Allogeneic hematopoietic cell transplantation after failed autologous transplant for lymphoma using TLI and anti-thymocyte globulin conditioning.

We describe 47 patients with lymphoma and failed prior autologous hematopoietic cell transplantation (HCT) who received TLI-ATG (anti-thymocyte globulin) conditioning followed by allogeneic HCT. Thirty-two patients had non-Hodgkin lymphoma (NHL; diffuse large B-cell lymphoma (n=19), T-cell NHL (n=6), mantle cell lymphoma (n=4) or other B-cell subtypes (n=3)), and 15 had Hodgkin lymphoma. The median follow-up was 4.9 (range, 2.1-11.9) years. The cumulative incidence of grade II-IV acute GvHD at day +100 was 12%, and the cumulative incidence of extensive chronic GvHD at 1 year was 36%. The 3-year cumulative incidences of overall survival (OS), PFS and non-relapse mortality (NRM) were 81%, 44% and 7%, respectively. Fifteen patients died (relapse, n=10; NRM, n=5). Among the 25 patients with relapse after allogeneic HCT, 11 (44%) achieved durable (>1 year) CRs following donor lymphocyte infusion or chemoradiotherapy. The majority of surviving patients (75%; n=24) were able to discontinue all immunosuppression. For patients with relapsed lymphoma after autologous HCT, allogeneic HCT using TLI-ATG conditioning is a well-tolerated, predominantly outpatient therapy with low NRM (7% at 3 years), a low incidence of GvHD, durable disease control and excellent OS (81% at 3 years).

Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation.

Thirty-eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34(+) hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti-thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34(+) cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long-term graft survival in all patients.

Requirement for interactions of natural killer T cells and myeloid-derived suppressor cells for transplantation tolerance.

The goal of the study was to elucidate the cellular and molecular mechanisms by which a clinically applicable immune tolerance regimen of combined bone marrow and heart transplants in mice results in mixed chimerism and graft acceptance. The conditioning regimen of lymphoid irradiation and anti-T cell antibodies changed the balance of cells in the lymphoid tissues to create a tolerogenic microenvironment favoring the increase of natural killer T (NKT) cells, CD4+ CD25+ regulatory T cells and Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs), over conventional T cells (Tcons). The depletion of MDSCs abrogated chimerism and tolerance, and add back of these purified cells was restorative. The conditioning regimen activated the MDSCs as judged by the increased expression of arginase-1, IL-4Rα and programmed death ligand 1, and the activated cells gained the capacity to suppress the proliferation of Tcons to alloantigens in the mixed leukocyte reaction. MDSC activation was dependent on the presence of host invariant NKT cells. The conditioning regimen polarized the host invariant NKT cells toward IL-4 secretion, and MDSC activation was dependent on IL-4. In conclusion, there was a requirement for MDSCs for chimerism and tolerance, and their suppressive function was dependent on their interactions with NKT cells and IL-4.

Tolerance and withdrawal of immunosuppressive drugs in patients given kidney and hematopoietic cell transplants.

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

The changed balance of regulatory and naive T cells promotes tolerance after TLI and anti-T-cell antibody conditioning.

The goal of the study was to determine how the changed balance of host naïve and regulatory T cells observed after conditioning with total lymphoid irradiation (TLI) and antithymocyte serum (ATS) promotes tolerance to combined organ and bone marrow transplants. Although previous studies showed that tolerance was dependent on host natural killer T (NKT) cells, this study shows that there is an additional dependence on host CD4(+)CD25(+) Treg cells. Depletion of the latter cells before conditioning resulted in rapid rejection of bone marrow and organ allografts. The balance of T-cell subsets changed after TLI and ATS with TLI favoring mainly NKT cells and ATS favoring mainly Treg cells. Combined modalities reduced the conventional naïve CD4(+) T cells 2800-fold. The host type Treg cells that persisted in the stable chimeras had the capacity to suppress alloreactivity to both donor and third party cells in the mixed leukocyte reaction. In conclusion, tolerance induction after conditioning in this model depends upon the ability of naturally occurring regulatory NKT and Treg cells to suppress the residual alloreactive T cells that are capable of rejecting grafts.

Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation.

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.

Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells.

We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.

Treatment of rheumatoid arthritis with total lymphoid irradiation: long-term survival.

OBJECTIVE: Total lymphoid irradiation (TLI) has been used to treat rheumatoid arthritis (RA) since the 1970s. This study reviews long-term (15-20-year) mortality outcomes of patients treated with TLI for RA at Stanford University Medical Center and compares these outcomes with those in patients treated with disease-modifying antirheumatic drugs (DMARDs). METHODS: Fifty-three patients with RA were treated with full-dose TLI at Stanford University Medical Center. All had failed previous therapy with gold salts and penicillamine. One hundred six control patients were selected from the Arthritis, Rheumatism, and Aging Medical Information Systems database and were matched with the patients for age, sex, disease duration, and mean Health Assessment Questionnaire (HAQ) score. Survival was analyzed using Kaplan-Meier methods and Cox proportional hazards regression. RESULTS: No significant difference in age and sex was found between TLI-treated patients and controls. TLI-treated patients had more education (mean 13.4 years versus 11.8 years; P = 0.016) and received more DMARDs prior to TLI (mean 2.1 versus 1.3; P = 0.0001). TLI-treated patients had lower mean HAQ scores at the time of TLI (2.0 versus 2.4; P = 0.0002). TLI had no significant overall effect on survival in treated patients compared with controls (P = 0.62). The survival curves appeared to cross over at approximately 11 years of followup, with better early survival in the TLI group and better late survival in the control group. There was a total of 25 deaths in the TLI group. There were 45 deaths in the control group, with causes of death available for 20 patients. There were 3 patients with lymphoma and 2 with myelodysplastic syndrome in the TLI group, and none in the control group. The most common cause of death in both groups was infection. CONCLUSION: TLI had no significant effect on overall survival, with trends toward higher early mortality in controls and trends toward higher late mortality in TLI-treated patients. Overall, there was no difference in mortality, but it appears that there may have been more lymphoproliferative malignancies in the TLI cohort. We would recommend that TLI be used cautiously for patients with refractory RA in whom the benefits outweigh the risks.

Rapid engraftment after allogeneic transplantation of density-enriched peripheral blood CD34+ cells in patients with advanced hematologic malignancies.

BACKGROUND: Acute graft versus host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Preclinical studies have suggested that a T-cell subset with a CD4-/CD8- double-negative (DN) T-cell phenotype is capable of suppressing GVHD. Double-negative T cells can be mobilized into the peripheral blood with granulocyte colony-stimulating factor (G-CSF) and enriched by density centrifugation. The current study was performed to study the feasibility and safety of applying a density gradient separation technique for enrichment of CD34+ and DN T cells, while depleting CD4+ and CD8+ single-positive (SP) T cells from peripheral blood progenitor cells (PBPCs) for the purpose of allogeneic transplantation. METHODS: Twenty-five patients with advanced hematologic malignancies were treated with a myeloablative preparative regimen consisting of fractionated total body irradiation, etoposide, and cyclophosphamide. Human leukocyte antigen identical donors were mobilized with G-CSF PBPC collected by apheresis. The apheresis product was applied to a single-step density gradient, and the low-density cell population was collected. The low-density cell population was infused as the sole source of allogeneic cells after myeloablative therapy. Graft versus host disease prophylaxis consisted of cyclosporine with or without prednisone. RESULTS: CD34 cell recovery was efficient with a median 72% yield, providing for a median CD34+ cell dose of 6.5 x 10(6)/kg (range,1.0- 13.9 x 10(6)/kg). CD3+CD4+ or CD3+CD8+ SP T cells were depleted by a median of 94.4% (range, 58.8- 99.2%), and the ratio of CD34+:SP T cells increased 10-fold. Double-negative T cells were depleted by 92% (range, 18.8- 99.4%), thus the ratio of DN:SP T cells increased less than 2-fold in 71% of apheresis samples tested. Hematopoietic engraftment was rapid, and there was no occurrence of graft failure in examinable patients. Median time to absolute neutrophil count greater than 0.5 x 10(9)/L and platelet count greater than 20 x 10(9)/L was 10.5 and 12 days, respectively. The incidence of Grade 2-4 acute GVHD was 26% (95% confidence interval [CI], 6-45%), although not all patients were examinable due to an unexpectedly high nonrecurrence mortality that at Day 180 was 62% (95% CI, 40-83%). CONCLUSIONS: These data suggest that T-cell subset manipulation via density gradient separation is a safe procedure and allowed rapid hematopoietic recovery. Selective enrichment of a donor DN T-cell subset was observed in only a few and was not associated with a reduced incidence of GVHD. However, the low-density selected cells still resulted in GVHD, and there was a high treatment-related mortality.

Clonable progenitors committed to the T lymphocyte lineage in the mouse bone marrow; use of an extrathymic pathway.

We searched for clonable committed T cell progenitors in the adult mouse bone marrow and isolated rare (approximately 0.05%) cells with the Thy-1hiCD2-CD16+CD44hiCD25-Lin- phenotype. In vivo experiments showed that these cells were progenitors committed only to reconstituting the T cell lineage of irradiated Ly5 congenic hosts. Reconstitution of the thymus was minimal compared with that of the bone marrow, spleen, and lymph nodes. At limiting dilutions, donor T cell reconstitution of the spleen frequently occurred without detectable donor cells in the thymus. Progenitors were capable of rapidly reconstituting athymic hosts. In conclusion, the clonable bone marrow progenitors were capable of T cell reconstitution predominantly by means of an extrathymic pathway.

Allogeneic bone marrow cells that facilitate complete chimerism and eliminate tumor cells express both CD8 and T-cell antigen receptor-alphabeta.

Nonmyeloablative host conditioning regimens have been used in clinical allogeneic bone marrow and hematopoietic progenitor transplantation to effectively treat lymphohematopoietic tumors and reduce early toxicity. However, severe graft-versus-host disease (GVHD) remains a major problem. The goal of the current study was to determine whether specific subsets of cells in allogeneic bone marrow transplants can effectively treat the BCL(1) B-cell lymphoma in nonmyeloablated BALB/c mouse hosts given a single dose of sublethal (450 cGy) total body irradiation, without inducing severe GVHD. The experimental results show that high doses of whole bone marrow cells from major histocompatiblity complex (MHC)-mismatched donors eliminate both normal and malignant host-type lymphohematopoietic cells without causing injury to nonlymphohematopoietic host tissues. The CD8(+)T-cell antigen receptor-alphabeta+ (TCRalphabeta+) T cells within the marrow transplants mediated the killing of the tumor cells via both perforin- and FasL-dependent pathways. Cells present in marrow transplants from either CD8-/- or TCRalpha-/- donors failed to eliminate malignant and normal host lymphohematopoietic cells. Addition of small numbers of blood mononuclear cells to the marrow inoculum caused lethal GVHD. Thus, the resident allogeneic bone marrow CD8(+) TCRalphabeta+ T cells had the unique capacity to eliminate the host lymphohematopoietic cells without nonlymphohematopoietic tissue injury. (Blood. 2001;97:3458-3465)

Tolerance, mixed chimerism and protection against graft-versus-host disease after total lymphoid irradiation.

Total lymphoid irradiation (TLI), originally developed as a non-myeloablative treatment for Hodgkin's disease, has been adapted for the induction of immune tolerance to organ allografts in rodents, dogs and non-human primates. Moreover, pretransplantation TLI has been used in prospective studies to demonstrate the feasibility of the induction of tolerance to cadaveric kidney allografts in humans. Two types of tolerance, chimeric and non-chimeric, develop after TLI treatment of hosts depending on whether donor bone marrow cells are transplanted along with the organ allograft. An advantageous feature of TLI for combined marrow and organ transplantation is the protection against graft-versus-host disease (GVHD) and facilitation of chimerism afforded by the predominance of CD4+ NK1.1(+) -like T cells in the irradiated host lymphoid tissues. Recently, a completely post-transplantation TLI regimen has been developed resulting in stable mixed chimerism and tolerance that is enhanced by a brief course of cyclosporine. The post-transplantation protocol is suitable for clinical cadaveric kidney transplantation. This review summarizes the evolution of TLI protocols for eventual application to human clinical transplantation and discusses the mechanisms involved in the induction of mixed chimerism and protection from GVHD.

Favorable treatment outcome in non-Hodgkin's lymphoma patients with "poor" mobilization of peripheral blood progenitor cells.

Our purpose was to evaluate the outcome and costs of high-dose chemotherapy and autologous peripheral blood progenitor cell (PBPC) transplantation in patients with the inability to mobilize sufficient numbers of PBPCs to allow rapid engraftment after PBPC transplantation. We treated 172 consecutive non-Hodgkin's lymphoma (NHL) patients with cyclophosphamide and granulocyte colony-stimulating factor followed by apheresis to collect PBPCs. The cells were separated on a Percoll gradient and purged with monoclonal antibodies and complement. The patients were categorized as "good" mobilizers if a collection of > or =2 x 10(6) CD34+ cells/kg was obtained (n = 138, 80%) or "poor" mobilizers if <2 x 10(6) CD34+ cells/kg were obtained (n = 34, 20%). With a median follow-up of 3.5 years, there is no statistically significant difference in actuarial event-free survival, overall survival, or relapse for good mobilizers compared with poor mobilizers. However, there was a trend toward increasing nonrelapse, transplantation-related mortality of 11.8% for poor mobilizers versus 3.6% for good mobilizers (P = .08) and early death from all causes including relapse within 120 days (poor 20.6% versus good 8.7%, P = .06). The total cost for bone marrow transplantation-related care was significantly higher, at $140,264 for poor mobilizers versus $80,833 for good mobilizers (P = .0001). The population of patients with NHL who mobilize PBPCs poorly into the circulation have a higher cost for posttransplant support. However, there is no significant difference in relapse, event-free survival, or overall survival for such patients compared with those who mobilize PBPCs easily.

Natural killer 1.1(+) T cells and "natural suppressor" T cells in the bone marrow.

Unusual cells in the bone marrow of mice and/or humans suppress immune responses and inhibit the mixed leukocyte reaction, graft-versus-host disease, and systemic autoimmunity. Previous studies showed that these "natural suppressor" T cells expressed the CD4(-)CD8(-) T-cell receptor-alphabeta(+) phenotype. More recent studies demonstrate that the latter cells express the natural killer 1.1 (NK1.1) marker and are members of the NK1.1(+) T-cell family that secrete high levels of IFN-gamma and IL-4 after initial activation. The suppressive activity of the bone marrow NK1.1(+) T cells is dependent on their rapid secretion of high levels of IL-4. This unique cytokine secretion is not observed in conventional NK1. 1(-) T cells and can downregulate the function of the latter cells.

Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity.

BACKGROUND: Previous studies showed the feasibility of inducing transplantation tolerance to cadaveric renal allografts in patients given pretransplant total lymphoid irradiation (TLI). Microchimerism has been theorized to be an important or necessary factor in long-term graft acceptance and tolerance in humans. METHODS: A cadaveric renal transplant recipient given pretransplant total lymphoid irradiation and withdrawn from immunosuppressive drugs more than 12 years ago was tested for microchimerism using a sensitive nested polymerase chain reaction technique, and for anti-donor reactivity using the mixed leukocyte reaction and an ELISA screen for anti-HLA antibodies. Donor and recipient were mismatched for all HLA-A, B, and DR antigens. RESULTS: The "tolerant" recipient had good graft function, no detectable donor-type cells in the blood by polymerase chain reaction analysis, vigorous reactivity to donor stimulator cells in the mixed leukocyte reaction, and no detectable serum anti-HLA antibodies. CONCLUSIONS: Operational tolerance to HLA-A, B, and DR mismatched organ allografts can be induced prospectively in humans for at least 12 years after withdrawal of immunosuppressive drugs. The allograft can be maintained in the absence of detectable donor microchimerism and in the presence of anti-donor reactivity in the mixed leukocyte reaction, suggesting that neither chimerism nor clonal deletion or anergy of recipient T cells to alloantigens presented by donor Class II HLA molecules is required for persistence of the tolerant state using this total lymphoid irradiation protocol.

Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice.

Since anti-CD1 TCR transgenic T cells can activate syngeneic B cells via CD1 to secrete IgM and IgG and induce lupus in BALB/c mice, we studied the role of CD1 in the pathogenesis of lupus in NZB/NZW mice. Approximately 20% of B cells from the spleens of NZB/NZW mice expressed high levels of CD1 (CD1high B cells). The latter subset spontaneously produced large amounts of IgM anti-dsDNA Abs in vitro that was up to 25-fold higher than that of residual CD1int/low B cells. T cells in the NZB/NZW spleen proliferated vigorously to the CD1-transfected A20 B cell line, but not to the parent line. Treatment of NZB/NZW mice with anti-CD1 mAbs ameliorated the development of lupus. These results suggest that the CD1high B cells and their progeny are a major source of autoantibody production, and activation of B cells via CD1 may play an important role in the pathogenesis of lupus.

Cyclosporine facilitates chimeric and inhibits nonchimeric tolerance after posttransplant total lymphoid irradiation.

BACKGROUND: Previous studies showed that Lewis rats given posttransplant total lymphoid irradiation, antithymocyte globulin, and a single infusion of ACI peripheral blood or bone marrow cells develop tolerance to ACI heart allografts. METHODS: To determine the effects of cyclosporine on these tolerance induction protocols, groups of Lewis hosts, given either ACI blood or marrow infusions, were given a 60-day course of daily cyclosporine immediately after the cell infusion. RESULTS: Cyclosporine treatment was associated with uniform graft rejection in the groups given an ACI blood transfusion, and was associated with uniform graft acceptance in the groups given an ACI bone marrow infusion. Studies of donor-type T and B cell chimerism in the host blood showed that cyclosporine facilitated chimerism in the hosts given ACI bone marrow cells, and stable chimerism over a 300-day observation period was predicted by detectable chimerism by day 30. None of the hosts given ACI blood cells developed chimerism. CONCLUSION: Cyclosporine facilitated long-term graft acceptance in a tolerization protocol that induced mixed chimerism, but prevented long-term graft acceptance in a tolerization protocol that did not induce chimerism.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow.

We have identified a rare ( approximately 0.05-0.1%) population of cells (Thy-1(hi)CD16(+)CD44(hi)CD2(-)TCRalphabeta(-)B220(-)M ac-1(-)NK1. 1(-)) in the adult mouse bone marrow that generates CD4(+) and CD8(+) TCRalphabeta(+) T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2(-)CD16(+)CD44(hi)TCRalphabeta(-) to CD2(+)CD16(int/-)CD44(int/-)TCRalphabeta(-) cells, and a later transition to CD4(+)CD8(+)TCRalphabeta(+) double-positive T cells that rapidly generate the CD4(+) and CD8(+) single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2(+)TCRalphabeta(-) stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.