IL-2 pathway blocking in combination with anti-CD154 synergistically establishes mixed macrochimerism with limited dose of bone marrow cells and prolongs skin graft survival in mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5x10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.     

Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen.

Mixed hematopoietic chimerism can be induced in mice receiving allogeneic bone marrow transplantation (BMT) after nonmyeloablative host conditioning with depletion T cells with of anti-T cell monoclonal antibodies (mAbs), low-dose (3 Gy) total-body irradiation (TBI), and local thymic irradiation (7 Gy). These mice are specifically tolerant to donor and host antigens. When nontolerant donor T cells are given to chimeras several months after BMT, full donor-type chimerism develops, but graft-vs.-host disease (GVHD) does not occur. The induction of such lymphohematopoietic GVH reactions without GVHD could provide an approach to separating graft-vs.-leukemia (GVL) from GVHD in patients with hematologic malignancies. To make the nonmyeloablative conditioning regimen described above more cytoreductive for such malignancies, we have now modified it by replacing TBI with cyclophosphamide (CP). Treatment with anti-CD4 and anti-CD8 mAbs on day -5, 200 mg/kg CP on day -1, and 7 Gy thymic irradiation on day 0 was only slightly myelosuppressive and allowed fully major histocompatibility complex (MHC)-mismatched (with or without multiple minor antigen disparities) allogeneic bone marrow to engraft and establish long-term mixed chimerism in 40 to 82% of recipients in three different strain combinations. The administration of nontolerant donor spleen cells at 5 weeks or at 5, 8, and 11 weeks posttransplant was capable of eliminating host hematopoietic cells, leading to full or nearly full donor chimerism in six of six and two of four chimeric animals in two different strain combinations. No clinical evidence of GVHD was observed in any recipients of these donor leukocyte infusions (DLI). These studies demonstrate that induction of mixed chimerism with nonmyeloablative conditioning followed at appropriate times by DLI might allow lymphohematopoietic GVH reactions, and hence GVL effects, to eliminate chronic hematologic malignancies without causing clinically significant GVHD.     

Photochemical treatment of donor lymphocytes inhibited their ability to facilitate donor engraftment or increase donor chimerism after nonmyeloablative conditioning or establishment of mixed chimerism.

Donor T-cells can provide a graft-versus-leukemia effect and help to promote donor engraftment after allogeneic BMT; however, these benefits can be outweighed by the ability of the cells to induce life-threatening GVHD. Photochemical treatment (PCT) of T-cells with S-59 psoralen and long-wavelength UV-A light can inhibit their proliferative capacity and significantly decrease their ability to induce acute GVHD after allogeneic BMT. PCT donor T-cells have been shown to facilitate donor engraftment in a myeloablative BMT model. In this study, we examined whether donor T-cells subjected to PCT ex vivo could retain the ability to facilitate engraftment or increase donor chimerism after nonmyeloablative BMT or after establishment of mixed hematopoietic chimerism. In a transplantation model in which mice were conditioned for BMT with sublethal (600 cGy) TBI, an infusion of PCT donor T-cells was unable to facilitate engraftment of donor BM. A BMT model was used in which a mixture of allogeneic and syngeneic marrow cells was infused into lethally irradiated recipients for establishment of mixed hematopoietic chimerism. The goal was to determine whether PCT donor splenocytes could increase levels of donor chimerism. Recipients of splenocytes treated with UV-A light only (no S-59 psoralen) and given at the time of BMT or in a donor lymphocyte infusion (DLI) had significantly higher levels of donor chimerism than did recipients of BM only. Although PCT donor splenocytes given at the time of BMT modestly increased donor chimerism, PCT donor splenocytes given in a DLI did not increase donor chimerism. A nonmyeloablative BMT model was employed for determining whether DLI given relatively late after BMT could increase donor chimerism. Recipient mice were conditioned for BMT with a combination of low-dose TBI (50 or 100 cGy) and anti-CD154 (anti-CD40L) monoclonal antibody for achievement of low levels of mixed chimerism. When control mixed chimeras were given a DLI 71 days after BMT, donor chimerism was significantly increased. In contrast, PCT of the donor cells eliminated the ability of the cells to increase donor chimerism after infusion. Together results from these 3 distinct BMT models indicate that PCT of donor T-cells significantly inhibited the ability of the cells to facilitate donor engraftment after nonmyeloablative BMT or to increase donor chimerism in mixed hematopoietic chimeras when the cells were administered in a DLI.     

Prognostic Utility of Routine Chimerism Testing at 2 to 6 Months after Allogeneic Hematopoietic Cell Transplantation

The utility of routine chimerism analysis as a prognostic indicator of subsequent outcomes after allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning regimens remains controversial. To address this controversy, routine chimerism test results at 2 to 6 months after HCT with myeloablative conditioning regimens were evaluated for association with subsequent risk of chronic graft-versus-host disease (GVHD), nonrelapse mortality (NRM), relapse, and overall mortality. Only 70 of 1304 patients (5%) had < 95% donor-derived cells in the marrow. Low donor chimerism in the marrow occurred more often in patients with low-risk diseases compared with those with higher-risk diseases and was significantly associated with a reduced risk of chronic GVHD. Among 673 patients evaluated, 164 (24%) had < 85% donor-derived T cells in the blood. Low donor T cell chimerism was more frequent in patients with low-risk diseases compared with those with higher-risk diseases, in those who received conditioning with busulfan compared with those who received conditioning with total body irradiation, and in those with lower-grade acute GVHD. Low donor T cell chimerism in the blood was significantly associated with a reduced risk of chronic GVHD but not with a reduced risk of relapse, NRM, or overall mortality. Routine testing of chimerism in the marrow and blood at 2 to 6 months after HCT with myeloablative conditioning regimens may be helpful in documenting engraftment in clinical trials, but provides only limited prognostic information in clinical practice.     

Nonmyeloablative conditioning with busulfan before matched littermate bone marrow transplantation results in reversal of the disease phenotype in canine leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD)-1, a primary immunodeficiency disease caused by molecular defects in the leukocyte integrin CD18 molecule, is characterized by recurrent, life-threatening bacterial infections. Myeloablative hematopoietic stem cell transplantation is the only curative treatment for LAD-1. Recently, canine LAD (CLAD) has been shown to be a valuable animal model for the preclinical testing of nonmyeloablative transplantation regimens for the treatment of children with LAD-1. To develop new allogeneic transplantation approaches for LAD-1, we assessed a nonmyeloablative conditioning regimen consisting of busulfan as a single agent before matched littermate allogeneic bone marrow transplantation in CLAD. Three CLAD dogs received busulfan 10 mg/kg intravenously before infusion of matched littermate bone marrow, and all dogs received posttransplantation immunosuppression with cyclosporin A and mycophenolate mofetil. Initially, all 3 dogs became mixed chimeras, and levels of donor chimerism sufficient to reverse the CLAD phenotype persisted in 2 animals. The third dog maintained donor microchimerism with an attenuated CLAD phenotype. These 3 dogs have all been followed up for at least 1 year after transplantation. These results indicate that a nonmyeloablative conditioning regimen with chemotherapy alone is capable of generating stable mixed chimerism and reversal of the disease phenotype in CLAD.     

Inhibition of Alloantigen‐Primed Memory CD4+ and CD8+ T Cells by Hematopoietic Chimerism in Mice

Donor‐reactive memory T cells present a special hurdle in transplantation. Although hematopoietic chimerism is effective for inducing donor‐specific tolerance, the effects on memory T cells are unclear. Here, we induced stable chimerism and tolerance in mice (Tolerance group, n = 6) by donor‐specific transfusion (DST) plus anti‐CD154 monoclonal antibody (mAb), avoiding the toxic myeloablative conditioning treatment to assist bone marrow transplantation (DST/aCD154&BMTx). We then transferred memory CD4⁺ or CD8⁺ T cells from donor antigen primed mice to the tolerance‐induced recipients 4 days after heart transplantation (Tol/CD4⁺ Tm group and Tol/CD8⁺ Tm group, n = 6, respectively), but neither of these memory T‐cell subsets had an effect on the permanent graft survival (median survival time > 100 days). The unaltered rate of memory T cells in spleen and anergy to donor antigen in vitro demonstrated that these memory T cells were well controlled. The chimerism‐promoting protocol DST/aCD154&BMTx produced an immune environment that included high levels of regulatory T cells (Tregs), microchimerism and TGF‐β, all of which may act in suppressing the donor‐reactive memory CD4⁺ or CD8⁺ T cells. These findings have potentially important implications for designing approaches to suppressing memory T cells for success of transplantation.     

Tolerance induction through simultaneous double bone marrow transplantation with two-signal blockade

Cotransplantation of donor bone marrow cells (BMCs) in allograft recipients is currently the most promising concept for clinical tolerance induction; however, it still has many difficulties in its successful performance due to the toxicity of the required host conditioning, the risk of engraftment failure, and the problem of graft-versus-host disease (GVHD), as well as the limited accessibility of donor bone marrow cells. Therefore, we performed the studies to determine whether BMCs from multi-donors are simultaneously engrafted and lead to induction of chimerism-based tolerance through the tolerogenic protocol of whom effectiveness we have shown in a previous study. Using a murine model, it was demonstrated that grafted BMCs from BALB/c (H-2(d)) and CBA mice (H-2(k)) establish mixed type and multi-lineage double chimerism and induce immunological donor-specific tolerance to fully MHC-mismatched skin allografts in host C57BL/6 mice (H-2(b)) receiving conditioning with Busulfan and treatment with the two-signal blockade comprised of anti-CD45RB and anti-CD154 monoclonal antibodies.     

Control of mouse graft-versus-host disease following allogeneic bone marrow transplantation by blocking the CD28/B7 signaling pathway with lentiviral vector-mediated RNA interference

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective way to cure hematological malignancies. However, graft-versus-host disease (GVHD) following transplantation limits the clinical application to some extent. The donor T lymphocytes play a central role in the occurrence and development of GVHD. Control of GVHD by inhibition of T cell proliferation by blocking the CD28/B7 signaling pathway with RNA interference has not been examined. In this study, we constructed a lentiviral vector carrying CD28 shRNA and generated genetically engineered splenocytes through transduction in a murine allogeneic bone marrow transplantation model. The survival and the occurrence of GVHD in transplanted mice were monitored every day. Liver, intestine, skin, and other tissues from the mice in each group were used for histological examination. We also determined plasma concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, and interferon gamma (IFN-γ). Recipient bone marrow from mice that had survived for an extended period was examined to detect chimerism. We succeeded in suppressing the expression of CD28 gene and controlling mouse GVHD following allogeneic bone marrow transplantation in the engineered spleen cell group. These suggest that blocking the CD28/B7 signal transduction pathway with lentiviral vector-mediated RNA interference effectively controlled the occurrence of mouse GVHD following allogeneic bone marrow transplantation. Its mechanism could be due to the inhibition of T cell proliferation and, simultaneously, the promotion of the differentiation of TH0 to TH2 cells, thereby reducing GVHD in the mouse transplantation model.     

Classical transplantation tolerance in the adult: the interaction between myeloablation and immunosuppression.

Allogeneic bone marrow transplantation in the neonate is an effective way of inducing permanent tolerance to donor tissue. To do the same in the immunocompetent adult requires immunosuppression to counter host-versus-graft alloreactivity. Conditioning with monoclonal antibodies (mAb) to CD4 and CD8 has been sufficient where donor and recipient are mismatched at only multiple "minor" histocompatibility loci, or at major histocompatibility complex (MHC) class I plus "minor" loci, but not where the mismatch involves the entire MHC. Tolerance across the MHC barrier requires extra conditioning with agents that happen to be both immunosuppressive and myeloablative, so obscuring the assessment of which effect is important. By using dimethylmyleran as a selective "space"-creating myeloablative agent, and CD4 plus CD8 mAb as sole immunosuppressive agents, we have been able to dissect the relative requirements for immunosuppression and myeloablation. We show here that transplantation tolerance could only be achieved when both types of agent were combined together so as to guarantee sufficient donor-type hemopoietic chimerism. We argue that the donor marrow, given sufficient space, will engraft and provide a sustained source of tolerogen overriding any host resistance that antibodies cannot control.     

Induction of Lethal Graft-versus-Host Disease by Anti-CD137 Monoclonal Antibody in Mice Prone to Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. We previously showed that anti-CD137 monoclonal antibody (mAb) can cure advanced cGVHD by inducing activation-induced cell death of donor T cells. In this study, we examined whether administration of anti-CD137 mAb can prevent the development of cGVHD after bone marrow transplantation (BMT) in mice conditioned with total body irradiation (TBI). We used the B10.D2→Balb/c (H-2^d) minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. A single injection of anti-CD137 mAb was administered immediately after BMT. In contrast to the results obtained from the curing model of cGVHD, anti-CD137 given simultaneously with BMT resulted in lethal GVHD. Histopathologic evaluation revealed inflammation and damage of target organs from acute GVHD (aGVHD) in anti-CD137-treated mice. Anti-CD137–induced lethal aGVHD required host cells, as well as irradiation and mature donor T cells. Apparently, anti-CD137 mAb rapidly induced activation of donor T cells and sustained their activation status under the inflammatory condition triggered by irradiation. When given on day 12 after irradiation and BMT, anti-CD137 mAb could still exacerbate GVHD, but when given on day 30, it could not. Our data demonstrate that anti-CD137 mAb can amplify inflammation induced by host preconditioning, subsequently resulting in lethal aGVHD; thus, alleviating irradiation-induced toxicity is critical to allow the use of anti-CD137 mAb as GVHD prophylaxis.     

Combination of intra-bone marrow-bone marrow transplantation and subcutaneous donor splenocyte injection diminishes risk of graft-versus-host disease and enhances survival rate

The combination of allogeneic bone marrow transplantation (allo-BMT) and donor lymphocyte infusion (DLI) is a useful method for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, there is a risk of inducing uncontrollable fatal graft-versus-host disease (GVHD). In fact, allo-BMT plus intravenous (IV)-DLI using donor splenocytes induces fatal GVHD in recipient mice. In this study, we examined the effects of the combination of intra-bone marrow (IBM)-BMT and the subcutaneous injection of donor splenocytes (SC-DLI) on the allo-BMT system. Recipient BALB/c mice were conditioned by sublethal irradiation (5 Gy), followed by IBM-BMT plus IV-DLI or SC-DLI in C57BL/6 mice. The IV-DLI group showed better engraftment of donor hemopoietic cells than the control group (without DLI) but showed fatal GVHD. The SC-DLI group, however, showed good reconstitution and mild GVHD. These results suggest that the combination of SC-DLI and IBM-BMT promotes the reconstitution of hemopoiesis and helps reduce the risk of GVHD.     

Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats

T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 x 10(9) plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 x 10(8)/rat) and SPLCs (5 x 10(7)/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts.     

Bendamustine with Total Body Irradiation Limits Murine Graft-versus-Host Disease in Part Through Effects on Myeloid-Derived Suppressor Cells

Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11b⁺Gr-1^high myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1^high cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11b⁺Gr-1⁺ myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.     

Prevention of lethal graft-vs.-host disease by a single low dose injection of anti-T cell monoclonal antibody to the allograft recipients.

We investigated the capacity of monoclonal antibody (mAb) treatment to prevent graft-vs.-host disease (GVHD) in lethally irradiated, allogeneically reconstituted mice, employing anti-T cell (subset) mAb and a fully allogeneic strain combination. In this strain combination, purified CD4+ cells were able to induce a lethal GVH reaction, whereas purified CD8+ cells were not. In the same strain combination, a single intraperitoneal injection of IgG2b anti-Thy-1 mAb, one day after reconstitution, caused a dose-dependent improvement of the survival. A single injection of a dose as low as 12.5 micrograms per mouse was already effective. Intravenous and intraperitoneal administration of the mAb appeared equally effective. For effective prevention of GVHD the treatment could be postponed until the 4th day after transplantation, but treatment delayed until day 6 was no longer effective. Treatment with IgG2b mAb specific for either helper or cytotoxic T cells also led to improvement of GVHD and survival, but was less effective than treatment with anti-Thy-1 mAb. Clinically, there was a difference in the effectiveness of anti-CD4 and anti-CD8 treatment, since symptoms of GVHD started earlier in the anti-CD8 treated group and the survival was better in the anti-CD4 treated group. These results press for prospective clinical studies employing anti-T cell mAb treatment early after allogeneic bone marrow transplantation, especially in HLA mismatched cases.     

Allogeneic Transplantation with Reduced-Intensity Conditioning for Hodgkin and non-Hodgkin Lymphoma: Importance of Histology for Outcome

Allogeneic stem cell transplantation (SCT) with reduced-intensity conditioning (RIC) has the potential to lead to long-term remissions for patients with lymphoma. However, the role of RIC SCT in the treatment of lymphoma is still unclear. Specifically, the relative benefit of RIC SCT across lymphoma histologies and the prognostic factors in this population are incompletely defined. We retrospectively analyzed the outcomes of 87 patients with advanced lymphoma who underwent RIC SCT at the Dana-Farber Cancer Institute over a 6-year period with a homogeneous conditioning regimen consisting of fludarabine and low-dose busulfan. Thirty-six patients had Hodgkin disease (HD) and 51 had non-Hodgkin lymphoma (NHL). Sixty-eight percent had undergone prior autologous transplantation. The 1-year cumulative incidence of nonrelapse mortality was 13%, and the 3-year cumulative incidence of progression was 49%. The incidence of grade 3-4 acute GVHD was 11%. The 2-year cumulative incidence of chronic GVHD was 68%, and its development was associated with a decreased risk of progression and an improved progression-free survival (PFS). Three-year overall survival (OS) was 56% for patients with HD, 81% for indolent NHL, 42% for aggressive NHL, and 40% for mantle cell lymphoma. The corresponding figures for 3-year PFS were 22%, 59%, 22%, and 30%, respectively. Multivariate analysis identified elevated pretransplantation lactate dehydrogenase (LDH) as an adverse factor for PFS, while indolent NHL histology was favorable. For OS, advanced age and elevated pretransplantation LDH were adverse factors, whereas indolent NHL histology was favorable. Low early donor chimerism was not predictive of poor outcome in univariate or multivariate analyses. Moreover, progression was not associated with loss of chimerism. These results emphasize the importance of lymphoma histology for patients undergoing RIC SCT, as well as the lack of relevance of donor chimerism for outcome in this patient population.     

Early regulation of CD8 T cell alloreactivity by CD4+CD25- T cells in recipients of anti-CD154 antibody and allogeneic BMT is followed by rapid peripheral deletion of donor-reactive CD8+ T cells, precluding a role for sustained regulation

While acquisition of regulatory function by CD4+CD25- T cells has been reported following antigenic stimulation, "naturally occurring" regulatory CD4+ T cells (Treg) are believed to express CD25. We examined the mechanisms involved in peripheral CD8 T cell tolerance by induction of mixed chimerism using non-myeloablative conditioning with low-dose (3 Gy) total body irradiation and anti-CD154 antibody. Recipient CD4+ T cells were initially required for the induction of CD8 cell tolerance, but were not needed beyond 2 weeks. Depletion of CD25+ Treg prior to bone marrow transplantation and blockade of IL-2 with neutralizing antibody did not impede tolerance induction. Tolerance was dependent on CTLA4, but not on IFN-gamma. In C57BL/6 mice containing a fraction of 2C TCR transgenic CD8+ T cells, which recognize the MHC class I alloantigen Ld, induction of chimerism with L(d+), but not Ld-, bone marrow cells led to deletion of peripheral 2C+ CD8+ cells within 1 week in peripheral blood and spleen. Complete deletion required the presence of recipient CD4+ T cells. Thus, a novel, rapid form of regulation by CD4+CD25- T cells permits initial CD8 T cell tolerance in this model. Rapid peripheral deletion of donor-specific CD8 T cells precludes an ongoing requirement for CD4 T cell-mediated regulation.     

Establishment of complete and mixed donor chimerism after allogeneic lymphohematopoietic transplantation: recommendations from a workshop at the 2001 Tandem Meetings of the International Bone Marrow Transplant Registry and the American Society of Blood and Marrow Transplantation.

Approaches to the measurement of lymphohematopoietic chimerism have evolved from laboratory research to important clinical tools. However, there has been no logical, consistent, and uniform set of recommendations for the measurement of chimerism in clinical transplantation. The National Marrow Donor Program and the International Bone Marrow Transplant Registry (IBMTR) sponsored a workshop to discuss the use of chimerism analysis after allogeneic transplantation. The workshop was organized in an effort to make reasonable recommendations regarding laboratory techniques, the types of specimens to be studied, and the frequency of analysis. The panel recommended the following guidelines: 1. Chimerism analysis should use sensitive, informative techniques. At present, short tandem repeats (STR) or variable number tandem repeats (VNTR) analysis is the approach most likely to give reproducible informative data. 2. Peripheral blood cells are generally more useful than bone marrow cells for chimerism analysis. 3. Lineage-specific chimerism should be considered the assay of choice in the setting of nonmyeloablative and reduced-intensity conditioning. 4. The use of T-cell depletion, nonmyeloablative or reduced-intensity conditioning, or novel graft-versus-host disease (GVHD) prophylactic regimens warrants chimerism analysis at 1, 3, 6, and 12 months, because interventions such as donor lymphocyte infusions may depend on chimerism status. 5. In nonmyeloablative transplantation, the early patterns of chimerism may predict either GVHD or graft loss. Therefore, more frequent (every 2-4 weeks) peripheral blood analysis may be warranted. 6. For nonmalignant disorders, chimerism generally should be measured 1, 2, and 3 months after transplantation. Interventions to enhance donor engraftment must be considered on a disease-specific basis in relation to concurrent GVHD and, ultimately, clinical rationale.     

Adult recipients of matched related donor blood cell transplants given myeloablative regimens including pretransplant antithymocyte globulin have lower mortality related to graft-versus-host disease: a matched pair analysis.

Because pretransplantation anti-thymocyte globulin (ATG) seems to reduce graft-versus-host-disease (GVHD) and treatment-related mortality (TRM) after unrelated donor bone marrow transplantation (BMT), we investigated this agent in matched related donor (MRD) blood cell transplantation (BCT). Fifty-four adults receiving rabbit ATG, cyclosporine A, and methotrexate with myeloablative conditioning and undergoing first MRD BCT were matched for disease and stage with 54 patients not given ATG. Most ATG-treated patients had fludarabine with oral (7) or i.v. busulfan (46) with total body irradiation (TBI) in 10. Control patients largely received TBI with VP16 (28) or oral busulfan with cyclophosphamide (15) or fludarabine (7). The ATG was given at a total dose of 4.5 mg/kg over 3 d, finishing on day 0. Rates of acute GVHD (aGVHD) grade II-IV, aGVHD grade III-IV, and chronic GVHD (cGVHD) were 19 +/- 5% versus 32 +/- 6% (P = .1), 6 +/- 3% versus 13 +/- 5% (P = NS), and 55 +/- 8% versus 96 +/- 3% (P = .002) in the ATG and control groups, respectively. Patients given ATG had fewer sites involved by cGVHD compared with the control group (mean 2.1 +/- 0.2 versus 2.8 +/- 0.2, P = .04). Non-relapse mortality (NRM) with and without ATG, respectively, was 4 +/- 3% versus 17 +/- 5% at 100 d and 9 +/- 4% versus 34 +/- 7% at 4 yr (P = .002). Deaths were GVHD related in 3 ATG-treated patients versus 14 controls (P = .007). Despite a trend to more relapse with ATG (43 +/- 7% versus 22 +/- 7% at 4 yr, P = 0.05), survival was 66 +/- 7% in the patients given ATG versus 50 +/- 7% in the controls (P = 0.046). This study indicates that myeloablative regimens incorporating fludarabine and oral or i.v. busulfan with pretransplantation ATG given to recipients undergoing MRD BCT may result in less cGVHD, lower TRM, and probably improved quality of life in survivors compared with previous protocols.     

Chimerism studies in HLA-identical nonmyeloablative hematopoietic stem cell transplantation point to the donor CD8(+) T-cell count on day + 14 as a predictor of acute graft-versus-host disease.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4(+) and CD8(+) T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4(+) and CD8(+) donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P =.0002 and P =.019, respectively). The rate of disappearance of recipient CD8(+) T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P =.016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8(+) T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P =.003).     

Host Lymphocyte Depletion as a Strategy to Facilitate Early Full Donor Chimerism after Reduced-Intensity Allogeneic Stem Cell Transplantation

Reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (RIC-alloHSCT) is associated with lower toxicity but higher rates of prolonged mixed chimerism than myeloablative conditioning. Decreased pretransplantation host T cell numbers are associated with less graft rejection and early full donor chimerism. To compensate for variability in pretransplantation host lymphocyte numbers and facilitate the achievement of rapid full donor chimerism, we tested a strategy of targeted lymphocyte depletion (TLD) using chemotherapy at conventional doses to provide cytoreduction and lymphocyte depletion before RIC-alloHSCT. In our study, 111 patients with advanced hematologic malignancies received 1 to 3 cycles of conventional-dose chemotherapy to reduce circulating lymphocytes to a predetermined level. Patients then underwent RIC-alloHSCT from HLA-matched siblings. Patients received a median of 2 cycles of TLD chemotherapy, resulting in a median 71% decline in CD4⁺ count. All patients engrafted; there were no late graft failures. By day +14, median CD3⁺ chimerism was 99% donor and was significantly associated with lower post-TLD CD4⁺ counts (P = .012). One- and 5-year treatment-related mortality were 15% and 21%, respectively. At 1-year follow-up, 66% of patients had achieved complete remission (CR) of which 92% were not in CR at the time of transplantation. Overall survival at 1 and 5 years post transplantation were 66% and 47%, respectively.