Malcolm K. Brenner

Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.

Reactivation of Epstein-Barr virus (EBV) after bone-marrow transplantation leads in many cases to lymphoproliferative disease that responds poorly to standard therapy and is usually fatal. To prevent or control this complication, we prepared EBV-specific cytotoxic T-lymphocyte (CTL) lines from donor leucocytes and infused them into ten allograft recipients. Three of the patients had shown signs of EBV reactivation, with or without overt lymphoproliferation, and the others received CTL infusions as prophylaxis. No patient developed any complication that could be attributed to the CTL infusions. In the three patients with EBV reactivation, EBV DNA concentrations (measured by semiquantitative polymerase chain reaction [PCR]), which had increased 1000-fold or more, returned to the control range within 3-4 weeks of immunotherapy. The most striking consequence was the resolution of immunoblastic lymphoma in a 17-year-old patient who received four CTL infusions (two 1 x 10(7)/m2 and two 5 x 10(7)/m2). Because the CTL had been genetically marked before infusion, we were able to show by PCR analysis that they persisted for 10 weeks after administration. EBV-specific donor-type T-cell lines seem to offer safe and effective therapy for control of EBV-associated lymphoproliferation.

Gene-marking to trace origin of relapse after autologous bone-marrow transplantation

Bone marrow harvested for autologous bone-marrow transplantation may contain residual malignant cells even when it is judged to be in remission. Genetic marking and subsequent detection of these cells in recipients would give useful information about the origin of relapse after transplantation. We transferred the neomycin-resistance gene into bone-marrow cells harvested from children with acute myeloid leukaemia in remission. Two patients have relapsed since reinfusion of the marked cells. In both, the resurgent blast cells contained the neomycin-resistance gene marker; thus, remission marrow can contribute to disease recurrence. This method of tracking malignant cells should enable the development of better marrow purging strategies.

Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients

The contribution of infused bone marrow cells to long-term haemopoietic recovery in patients undergoing autologous bone marrow transplantation is unknown. Such information would help to clarify the role of this procedure in cancer therapy and would aid in the development of strategies to reduce the risk of subsequent aplasia. By transferring a neomycin resistance marker gene into the marrow cells of 20 patients before transplantation, we were able to trace the pattern of haemopoietic reconstitution postinfusion. The marker gene was present and expressed in all haemopoietic lineages in vivo in 15 of 18 evaluable patients at 1 month post-transplantation, in 8 of 9 patients at 6 months, and in 5 of 5 at 1 year. The marker has remained detectable for up to 18 months--the duration of our study. Our findings indicate that harvested bone marrow consistently contributes to long-term multilineage recovery of haemopoiesis after autologous marrow transplantation in cancer patients. These results provide a rationale for the continued exploration of more ablative preparative regimens with single or sequential autologous marrow transplants.

Early identification of Epstein-Barr virus-associated post-transplantation lymphoproliferative disease

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a common, usually fatal, complication developing after transplantation of bone marrow from HLA-mismatched or HLA-matched unrelated donors. Prompted by recent reports of successful treatment of EBV-LPD, we investigated methods which could result in early identification of patients at high risk for this disorder, thus improving the likelihood of successful therapeutic interventions. Both the outgrowth of transformed B lymphocytes ex vivo (100% correlation) and the detection of EBV DNA by a PCR method (80% correlation) showed statistically significant association with the histopathological diagnosis of EBV-LPD. Because these abnormalities can be detected prior to the onset of clinical disease. It should now be possible to use a combination of the methods described here to identify patients at high risk of developing EBV-LPD, thus enabling early therapeutic intervention.

Outcomes of transplantation with matched-sibling and unrelated-donor bone marrow in children with leukaemia

BACKGROUNDnFor most conditions amenable to bone-marrow transplantation, grafts from HLA-matched but unrelated donors have yielded poorer results than those obtained from matched-sibling donors. We assessed this pattern in the light of improvements in donor selection and post-transplant supportive care.nnnMETHODSnWe reviewed transplant outcome in 103 consecutive patients with childhood leukaemia who underwent allogeneic bone-marrow transplantation with HLA-matched sibling marrow (n = 52) or matched unrelated donor marrow (n = 51) between May, 1990, and March, 1996, at St Jude Childrens Research Hospital.nnnFINDINGSnAnalysis of engraftment, frequency of procedure-related complications, and disease-free survival revealed no advantage from use of matched-sibling marrow. The 2-year disease-free survival estimate for standard-risk recipients of matched-sibling marrow was 81 [8.1]% compared with 73 [12.1]% in the unrelated donor marrow group (p = 0.77). In the high-risk category, patients with a matched-sibling donor had a 2-year disease-free survival of 31 [11.6]%, compared with 32 [15.1]% among recipients of matched unrelated donor marrow (p = 0.87).nnnINTERPRETATIONnWe believe this improved result with unrelated donor marrow is a consequence of recent innovations in histocompatibility matching, prevention of graft-versus-host disease (GvHD), and antiviral prophylaxis. We suggest that such grafts can now be used in patients at both standard and high risk without compromising treatment outcome.

Graft rejection following HLA matched T-lymphocyte depleted bone marrow transplantation

Summary. Bone marrow graft rejection following HLA‐matched bone marrow transplantation (BMT) for leukaemia has been a rare problem. However, with the introduction of T‐lymphocyte depleted BMT, graft rejection is recognized as a new complication. At the Royal Free Hospital (RFH) in London T‐depletion is achieved using two monoclonal antibodies with complement mediated lysis. The methodology was extended to other centres and in total 56 patients have received T‐depleted, HLA matched BMT. Twelve of 56 patients have had graft rejection. At the RFH three of 41 (7%) patients have had rejection whereas at collaborating centres nine of 15 (60%) patients have had rejection. We have investigated these rejections in order to identify factor(s) responsible. Rejection was not restricted by patient or donor characteristics, nor disease status. Patient management, chemotherapy conditioning, efficiency of T‐depletion, graft versus host disease (GvHD), and infection post BMT, were not consistently implicated. The major difference between the RFH and all other centres was in the radiotherapy (RT) conditioning: The RFH prescribed a single fraction of 7‐5 Gy total body irradiation (TBI) whilst collaborating centres gave 10 or 12 Gy fractionated TBI. We conclude that the different incidence of rejection (7% v. 60%) relates primarily to the RT conditioning although the mechanisms(s) of rejection remain unknown. We conclude that where T‐depleted BMT is used, compensation by more intensive RT conditioning is required in order to avert graft rejection.

TRANSFER OF A FUNCTIONING HUMORAL IMMUNE SYSTEM IN TRANSPLANTATION OF T-LYMPHOCYTE-DEPLETED BONE MARROW

To test the effect of transplantation of T-cell-depleted bone marrow on recipient immune function the results of pre-transplantation immunisation with tetanus toxoid and hepatitis-B vaccine were studied in 38 donor-recipient pairs. Immunisation of the donor alone resulted in transfer of an antibody response to the recipient; immunisation of both donor and recipient resulted in potentiation of the antibody response in both magnitude and duration. These findings indicate that donor T-cell-depleted marrow can transfer humoral immunity to the recipient and that appropriate pre-transplant immunisation schedules may be of benefit to the recipient.

IMMUNE DONORS CAN PROTECT MARROW-TRANSPLANT RECIPIENTS FROM SEVERE CYTOMEGALOVIRUS INFECTIONS

To study the importance of transferred immunity against cytomegalovirus (CMV) in allogeneic, HLA-matched, T-cell-depleted bone-marrow transplantation, the incidence, severity, and outcome of CMV infections were studied in 40 CMV-seropositive recipients in relation to the donors immunity against CMV. There was no significant difference in the incidence of CMV infections between recipients of seropositive (n = 27) and seronegative (n = 13) marrow. However, the incidence of CMV pneumonitis (8/13 compared with 4/27; p less than 0.001) and the mortality attributable to CMV infection (6/13 compared with 1/27, p less than 0.01) were significantly greater in the group with seronegative donors than in those with seropositive donors. Multivariate regression analysis showed that recipients of seronegative marrow had a fifteen-fold greater risk of CMV pneumonitis and a fifty-fold increase in risk of a fatal CMV infection than recipients of seropositive marrow. Thus, after T-cell depletion CMV-seropositive marrow protects seropositive recipients against severe CMV infections; whenever possible, therefore, such recipients should be given marrow from seropositive donors. Ultimately, active immunisation of CMV-seronegative donors might help to protect seropositive recipients of T-cell-depleted marrow transplants against severe CMV infections.

Natural killer cell activity following T‐cell depleted allogeneic bone marrow transplantation

Summary. We have examined the recovery of natural killer (NK) cell function in seven recipients of MHC matched T cell depleted bone marrow allografts. NK cell activity against the erythroblastoid line K562 recovers 2–3 weeks after transplantation. Recipients also show a high level of killing of the T cell target HSB2 and of EBV transformed lymphoblastoid cell lines (B‐LCL). This activity peaks at 4–6 weeks and declines towards normal by 12–14 weeks after transplantation. Although killing of HSB2 and B‐LCL is usually the property of activated NK cells, few of these patients had any obvious ‘trigger’ of such activation: none had CMV infection, there were no episodes of graft rejection, and only two patients had mild and transient grade I graft versus host disease (GvHD). We conclude that T cell depletion does not affect the reconstitution of NK cell function and that NK cell activation occurs after T depleted bone marrow transplantation even in the absence of clinically detectable GvHD, graft rejection or CMV infection.

Autoimmune disease induced by dendritic cell immunization against leukemia

Induction of an optimal immune response will likely be a prerequisite for successful immunotherapy of human leukemias and other malignancies. Dendritic cells are highly effective at inducing an immune response to antigens to which the host is unresponsive, while transgenic expression of the costimulator molecule CD40 ligand (gp39/CD154) and the T cell growth factor interleukin 2 (IL2) are also able to augment immune responsiveness. We therefore investigated whether a combination of these two distinctive approaches to immunostimulation could safely increase the anti-tumor immune response compared to each stimulus alone. We injected BALB/CBYJ mice with syngeneic dendritic cells (DC) exposed to A20 lymphoblastic leukemia cell-derived peptides and proteins which had been acid-eluted from the cell surface. In additional mice, the pulsed DC were mixed with genetically modified syngeneic fibroblasts that were expressing CD40 ligand or secreting interleukin 2 (IL2). Three days after their third, weekly, vaccination, they were challenged with parental A20 cells. Tumor growth was suppressed by responses to pulsed DC alone (P < 0.02). This suppression was further enhanced when pulsed DC were coinjected with fibroblasts expressing CD40 ligand and IL2 (P < 0.0005 compared to DC alone) even though CD40 ligand and IL2-expressing fibroblasts alone offered no significant protection in this model. Mice receiving the full complement of immunostimulants either failed to develop visible tumors or developed small tumors which quickly necrosed and regressed, allowing the mice to become long term tumor-free survivors. Antibody mediated depletion of either CD4+ or CD8+ T-cell subset significantly reduced the level of protection afforded by the vaccination. However, it became evident that this intensive stimulation of the immune system lead not only to tumor eradication but also to destruction of cells bearing normal self antigens. Hence, 60 days after challenge with A20 cells all mice in the DC/IL2/CD40 ligand group developed a severe, systemic autoimmune disorder that resembled graft versus host disease and manifest itself by significant peripheral blood cytotoxicity against autologous fibroblasts, blood dyscrasias, gross hepatosplenomegaly, cachexia and fur loss. This phenomenon depended on CD8+ cytotoxic T lymphocytes. Our results therefore suggest that the most effective strategies of immunotherapy against leukemia may also exceed the threshold of anergic cells, leading to a loss of self tolerance to normal self-antigens and the induction of an CD8+ anti-self effector response.