Hematopoietic donor chimerism and graft-versus-myeloma effect in relapse of multiple myeloma after allogeneic bone marrow transplantation

 A large group of patients relapsing after allogeneic bone marrow transplantation (BMT) have obtained remission after infusion of leukocytes from their original donor, suggesting a graft-versus-myeloma effect. However, side effects such as graft-versus-host disease and myelosuppression are severe, and sometimes fatal, complications of this therapeutic approach. Previously we demonstrated that patients with leukemia who lack donor hematopoiesis in relapse after BMT experience severe and lasting aplasia after infusion of donor leukocytes. In two patients – one with extramedullary and one with marrow relapse after a sex-mismatched transplantation – we analyzed hematopoietic chimerism by cell sorting and bone marrow cultures. CD34-positive cells, CD4-CD8-positive cells, committed progenitors, and LTC-IC were of donor origin, as demonstrated by two-color fluorescence in situ hybridization (FISH). Additionally, in relapse complete donor T-cell chimerism was seen. In contrast, plasma cells were of recipient origin in the patient who had a relapse in the bone marrow. Both patients were treated with infusions of donor leukocytes from their original donor. Neither patient suffered myelosuppression, and one achieved a stable complete remission. Received: February 26, 1999 / Accepted: April 14, 1999     

Immunologic effects of prophylactic donor lymphocyte infusion after allogeneic marrow transplantation for multiple myeloma

Reconstitution of T-cell immunity after bone marrow transplantation (BMT) is often delayed, resulting in a prolonged period of immunodeficiency. Donor lymphocyte infusion (DLI) has been used to enhance graft-versus-leukemia activity after BMT, but the effects of DLI on immune reconstitution have not been established. We studied 9 patients with multiple myeloma who received myeloablative therapy and T-cell-depleted allogeneic BMT followed 6 months later by infusion of lymphocytes from the same donor. DLI consisted of 3 x 10(7) CD4(+) donor T cells per kilogram obtained after in vitro depletion of CD8(+) cells. Cell surface phenotype of peripheral lymphocytes, T-cell receptor (TCR) V beta repertoire, TCR rearrangement excision circles (TRECs), and hematopoietic chimerism were studied in the first 6 months after BMT and for 1 year after DLI. These studies were also performed in 7 patients who received similar myeloablative therapy and BMT but without DLI. Phenotypic reconstitution of T and natural killer cells was similar in both groups, but patients who received CD4(+) DLI developed increased numbers of CD20(+) B cells. TCR V beta repertoire complexity was decreased at 3 and 6 months after BMT but improved more rapidly in patients who received DLI (P =.01). CD4(+) DLI was also associated with increased numbers of TRECs in CD3(+) T cells (P <.001) and with conversion to complete donor hematopoiesis (P =.05). These results provide evidence that prophylactic infusion of CD4(+) donor lymphocytes 6 months after BMT enhances reconstitution of donor T cells and conversion to donor hematopoiesis as well as promoting antitumor immunity.     

A randomized multicenter comparison of CD34(+)-selected progenitor cells from blood vs from bone marrow in recipients of HLA-identical allogeneic transplants for hematological malignancies

OBJECTIVE: Peripheral blood progenitor cells (PBPC) have been established as an alternative source of hematopoietic stem cells for allogeneic transplantation, but an increased incidence of both acute and chronic graft-vs-host disease (GVHD) has become apparent. We performed a prospective randomized trial comparing bone marrow transplantation (BMT) vs PBPC transplantation (PBPCT) using CD34(+) selection for T-cell depletion (TCD) in both study arms. PATIENTS AND METHODS: Between January 1996 and October 2000, 120 patients with a diagnosis of acute leukemia, myelodysplasia, multiple myeloma, or lymphoma were randomized to receive either filgrastim-mobilized PBPC or BM from HLA-identical sibling donors after standard high-dose chemoradiotherapy. Patient characteristics did not differ between study arms. RESULTS: Recipients of PBPC received more CD3(+) T cells (median: 3.0 vs 2.0 x 10(5)/kg, p<0.0001) and more CD34(+) cells (median: 3.6 vs 0.9 x 10(6)/kg, p<0.0001). Neutrophil and platelet recoveries occurred significantly faster after PBPCT. The cumulative incidence of acute GVHD grades II-IV was 37% after BMT vs 52% after PBPCT and was most significantly (p=0.007) affected by the number of CD3(+) T cells in the graft. Acute GVHD appeared strongly associated with increased treatment-related mortality (TRM) in a time-dependent analysis. Higher numbers of CD34(+) cells were associated with less TRM. With a median follow-up of 37 months (range: 12-75), overall survival at 4 years from transplantation was 60% after BMT and 34% for recipients of PBPCT (p=0.04), which difference was largely due to increased GVHD and TRM in PBPC recipients receiving T-cell dosages greater than 2 x 10(5)/kg. CONCLUSION: Outcome following T cell-depleted PBPCT critically depends on the number of CD3(+) T cells, whereby high T-cell numbers may blunt a favorable effect of higher CD34(+) cell numbers.     

Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation

The "conventional" NK1.1(-) T cells from mouse blood and marrow were compared with regard to surface receptors, cytokine secretion, and function. Most blood NK1.1(-) CD4(+) and CD8(+) T cells expressed the naive CD44(int/lo)CD62L(hi)CD45RB(hi) T-cell phenotype typical of those in the peripheral lymphoid tissues. In contrast, most marrow NK1.1(-) CD4(+) and CD8(+) T cells expressed an unusual CD44(hi)CD62L(hi)CD45RB(hi) phenotype. The blood NK1.1(-) CD4(+) T cells had a naive T-helper cytokine profile and a potent capacity to induce lethal graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host bone marrow transplantation model. In contrast, the marrow NK1.1(-) CD4(+) T cells had a Th0 cytokine profile and failed to induce lethal GVH disease, even at 20-fold higher numbers than those from the blood. NK1.1(-) CD8(+) T cells from the blood but not the marrow induced lethal GVH disease. Nevertheless, the marrow NK1.1(-) CD8(+) T cells induced potent antitumor activity that was augmented by marrow NK1.1(-) CD4(+) T cells and facilitated hematopoietic progenitor engraftment. The inability of marrow CD4(+) and CD8(+) T cells to induce GVH disease was associated with their inability to expand in the blood and gut of allogeneic recipients. Because neither the purified marrow CD4(+) or CD8(+) T cells induced GVH disease, their unique features are desirable for inclusion in allogeneic bone marrow or hematopoietic progenitor transplants.     

Kinetic of regulatory CD25high and activated CD134+ (OX40) T lymphocytes during acute and chronic graft-versus-host disease after allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) is still a major complication after allogeneic stem cell transplantation. In murine models, freshly isolated or ex vivo expanded CD4(+)CD25(high) regulatory T cells (Treg) are able to ameliorate GVHD while maintaining graft-versus-leukaemia reactions. However, in the human setting, prospective studies of this population and its interaction with activated non-regulatory CD134(+) (OX40) lymphocytes during post-transplant follow-up are lacking. In this study, we prospectively quantified CD4(+)CD25(high) and activated CD134(+) lymphocytes in 119 peripheral blood samples from 35 consecutive patients who underwent allogeneic bone marrow transplantation (BMT). Fifty-five samples obtained less than 100 d after allogeneic BMT, were not statistically different regarding CD4(+)CD25(high) Treg or CD134(+) lymphocytes compared with those obtained from patients with (n = 35) or without (n = 20) acute GVHD. Chronic GVHD was associated with a small, but not statistically significant, increase in the number of Treg (9.9 vs. 6.7 x 10(6)/L). However, the CD134/CD25(high) ratio was significantly higher during chronic GVHD (cGHVD) when compared with either patients without cGVHD (67.7 +/- 40.3 vs. 4.0 +/- 0.9, P < 0.01) or cGVHD after treatment (67.7 +/- 40.3 vs. 3.7 +/- 0.8, P < 0.01). Our findings suggest that the suppressive activity of CD4(+)CD25(high) Treg could be abrogated in vivo during cGVHD by CD134 expression in a much higher number of activated donor T lymphocytes. In addition to CD4(+)CD25(high)ex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.     

Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation

Allogeneic bone marrow transplantation (BMT) is a potentially curative treatment for both inherited and acquired diseases of the hematopoietic compartment; however, its wider use is limited by the frequent and severe outcome of graft-versus-host disease (GVHD). Unfortunately, efforts to reduce GVHD by removing donor T cells have resulted in poor engraftment and elevated disease recurrence. Alternative cell populations capable of supporting allogeneic hematopoietic stem/progenitor cell engraftment without inducing GVHD could increase numbers of potential recipients while broadening the pool of acceptable donors. Although unfractionated CD4(+) T cells have not been shown to be an efficient facilitating population, CD4(+)CD25(+) regulatory cells (T-reg's) were examined for their capacity to support allogeneic hematopoietic engraftment. In a murine fully major histocompatibility complex (MHC)-mismatched BMT model, cotransplantation of donor B6 T-reg's into sublethally conditioned BALB/c recipients supported significantly greater lineage-committed and multipotential donor progenitors in recipient spleens 1 week after transplantation and significantly increased long-term multilineage donor chimerism. Donor engraftment occurred without GVHD-related weight loss or lethality and was associated with tolerance to donor and host antigens by in vitro and in vivo analyses. Donor CD4(+)CD25(+) T cells may therefore represent a potential alternative to unfractionated T cells for promotion of allogeneic engraftment in clinical hematopoietic cell transplantation.     

Regulatory dendritic cells protect against cutaneous chronic graft-versus-host disease mediated through CD4+CD25+Foxp3+ regulatory T cells

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity and mortality in allogeneic bone marrow transplantation (alloBMT). However, effective strategies for the treatment of cGVHD have not been established. In this study, we examined the therapeutic utility of modified dendritic cells (DCs) with a greater capacity to regulate immune responses than previously known tolerogenic DCs, regulatory DCs (DC(regs)), in the major histocompatibility complex-compatible, and multiple minor histocompatibility antigen-incompatible model of cGVHD in alloBMT. Treatment of the recipient mice after alloBMT with the recipient-type DC(regs) led to greater suppression of the incidence and severity of cutaneous cGVHD than rapamycin, whereas treatment with the recipient-type mature DCs promoted the pathogenesis. Analysis of the recipient mice suggested that the protective effect of the recipient-type DC(regs) involved the peripheral generation of alloreactive CD4(+)CD25(+)Foxp3(+)regulatory T (T(R)) cells from donor-derived CD4(+)CD25(-)Foxp3(-) T cells. Thus, immunotherapy with DC(regs) is a promising strategy for the treatment of cGVHD in alloBMT mediated through the induction of a dominant tolerance involving CD4(+)CD25(+)Foxp3(+) T(R) cells.     

CD8-depleted donor lymphocyte infusion as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation

Donor lymphocyte infusions can reinduce complete remission in the majority of patients with chronic myelogenous leukemia (CML) who relapse into chronic phase after allogeneic bone marrow transplantation (BMT). Such infusions are associated with a high incidence of graft- versus-host disease (GVHD) and marrow aplasia. BMT using selective depletion of CD8+ lymphocytes from donor cells reduces the incidence of GVHD without an increase in leukemia relapse. We hypothesized that infusion of CD8-depleted donor peripheral blood lymphocytes could also reinduce complete remissions with a lesser potential to produce symptomatic GVHD in patients with CML who relapsed after allogeneic BMT. Ten patients with Ph(+) CML who relapsed a median of 353 days after BMT (range, 82 to 1,096 days) received donor lymphocyte infusions depleted of CD8+ cells. Nine patients received a single infusion and 1 received two infusions. Four patients were treated while in chronic phase with clonal evolution, 2 during accelerated phase, 3 during blast crisis, and 1 in a cytogenetic relapse. A mean of 0.9 +/- 0.3 x 10(8) mononuclear cells/kg were infused, containing 0.6 +/- 0.4 x 10(6) CD3+CD8+ cells/kg. Six patients achieved hematologic and cytogenetic remission at 4, 8, 11, 15, 39, and 54 weeks after lymphocyte infusion. Two patients developed > or = grade II acute GVHD, and 1 patient developed mild chronic GVHD. We conclude that donor lymphocyte infusions depleted of CD8+ cells can induce remissions with a low rate of severe acute GVHD in patients with CML who relapse after allogeneic BMT, supporting the hypothesis that CD8+ lymphocytes are important effectors of GVHD, but may not be essential for the graft-versus- leukemia effect against this disease. Further controlled studies are required to confirm these preliminary observations.     

Human gammadelta(+) T lymphocytes have in vitro graft vs leukemia activity in the absence of an allogeneic response

Refractory acute lymphoblastic leukemia (ALL) is often incurable, and relapse rates following allogeneic bone marrow transplantation (BMT) remain high. We have reported that patients who develop increased numbers of gammadelta(+) T cells soon after BMT are significantly less likely to relapse. We now show in seven donor/recipient pairs that donor-derived Vdelta1(+)CD4(-)CD8(-)gammadelta(+) T cells are activated and proliferate in response to recipient primary ALL blasts. In addition, these cells have been shown to bind and lyse the recipient ALL blasts. Separately, gammadelta(+) T cells proliferate poorly or not at all in mixed lymphocyte culture against HLA-mismatched unrelated stimulator cells. These observations suggest that allogeneic gammadelta(+) T cells could be an effective immunotherapeutic strategy against refractory disease without the risk of graft-versus-host disease.     

Cytogenetic events after bone marrow transplantation for chronic myeloid leukemia in chronic phase

Forty-eight patients treated by allogeneic bone marrow transplantation (BMT) for Philadelphia (Ph) chromosome-positive chronic myeloid leukemia in chronic phase had serial cytogenetic studies of marrow performed at intervals after transplant. Twenty patients received marrow cells from donors of opposite sex. Ph+ marrow metaphases were identified in 24 of 48 (50%) of patients after BMT; they were first seen early (within 1 year) in 16 cases and late (greater than 1 year after BMT) in eight cases. Ph-positivity after BMT occurred more commonly in recipients of T-depleted than nondepleted marrow (19 of 28 v 5 of 20). In 4 cases the Ph+ metaphases were found only transiently after BMT; in 11 cases the Ph+ metaphases have persisted but hematologic relapse has not ensued; in 9 cases the finding of Ph+ metaphases coincided with or preceded hematologic relapse. Chromosomes in cells of donor origin had morphological abnormalities in two cases. No relapses were identified in cells of donor origin. Our data suggest that the relationship between cells of recipient and donor origin is complex: cure of leukemia may depend on factors that operate for some months or years after BMT.     

Major histocompatibility complex-mismatched allogeneic bone marrow transplantation using perforin and/or Fas ligand double-defective CD4(+) donor T cells: involvement of cytotoxic function by donor lymphocytes prior to graft-versus-host disease pathogenesis.

Experimental allogeneic bone marrow transplantation (BMT) models using cytotoxic single-deficient (perforin/granzyme or Fas ligand [FasL]) and cytotoxic double-deficient (cdd) CD4(+) donor T cells have previously demonstrated roles for both effector pathways in graft-versus-host disease (GVHD). In the present study, the role of CD4-mediated antihost cytotoxicity in a GVH response is further examined across a complete major histocompatibility complex class I/II mismatch. As predicted, a double cytotoxic deficiency resulted in a clear delay in GVH-associated weight loss, clinical changes, and mortality. Interestingly, analysis of donor T-cell presence in 5.5-Gy recipients soon after BMT demonstrated that the double cytotoxic deficiency resulted in a marked decrease in donor CD4 numbers. Transplantation of singularly perforin- or FasL-deficient donor CD4(+) T cells demonstrated that the absence of FasL was responsible for the markedly diminished CD4 number in recipient lymph nodes and spleens soon after BMT. However, increasing recipient total body irradiation conditioning (11.0 Gy) abrogated the decrease in FasL-defective B6-cdd and B6-gld CD4 numbers. Thus, the decrease was not a result of inherent CD4 defects, but was probably attributable to host resistance. Consistent with these observations, transplantation into 11.0-Gy recipients resulted in identical GVH lethality by equal numbers of B6 wild-type, B6-cdd, and B6-gld CD4(+) T-cell inoculum. In total, the findings indicate that aggressive host conditioning lessens the requirement for donor CD4(+) cytotoxic function in GVH responses soon after BMT. The present results thus support the notion of a role for cytotoxic effector function in donor CD4(+) T cells prior to GVH-induced tissue injury.     

Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation

The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part because of immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilms Tumor 1 (WT1) peptides, induces a T-cell population that is tumor antigen specific. We determined whether allogeneic BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent antitumor activity than either therapy alone. WT1 peptide vaccinations of healthy donor mice induced CD8(+) T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that peptide immunization was effective as a prophylactic vaccination before tumor challenge, yet was ineffective as a therapeutic vaccination in tumor-bearing mice. BMT from vaccinated healthy MHC-matched donors, but not syngeneic donors, into recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of FBL3 leukemia. The transfer of total CD8(+) T cells from immunized donors was more effective than the transfer of WT1-tetramer(+)CD8(+) T cells and both required CD4(+) T-cell help for maximal antitumor activity. These findings show that WT1 peptide vaccination of donor mice can dramatically enhance GvT activity after MHC-matched allogeneic BMT.     

Paradoxical effects of interleukin-18 on the severity of acute graft-versus-host disease mediated by CD4+ and CD8+ T-cell subsets after experimental allogeneic bone marrow transplantation

Administration of exogenous interleukin-18 (IL-18) regulates experimental acute graft-versus-host disease (GVHD) in a Fas-dependent manner when donor CD4(+) T cells are required for mortality after experimental allogeneic bone marrow transplantation (BMT). However, CD4(+) and CD8(+) T cells can induce acute GVHD after clinical allogeneic BMT, and the role of IL-18 in CD8(+)-mediated acute GVHD is unknown. We, therefore, determined the role of IL-18 in GVHD mediated by CD4(+) or CD8(+) T cells across major histocompatibility complex (MHC) class II- and class I-disparate allogeneic BMT, respectively. Administering IL-18 significantly increased survival in CD4(+)-mediated GVHD but reduced survival in CD8(+)-mediated GVHD. This increase in deaths was associated with significantly greater clinical, biochemical, and histopathologic parameters of GVHD damage and was independent of Fas expression on donor T cells. Administering IL-18 significantly enhanced allospecific cytotoxic function and expansion of CD8(+) cells. Endogenous IL-18 was critical to GVHD mediated by CD8(+) donor T cells because IL-18 receptor-deficient donors caused significantly less GVHD but exacerbated CD4(+)-mediated, GVHD-related death. Furthermore, administering anti-IL-18 monoclonal antibody significantly reduced CD8(+)-mediated, GVHD-related death. Together these findings demonstrate that IL-18 has paradoxical effects on CD4(+) and CD8(+) cell-mediated GVHD.     

Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.     

Transduction of donor hematopoietic stem-progenitor cells with Fas ligand enhanced short-term engraftment in a murine model of allogeneic bone marrow transplantation

Fas-mediated apoptosis is a major physiologic mechanism by which activated T cells are eliminated after antigen-stimulated clonal expansion generates a specific cellular immune response. Because activated T cells are the major effectors of allograft rejection, we hypothesized that genetically modifying allogeneic bone marrow (BM) cells prior to transplantation could provide some protection from host T-cell attack, thus enhancing donor cell engraftment in bone marrow transplantation (BMT). We undertook studies to determine the outcome of lentiviral vector-mediated transduction of Fas ligand (FasL) into lineage antigen-negative (lin(-)) mouse BM cells (lin(-) BMs), in an allogeneic BMT model. FasL-modified lin(-) BMs killed Fas-expressing T cells in vitro. Mice that received transplants of allogeneic FasL(+) lin(-) BMs had enhanced short-term engraftment, after nonmyeloablative conditioning, as compared to controls. We observed no major hepatic toxicity or hematopoietic or immune impairment in recipient mice at these time points. These results suggest potential therapeutic approaches by manipulating lymphohematopoietic stem-progenitor cells to express FasL or other immune-modulating genes in the context of BMT.     

Rapidly proliferating CD44hi peripheral T cells undergo apoptosis and delay posttransplantation T-cell reconstitution after allogeneic bone marrow transplantation

Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-X(L) expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4(+) and CD8(+) T cells. Transplantation of RAG-2-eGFP-transgenic BM revealed that proliferating eGFP(lo)CD44(hi) donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFP(hi)CD44(lo) recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester-labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44(hi) phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.     

Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease

Prolonged immunodeficiency after allogeneic bone marrow transplantation (BMT) causes significant morbidity and mortality from infection. This study examined in murine models the effects of interleukin-7 (IL-7) given to young and middle-aged (9-month-old) recipients of major histocompatibility complex (MHC)-matched or -mismatched allogeneic BMT. Although administration of IL-7 from day 0 to 14 after syngeneic BMT promoted lymphoid reconstitution, this regimen was ineffective after allogeneic BMT. However, IL-7 administration from day 14 (or 21) to 27 after allogeneic BMT accelerated restoration of the major lymphoid cell populations even in middle-aged recipients. This regimen significantly expanded donor-derived thymocytes and peripheral T cells, B-lineage cells in bone marrow and spleen, splenic natural killer (NK) cells, NK T cells, and monocytes and macrophages. Interestingly, although recipients treated with IL-7 had significant increases in CD4(+) and CD8(+) memory T-cell populations, increases in naive T cells were less profound. Most notable, however, were the observations that IL-7 treatment did not exacerbate graft-versus-host disease (GVHD) in recipients of an MHC-matched BMT, and would ameliorate GVHD in recipients of a MHC-mismatched BMT. Nonetheless, graft-versus-leukemia (GVL) activity (measured against 32Dp210 leukemia) remained intact. Although activated and memory CD4(+) and CD8(+) T cells normally express high levels of IL-7 receptor (IL-7R, CD127), activated and memory alloreactive donor-derived T cells from recipients of allogeneic BMT expressed little IL-7R. This might explain the failure of IL-7 administration to exacerbate GVHD. In conclusion, posttransplant IL-7 administration to recipients of an allogeneic BMT enhances lymphoid reconstitution without aggravating GVHD while preserving GVL.     

Engraftment of DLA-haploidentical marrow with ex vivo expanded, retrovirally transduced cytotoxic T lymphocytes.

Genetically modified donor T cells with an inducible "suicide" gene have the potential to improve the safety and availability of allogeneic hematopoietic stem cell transplantation by enhancing engraftment and permitting control of graft-versus-host disease (GVHD). However, several clinical studies of gene-modified T cells have shown limited to no in vivo function of the ex vivo expanded T cells. Using the well-established dog model of allogeneic marrow transplantation, the question was asked if retrovirally transduced, donor derived, ex vivo expanded cytotoxic T lymphocytes (CTLs) that are recipient specific could enhance engraftment of dog leukocyte antigen (DLA)-haploidentical marrow following a single dose of 9.2 Gy total body irradiation and no postgrafting immunosuppression. In this setting, only 4 of 11 control recipients of DLA-haploidentical marrow without added CTLs engrafted. CTLs did not enhance engraftment of CD34(+) selected peripheral blood stem cells. However, recipient-specific CTLs enhanced engraftment of DLA-haploidentical marrow in 9 of 11 evaluable recipients (P =.049). All dogs that engrafted developed multiorgan GVHD. To facilitate in vivo tracking, 8 dogs received CTLs transduced with a retroviral vector encoding green fluorescent protein (GFP) and neomycin phosphotransferase (neo). Recipients that engrafted had sharp increases in the numbers of circulating GFP(+) CTLs on days +5 to +6 after transplantation. GFP(+) CTLs isolated from blood were capable of recipient-specific lysis. At necropsy, up to 7.1% of CD3(+) cells in tissues were GFP(+) and polymerase chain reaction in situ hybridization for neo showed infiltration of transduced CTLs in GVHD-affected organs. These results show that ex vivo expanded, transduced T cells maintained in vivo function and enhanced marrow engraftment.     

Influence of CD34(+) marrow cell dose on outcome of HLA-identical sibling allogeneic bone marrow transplants in patients with chronic myeloid leukaemia

In order to study the influence of bone marrow CD34(+) cell dose on the outcome of allogeneic bone marrow transplantation (BMT), we analysed the results of BMT from HLA-identical siblings donors in 50 patients with chronic myeloid leukaemia (CML). The median numbers of nucleated cells (NC) and CD34(+) cells infused were 2.18 x 10(8)/kg (0.05-4.14 x 10(8)/kg) and 3.12 x 10(6)/kg (0.35-8.52 x 10(6)/kg), respectively. All patients engrafted. In univariate analysis, there was no correlation between the number of CD34(+) cells infused and the time to neutrophil recovery (P = 0.17). The Kaplan-Meier estimate of grade II-IV acute graft-versus-host disease (GVHD) at day 100 was 53 +/- 14% and 2-year survival was 46 +/- 15%. A number of CD34(+) cells infused greater than the median was the main factor increasing survival (P = 0.0006) and decreasing 100 day transplant-related mortality (P = 0.009). Patient-, disease- and transplant-related characteristics were not statistically different among patients receiving more or less than the median number of CD34(+) cells. The rate of infectious deaths was significantly higher in patients receiving less than 3.12 x 10(6) CD34/kg (48% vs 16%, P = 0.01). In a multivariable analysis, two factors associated with increased risk of death were advanced disease status at transplant (HR: 2.5 (95% CI: 1.09-5.75), P = 0.03) and a lower number of marrow CD34(+) cells infused/kg (HR: 4.55 (95% CI: 1.87-10.90), P = 0.0008).     

T-cell--depleted allogeneic bone marrow transplantation followed by donor lymphocyte infusion in patients with multiple myeloma: induction of graft-versus-myeloma effect

Previous trials of allogeneic bone marrow transplantation (BMT) in patients with multiple myeloma (MM) have demonstrated high response rates but also high transplantation-related mortality (TRM) and high relapse rates. Exploitation of this strategy remains of interest because donor lymphocyte infusions (DLIs) can induce a potent graft-versus-myeloma (GVM) effect. CD6 T-cell--depleted allogeneic BMT was combined with prophylactic CD4(+) DLI administered 6 to 9 months after BMT in an effort to reduce TRM and to induce a GVM response after BMT. Twenty-four patients with matched sibling donors and chemotherapy-sensitive disease underwent BMT. CD6 T-cell depletion of donor bone marrow was the sole method of graft-versus-host disease (GVHD) prophylaxis. GVHD after BMT was minimal, 1 (4%) grade III and 4 (17%) grade II GVHD. Fourteen patients received DLI, 3 in complete response and 11 with persistent disease after BMT. Significant GVM responses were noted after DLI in 10 patients with persistent disease, resulting in 6 complete responses and 4 partial responses. After DLI, 50% of patients developed acute (> or = II) or extensive chronic GVHD. Two-year estimated overall survival and current progression-free survival (PFS) for all 24 patients is 55% and 42%, respectively. The 14 patients receiving DLI had an improved 2-year current PFS (65%) when compared with a historical cohort of MM patients who underwent CD6-depleted BMT survived 6 months with no GVHD and did not receive DLI (41%) (P =.13). Although this study suggests that prophylactic DLI induces significant GVM responses after allogeneic BMT, only 58% of patients were able to receive DLI despite T-cell--depleted BMT. Therefore, less toxic transplantation strategies are needed to allow a higher proportion of patients to receive DLI and the benefit from the GVM effect after transplantation. (Blood. 2001;98:934-939)