T cells in the graft-versus-leukemia effect (GVL) was investigated. AKR mice (H2k, Mls1a, Thy1.1) received TBI (9,5 Gy) and T cell-depleted (TCD) C3H (H2k, Mls2a, Thy1.2) BM alone (BM chimeras), or TCD BM together with immunocompetent C3H spleen cells at the time of BM transplantation (BM+SP chimeras), or TCD BM and 3 weeks later C3H spleen cells (DLI chimeras). Chimerism and T lymphocyte subsets were scored using FACS and anti-Thy, anti-Vbeta6, anti-IL2-beta receptor, anti-CD4, anti-CD3, and anti-CD8 mAbs. Leukemia challenge consisted of 5 x 10(6) AKR T cell lymphoma (BW4157) cells injected i.v. ASGM1 positive (ASGM1+) cells and T cells were depleted using anti-ASGM1 or anti-Thy1.2 antibodies, respectively. Immune tolerance was studied using MLR and CML tests. BM + SP chimeras developed acute and lethal GVHD, whereas DLI chimeras were totally free from GVHD. In DLI chimeras, host-reactive cytotoxic T cells (CTL) could not be induced and host-reactive CD8Vbeta6 cells were deleted whereas CD4Vbeta6 cells and MLR reactivity persisted temporarily. In contrast, in BM+SP chimeras, anti-host CTL were easily generated and an expansion of both host-reactive CD8Vbeta6 and CD4Vbeta6 T cells was found as well as high anti-host MLR reactivity. Depletion of either ASGM1 + cells or T cells from the DLI inoculum resulted in an abrogation of GVL reactivity, suggesting that both cell populations were involved in the protection against BW4157 leukemia. Three weeks after DLI, the GVL effect waned which correlated with the disappearance of host-reactive CD4 cells and MLR reactivity. In minor antigen mismatched BM chimeras, anti-host reactivity after DLI is characterized by (1) an absence of clinical GVHD, (2) clonal deletion of host-reactive CD8 cells, (3) an absence of anti-host CTL induction, and ( 4) a temporary persistence of host-reactive CD4 T cells and of MLR reactivity. In addition, either donor ASGM1+ cells or an interaction between these cells and T cells contribute to the GVL effect.

Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies

Mixed lymphohematopoietic chimerism can be induced in mice with bone marrow transplantation (BMT) after a nonmyeloablative preparative regimen that includes cyclophosphamide, anti-T-cell antibody therapy, and thymic irradiation. These mixed chimeras are resistant to the induction of graft-versus-host disease (GVHD) after delayed donor leukocyte infusions (DLIs), despite a potent lymphohematopoietic graft-versus-host reaction that converts the mixed chimeric state to a full donor one. Based on this animal model, we initiated a trial of nonmyeloablative therapy with HLA-matched or -mismatched donor BMT and DLI for refractory hematologic malignancies. Twenty-one of 36 patients enrolled in this trial received a genotypically (n = 20) or phenotypically (n = 1) HLA-matched donor transplant; results reported here are for those patients only. Preparative therapy consisted of cyclophosphamide in doses of 150 to 200 mg/kg; peritransplant antithymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal radiation therapy); and cyclosporine. Eighteen of 20 evaluable patients developed persistent mixed lymphohematopoietic chimerism as defined by >1% donor peripheral white blood cells until at least day 35 posttransplantation. Ten patients received prophylactic DLI beginning 5 to 6 weeks after BMT for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-leukemia effect. Fourteen of 20 evaluable patients (70%) achieved an antitumor response; 8 of these responses were complete, and 6 were partial. Of the 8 evaluable patients who received prophylactic DLI, 6 showed conversion to full donor chimerism. Five of the 9 evaluable patients (56%) who received prophylactic DLI achieved a complete response, compared with 3 of 11 patients (27%) who did not receive prophylactic DLI. Currently 11 patients are alive, and 7 of these are free of disease progression at a median follow-up time of 445 days (range, 105-548 days) posttransplantation. Transplantation-related complications included cyclophosphamide-induced cardiac toxicity in 3 of 21 patients (14%) and grade II or greater GVHD in 6 patients (29%). One patient (5%) died from a complication of BMT, and 1 patient (5%) died from GVHD after 2 prophylactic DLIs were given for conversion of chimerism. In summary, mixed lymphohematopoietic chimerism was reproducibly induced after a novel nonmyeloablative preparative regimen incorporating chemotherapy, peritransplant antithymocyte globulin, and thymic irradiation, allowing for early administration of DLI in 10 of 21 patients. After treatment, striking antitumor responses were observed in the majority of patients with chemotherapy-refractory hematologic malignancies. Biol Blood Marrow Transplant 2000;6(3A):309-20.

Effect of sublethal total body irradiation on acute graft-versus-host disease and graft-versus-leukemia effect in SCID mice.

SCID mice were transplanted with H-2-incompatible C3H/He splenocytes with or without previous TBI with 2Gy to evaluate the influence of sublethal TBI on GVHD and on the graft-versus-leukemia (GVL) effect. Transplantation immediately after TBI induced lethal GVHD, but delayed donor leukocyte infusion (DLI) 5 days after TBI reduced the severity of the GVHD. SCID mice inoculated with L1210 cells after TBI received a DLI 5 days after TBI to induce the GVL effect. Survival of these mice was longer than that of control nonirradiated mice. Serum levels of tumor necrosis factor-alpha, IL-1alpha, IL-6, IL-2 and IFN-gamma were significantly elevated, and they reached maximum levels at 5 days post-transplantation. Except for IFN-gamma, all cytokine levels were higher in irradiated mice than those in nonconditioned mice. Cytotoxicity against L1210 cells mediated by splenocytes from irradiated recipients was greater than that mediated by effector cells from nonirradiated mice. All the irradiated mice survived more than 120 days after L1210 rechallenge, while all nonirradiated mice died of leukemia within 5 weeks. In conclusion, compared with control mice infused with donor splenocytes without previous TBI, SCID mice which received sublethal TBI and DLI showed superior cytotoxicity against L1210 cells and survived longer without severe GVHD.