From leukocyte reduction to leukocyte transfusion: the immunological effects of transfused leukocytes

BaillieÁre's Clinical Haematology Vol. 13, No. 4, pp. 585±600, 2000

doi:10.1053/beha.2000.0101, available online at http://www.idealibrary.com on

6 From leukocyte reduction to leukocyte transfusion: the immunological e€ects of transfused leukocytes* Jong-Hoon Lee{

MD

Chief Blood and Plasma Branch, Division of Blood Applications, Oce of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland, USA

Harvey G. Klein

MD

Chief Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA

In transfusion medicine, mononuclear leukocytes have been studied more often as contaminants of red blood cells or platelets responsible for adverse transfusion outcomes than as therapeutic cells; leukocyte transfusion has been e€ective in augmenting recipient immunity only in limited clinical situations. Studies in leukocyte reduction and leukocyte transfusion have progressed separately as if the leukocytes' adverse and therapeutic e€ects result from di€erent immunological mechanisms. With growing clinical experience, however, it is increasingly clear that some adverse immune e€ects may be exploited for therapeutic bene®t. Advances in clinical immunology, understanding of the variety of cells and functions in the leukocyte fraction of blood, and blood component preparation technology may lead to new ways of deriving immunological bene®t from transfused blood leukocytes while minimizing their adverse e€ects. This chapter reviews the current uses of leukocyte reduction and mononuclear leukocyte transfusion, with an emphasis on the relationship between transfusionassociated graft-versus-host disease and donor lymphocyte infusion in controlling relapsed leukaemias. Key words: leukocyte reduction; leukocyte transfusion; immunomodulation; transfusionassociated graft-versus-host disease; donor lymphocyte infusion; graft-versus-leukaemia e€ect.

The safety of blood transfusion has been a major concern for the transfusion community for the past three decades. In comparison, the ecacy of blood transfusion has received relatively little attention. When blood components are collected and transfused according to established standards1±3, there has been little reason to question the *The views of the authors represent scienti®c opinion and should not be construed as opinion or policy of the United States Food and Drug Administration or of the National Institutes of Health. {Address for correspondence: Food and Drug Administration, HFM-375, 1401 Rockville Pike, Rockville, MD 20852-1448, USA. 1521±6926/00/040585‡16 $12.00/00

c 2000 Harcourt Publishers Ltd. *

586 J.-H. Lee and H. G. Klein

role of red blood cells in oxygen delivery, or the role of platelets, plasma, and cryoprecipitate in e€ecting haemostasis in selected patients with bleeding diathesis. With the recent advent of recombinant cytokines, granulocyte transfusion to treat bacterial or fungal infection is undergoing intensive study. However, the role of leukocytes in immune modulation has not been well accepted and continues to receive relatively little attention. Although leukocytes have often been the subject of clinical investigation as contaminants of red blood cells and platelets responsible for a variety of adverse transfusion e€ects, the current indications for reducing the leukocyte content of blood components do not include the avoidance of recipient immunosuppression. As a therapy, leukocyte transfusion has been clinically e€ective in augmenting recipient immunity only in limited clinical situations. With growing understanding of the variety of cells and functions contained in the leukocyte fraction of blood, it is increasingly clear that leukocytes' immune e€ects may be exploited for therapeutic bene®t. This chapter discusses the clinical uses of leukocyte reduction and mononuclear leukocyte transfusion, and explores how the opposing clinical uses may be mechanistically related. LEUKOCYTE REDUCTION The growing list of adverse transfusion outcomes attributed to leukocytes in blood components has stimulated a recent international trend towards pre-storage universal leukocyte reduction (ULR). `Leukocyte reduction' is de®ned typically as a bloodprocessing step for reducing the leukocyte content of whole blood, red blood cell, or platelet units to 5  106 (1  106 in Europe) residual cells, or fewer, per unit, and ULR as the routine application of the blood-processing step to all units of whole blood, red blood cells, and platelets prior to storage. (The di€erence between the United States and the European leukocyte reduction standards is more apparent than real; when one considers the actual fraction of blood units that meets the respective standard under direct quality control testing, the two standards specify equivalent blood safety criteria.) Canada, the United Kingdom, Ireland, France, Germany and Portugal have either implemented or are in the process of implementing ULR, and several other countries, including the United States, are considering implementation. The reasons for adopting ULR as a blood policy vary among the national blood authorities; as might be expected, the degree of public concern has been as important as the availability of an adequate scienti®c basis. Selected indications for which leukocyte reduction may be clinically considered are further discussed below (Table 1). Febrile transfusion reactions A febrile, non-haemolytic transfusion reaction (FNHTR) is de®ned as a self-limited rise in patient temperature of 18C or more in temporal association with transfusion and in the absence of other recognizable causes for the fever.4 Its frequency depends on the type of blood component used: 1% with red blood cells or whole blood5, and up to 30% with platelets.6 The 30-fold disparity in the frequency of FNHTR may re¯ect unrecognized extrinsic causes (e.g. occult bacterial contamination of room-temperature stored component) as well as causes intrinsic to blood component type. FNHTR may be eliminated by using blood components that have been leukocyte-reduced prior to storage. The immunological mechanisms proposed to date implicate donor leukocytes and their elaboration of in¯ammatory cytokines, including the interleukins

E€ects of transfused leukocytes 587 Table 1. Leukocyte reduction in the prevention of adverse transfusion e€ects.a Adverse e€ects of transfused leukocytes as indications for leukocyte reduction Regarded as e€ective Febrile reaction Alloimmunization Leukotropic virus transmission Regarded as ine€ective TA-GvHD Controversial Transfusion-related immunosuppression Latent virus reactivation Transfusion-related acute lung injury a

Adverse e€ects of the transfused leukocytes may have therapeutic roles. Mechanistically, transfusionassociated graft-versus-host disease (TA-GvHD) appears to be related to the graft-versus-leukaemia e€ect of donor lymphocyte infusion therapy, as does transfusion-related immunosuppression to the induction of graft tolerance by blood (leukocyte) transfusion. Not all controversial indications for leukocyte reduction are listed here.

(IL-1, IL-6 and IL-8) and tumour necrosis factor7, which, in turn, mediate fever by upregulating prostaglandin synthesis at the hypothalamus.8 Although not lifethreatening, FNHTR may cause signi®cant patient morbidity and complicate patient management. Alloimmunization against platelet transfusion Three antigen systems are important to the immunological compatibility of platelets: class I human leukocyte antigens (HLA), human platelet antigens (HPA), and ABO blood group antigens.9 Of these, HPA incompatibility occurs rarely in transfusion practice and ABO incompatibility is typically well-tolerated by the transfusion recipient, leaving HLA incompatibility as the major challenge in overcoming the immune refractory state to platelet transfusion. Once broadly sensitized to multiple HLA antigens, the thrombocytopenic patient typically does not bene®t from a posttransfusion platelet increment, a situation in as many as two-thirds of chronic transfusion recipients.10 A reduction in the number of contaminant leukocytes expressing both class I and class II HLA antigens decreases the likelihood of recipient HLA allosensitization, presumably because the presentation of both class I and class II antigens is necessary for immune recognition.9 The consistent use of leukocytereduced blood components may eliminate recipient HLA sensitization, but the leukoreduction threshold to achieve this aim has not yet been determined. Cytomegalovirus transmission When transmitted to the immunocompromised transfusion recipient, cytomegalovirus (CMV) may cause signi®cant morbidity and mortality (gastroenteritis, retinitis,

588 J.-H. Lee and H. G. Klein

pneumonitis). The exclusive use of CMV seronegative blood may decrease the chance of transmitting CMV by as much as a tenfold, down to 2±4% from 20±40% for CMVuntested units.11 CMV transmission may be prevented also by using leukocyte-reduced blood because CMV resides on or within leukocytes. Although leukocyte reduction has not been demonstrated conclusively to be equivalent to CMV seronegativity, the use of leukocyte-reduced units is an e€ective alternative when seronegative units are not readily available. Immunosuppression Experimental data suggest that allogeneic blood transfusion may induce clinical immunosuppression, which, in turn, may lead to an increased susceptibility of the transfusion recipient to tumour recurrence, bacterial infection, or re-activation of latent viruses.12±16 When injected with ®brosarcoma cells, mice and rabbits that received allogenic blood became more susceptible to pulmonary metastases than did control animals given syngeneic blood. The apparent tumorigenic e€ect may be leukocyte-mediated: the use of leukocyte-reduced allogeneic blood appears to abrogate the e€ect, but only when blood leukocyte content was reduced prior to blood storage. The immune mechanisms responsible for the immunosuppressive e€ect have not been clearly elucidated but may involve disruption of cytokine balance, clonal deletion of T cells, activation of immune regulatory cells, or the generation of soluble factors.12 Recently, Ghio et al13 have suggested that soluble HLA class I and Fas ligand molecules may be released from leukocytes during blood storage to mediate clinically signi®cant immunosuppression in the transfusion recipient. These observations collectively suggest that donor±recipient antigenic disparity impairs recipient immune function, but attempts to ®nd clinical outcome correlates have been inconclusive14±16 and a demonstration of this important e€ect may be possible only through conducting a prospective randomized clinical trial. Transfusion-associated graft-versus-host disease Leukocyte engraftment and the obliteration of recipient immunity may be considered as an extreme manifestation of the immunosuppressive e€ect of blood transfusion. Allogeneic lymphocytes retain the ability to engraft and proliferate after transfusion if they are not removed by the recipient's cytotoxic T cells, typically within 2 days after transfusion.17 On rare occasions, recipient immunity may allow sucient donor lymphocyte engraftment to permit transfusion-associated graft-versus-host disease (TA-GvHD), a condition that is typically lethal within 1 month of clinical recognition.18 Clinical situations that predispose a transfusion recipient to TA-GvHD include: (1) haematopoietic transplantation, (2) haematological malignancies or solid cancers, (3) T cell de®ciency or dysfunction, (4) intrauterine or neonatal transfusion, (5) use of blood from blood relatives, and (6) use of blood from unrelated donors when HLAmismatched only in the direction of donor to recipient (homozygous donor for an extended HLA haplotype and heterozygous recipient for the HLA haplotype). The probability of HLA-mismatch only in the donor-to-recipient direction has been estimated to be as high as one in 20 000 random allogeneic transfusions19, but clinically recognizable TA-GvHD occurs much less frequently. Over the last decade, only about seven fatal TA-GvHD cases have been reported to the United States Food and Drug Administration per year.

E€ects of transfused leukocytes 589

Ta-GvHD di€ers most notably from its counterpart following allogeneic haematopoietic transplantation (AHT) in that the engrafting lymphocytes originate from allogeneic blood rather than from the successful haematopoietic graft: the lymphocytes' immune attack against the host includes host haematopoiesis to result in lethal pancytopenia, in addition to the expected dermatological, pulmonary, hepatic and gastrointestinal involvement.20 The de®nitive method for preventing TA-GvHD is irradiation of cellular blood components to inactivate donor lymphocytes; leukocyte reduction is not e€ective, and TA-GvHD occurs with currently achievable levels of residual leukocytes. The criteria for using irradiated blood typically include the clinical situations listed above, but may di€er from centre to centre. Little is known about the fate of transfused leukocytes in the typical transfusion recipient. Using quantitative allele-speci®c polymerase chain reaction (PCR) technique, Lee et al21 recently studied the survival kinetics of transfused leukocytes in 10 trauma patients who received multiple units of fresh (less than 2-week storage) red blood cells derived from multiple allogeneic donors. None of the units were irradiated or leukocyte-reduced. In seven of the 10 patients, multilineage (CD4, CD8, CD15, CD19) cell proliferation resulted in peripheral blood concentrations of 10 to 100 cells per microlitre, and donor cells persisted in the recipient's circulation for up to 1.5 years. Cell proliferation typically included the CD15- and CD19-positive committed haematopoietic progenitor cells. Up to 5% of circulating leukocytes were donorderived in some patients. None of the patients showed evidence of TA-GvHD. The results of this transfusion study are consistent with recent observations about maternal-fetal microchimerism in humans.22,23 Both fetal and maternal leukocytes may cross the placenta to engraft and persist in the respective opposite circulation for decades following pregnancy. The initial observation in patients with immune de®ciency disorders has been con®rmed in immune competent subjects.23 The long-term, bidirectional mother±child microchimerism is intriguing but perhaps not unexpected because pregnancy is a state of mutual fetal-maternal allogeneic tolerance. That transfusion-induced microchimerism may also persist long term is more surprising, and its recent demonstration suggests that the immunosuppressive e€ects of blood transfusion are mechanistically related to TA-GvHD. Understanding the immunological basis for the apparent mutual tolerance between lymphocytes of allogeneic pairs remains as a major challenge, and perhaps the key, to unravelling the immunotherapeutic potential of leukocyte transfusion. LEUKOCYTE TRANSFUSION While advances in blood ®ltration technology have enabled increasingly more complete removal of unwanted leukocytes from whole blood, red blood cells, or platelets, parallel advances in blood collection technology have allowed the generation of increasingly purer leukocyte preparations in greater doses. In particular, the availability of recombinant cytokine growth factors and the re®nement of automated blood cell separators have facilitated the exploration of the leukocytes' immunotherapeutic potential to control infections, augment anti-tumour immunity, prevent graft rejection, and to correct abnormal immune regulation. Evidence is growing that the adverse immune e€ects do have bene®cial counterparts, and that these adverse e€ects may indeed be exploited as immunotherapy. Recent success of donor lymphocyte infusions (DLI) serves as compelling evidence. The therapeutic potential of DLI for controlling relapsed chronic myelogenous leukemia (CML), arguably one of the most important clinical

590 J.-H. Lee and H. G. Klein

advances in haematopoietic transplantation, appears to result from an immune mechanism akin to the potentially lethal transfusion complication, TA-GvHD. Donor lymphocyte infusion and graft-versus-leukaemia The observation that host haematopoiesis is a potential target of lymphocyte immune attack in TA-GvHD supports the concept that donor-derived lymphocytes (DDL) may be used to attack the host's leukaemic haematopoiesis when AHT has been performed to treat leukaemia. The in vitro and animal data suggestive of the graft-versusleukaemia e€ect (GvL)24 have been consistent with clinical observations: post-AHT graft-versus-host disease (GvHD) appears to protect against leukaemic relapse25±28, and attempts to control GvHD by reducing the T cell content of the haematopoietic graft result in increased rates of leukaemic relapse.29±31 In 1990 Kolb et al32 provided direct clinical evidence for GvL: DDL administered in conjunction with alpha-interferon (IFN-a) induced cytogenetic remission in three patients with CML in relapse following AHT. This initial clinical demonstration of GvL has been since repeatedly con®rmed, both in Europe (European Group for Blood and Marrow Transplantation: 168 patients, 111 evaluable with CML)33,34, and in North America (140 patients, 55 evaluable with CML)35 at over 50 transplant centres (Table 2). The studies collectively show that, in relapsed chronic-phase CML post-AHT, DLI induce durable clinical remission at approximately 75% response rate. The time to remission ranges from 1 to 9 months with a mean of 3 months, and the probability of remaining in remission at 3 years approaches 90%.33±36 Further, limited experience in 33 DLI-responsive CML patients suggests that: (1) clinical remission is typically not seen within the ®rst month after DLI, (2) the 30% probability of achieving clinical remission at 2 months increases to 90% by 5 months, and (3) nearly all responses are seen within 8 months.35,37 A lower level of disease activity at DLI intervention appears to predict a more favourable outcome: response rates for patients receiving DLI in early relapse (cytogenetic or molecular) may approach 100%.33±38 The response rate, however, may not predict response quality or durability. Molecular remission (inability to detect bcrabl mRNA by PCR) has been achieved in nearly all patients entering clinical remission, including those with haematological relapse. The host circulation, often chimeric during chronic-phase relapse, typically converts to contain cells only of donor origin with the gradual disappearance of the Philadelphia chromosome. Lymphocytes appear to respond before other cell lines, with granulocytes converting last39; in relapse, Table 2. Clinical response rates of leukaemias in relapse following AHT to DLI as adoptive immunotherapy.a,b Relapsed leukaemias North American experience European experience Combined experience CML-cp CML-ap AML ALL a

76% 28% 15% 18%

(37) (18) (39) (11)

74% 7% 24% 5%

(97) (14) (37) (20)

75% 19% 20% 10%

(134) (32) (76) (31)

AHT ˆ allogeneic hoematopoietic transplantation; DLI ˆ donor lymphocyte infusion; CML-cp ˆ chronic myelogenous leukaemia in chronic phase relapse; CML-ap ˆ chronic myelogenous leukaemia in accelerated phase relapse; AML ˆ acute myelogenous leukaemia; ALL ˆ acute lymphocytic leukaemia. b The number of patients assessed is given in parenthesis following the corresponding clinical response rate. Haematopoietic grafts and DDL components were harvested typically from an HLA-identical sibling.

E€ects of transfused leukocytes 591

lymphopoiesis appears to lag behind erythropoiesis or granulopoiesis.37 The lineagespeci®c kinetics appear to re¯ect overall cell numbers and cell turnover rather than lineage-speci®c immunological mechanisms. Despite the response rate approaching 100% in early disease, early therapy may not improve overall clinical outcome: (1) the natural history of early disease has not been well de®ned and early relapse may not always progress to haematological relapse, (2) DLI therapy is associated with substantial GvHD, (3) a positive response in haematological relapse appears comparable in quality to that in early relapse. Future studies may identify patient subsets that are una€ected by DLI intervention, with either favourable or poor clinical prognosis. Donor lymphocyte infusion and graft-versus-host disease In the setting of DLI, there is no distinction between GvHD and TA-GvHD because the donor lymphocytes are autologous to the recipient's reconstituted marrow. DLIassociated GvHD has been observed in up to 80% of patients. Organ involvement may be milder than expected (particularly skin), possibly because DLI is typically not preceded by a toxic conditioning regimen.33±38 Myelosuppression seen in up to 50% of patients appears to result from an insucient reserve of donor-derived haematopoiTable 3. A comparison of graft-versus-host disease associated with allogeneic haematopoietic transplantation, blood transfusion, and donor lymphocyte infusion.a,b GvHD

AHT

Transfusion

DLI

Occurrence Time frame Pancytopenia Mortality

Frequent (70%) 5±10 weeks Rare 10±20%

Rare 0±5 weeks Typical 80±100%

Frequent (580%) 44 weeks Infrequent 20±30%

a

GvHD ˆ graft-versus-host disease; AHT ˆ allogeneic haematopoietic transplantation; DLI ˆ donor lymphocyte infusion. b The clinical features of DLI-GvHD, similar to AHT-GvHD and distinct from transfusion-GvHD, re¯ect that the transfusion of donor-derived lymphocytes is an extension of transplantation which reinforces the haematopoietic graft. In transfusion-GvHD, the lymphocytes present in allogeneic blood as cellular contaminants mount an immune attack against the recipient, including the recipient's bone marrow.

esis, and may be reversed by infusing donor-derived haematopoietic progenitor cells.40,41 GvHD has been responsible for much of the 25% mortality rate seen in patients receiving DLI therapy (Table 3).33±36 Can GvL be separated from GvHD? DLI-associated GvHD is expected to overlap with GvL. Attempts to separate GvL from GvHD have included the depletion of T cells or T cell subsets from DDL42±45, as well as the expansion of CD446 or NK47,48 cells using cytokine growth factors, either in vitro or in the recipient. The antileukaemic e€ect of IFN-a appears not to signi®cantly augment GvL35, but IL-2 may prove important despite its toxicity.47,48 Low-dose continuous infusion of IL-2 in conjunction with T cell-depleted DLI (with or without prior in vitro IL-2 activation) has been e€ective in reducing GvHD while presumably fully retaining GvL.49±50 IL-2 has been studied extensively in the adoptive immunotherapy of selected solid cancers51±53; its role in the therapy of haematological malignancies in conjunction with DLI is receiving increasing investigative attention.

592 J.-H. Lee and H. G. Klein

There is no conclusive evidence to date, however, that GvHD is separable from GvL; the two opposing immune e€ects appear to result from a common mechanism, and a reduction in GvHD without compromising GvL may be dicult. Cell selection on the basis of antigen speci®city may prove more successful than T cell subset selection or cytokine manipulation. Falkenburg et al54 recently reported the ®rst successful treatment of a patient in accelerated CML relapse, who failed earlier DLI therapy, using in vitro expanded leukaemia-speci®c lymphocytes. Presumably, the in vitro cell selection for cytotoxicity and inhibitory activity against CML cells followed by culture restored CML antigen-presenting activity critical for GvL, typically already present in vivo in chronic-phase CML but lacking in accelerated-phase CML.55±57 The speci®c antigens have not been identi®ed and may turn out to be minor HLA antigens with tissue distribution restricted largely to blood cells.58±61 In this context, the separation of GvL and GvHD may be thought of as the targeting of GvHD to haematopoietic cells, a therapeutic concept in leukocyte transfusion therapy which parallels the current understanding of TA-GvHD. Di€erential antigen expression among cells of the haematopoietic system58±65 has been studied recently in an e€ort to understand the di€erences in the antileukaemic e€ect of DLI. These laboratory and clinical observations provide some early insight as to how DLI may be made more e€ective in the future.

Epstein±Barr virus lymphoproliferative disorders The experience with Epstein±Barr virus lymphoproliferative disorders (EBV-LPD) occurring after marrow transplantation illustrates the potential to separate GvL from GvHD clinically, even if the two phenomena result from inseparable pathogenetic mechanisms. EBV-LPD complicate T cell-depleted allogeneic marrow transplantation at a rate of approximately 10%. The secondary lymphomas respond readily to DLI at a cell dose of up to one log lower than that typically used to treat the primary leukaemic disease. Sustained clinical remission with only mild associated GvHD has been consistently observed, often without additional maintenance therapy.66±68 Despite an incomplete understanding of the pathogenetic mechanisms underlying GvL and GvHD, Bonini et al69 have successfully separated the two clinical phenomena using gene-modi®ed DDL. In a patient with an aggressive EBV-induced B cell lymphoma of donor origin as a complication of T cell-depleted marrow transplantation, DLI (1.5  106 lymphocytes/kg) using cells transduced with thymidine kinase gene (fused with a marker gene) resulted in prompt remission of disease within 2 weeks. Acute GvHD occurring at 4 weeks responded dramatically to the intravenous administration of ganciclovir (2 doses of 10 mg/kg) and the number of circulating DDL also promptly decreased. Despite technical demands and substantial safety concerns, the use of manipulated DDL may allow control of cell function as clinically needed based on time courses associated with GvL and GvHD. The potential of gene-modi®ed DDL as a routine clinical tool against GvHD is being increasingly explored.69±73 As a tumour resulting from infection by a transforming virus, the insight into the mechanisms of anti-tumour activity in EBV-LPD may be applicable also to the adoptive immunotherapy of viral diseases, including CMV disease74, hepatitis B75, and HIV disease.76,77 Of these, the relatively frequent CMV disease may be of particular importance in marrow transplantation to treat leukaemias, against which DLI using CMVspeci®c cytotoxic T cells may provide reliable prophylaxis or therapy.

E€ects of transfused leukocytes 593

Cytomegalovirus disease Walter et al74 has successfully used in vitro-stimulated and culture-expanded donorderived CMV-speci®c cytotoxic T cells to adoptively facilitate the reconstitution of cellular immunity against CMV in 11 of 14 allogeneic marrow transplant patients. The DLI therapy consisted of four escalating cell doses (0.33, 1.0, 3.3 and 10.0  108 cells) administered at weekly intervals beginning at day 30 to 40 after transplantation. DLIassociated toxicity, CMV disease, and CMV viraemia were not observed. The study begins to suggest that in comparison with GvL, graft-versus-CMV e€ect may be more readily separable from GvHD. Donor-derived lymphocytes as a blood component The lymphocyte dose appears not to be critical for GvL. Nucleated cell doses that range from 0.34 to 12.3  108 cells/kg have been equally e€ective32±35, and an ecacy threshold has not been clearly de®ned. The cell dose is likely to be not as important as the immune interactions in the recipient that allow successful proliferation to a cell concentration e€ective against the existing leukaemic burden. HLA-compatibility, lymphocyte subset composition and balance, and the appropriate cytokine milieu may be more critical than the overall cell dose. Nevertheless, a total dose of 5 (2 to 8)  108 mononuclear cells/kg are typically collected from the original HLA-matched sibling using an automated blood separator in generating 3 DDL units as immunotherapeutic blood components in as many collection procedures over 1 week. Between 1 and 2  108 mononuclear cells/kg containing 50±60% T cells are collected as a dose unit at each 3-hour leukapheresis procedure that resembles a standard plateletpheresis donation (Table 4). Few adverse donor e€ects have been associated with lymphocytapheresis beyond mild citrate toxicity in about a third of the donors. The design of DDL collection schedule and the choice of instrumentation have been guided by practical Table 4. Lymphocyte content as cellular contaminants of major blood components relative to the typical dose in donor-derived lymphocytes.a,b Blood component

ML/unit

LLC

PLT-LR RBC-LR PLT RBC Whole blood Granulocytes DDL

55  106 55  106 1±3  108 1±3  109 1±3  109 1±3  1010 1±3  1010

0 0 2 3 3 4 4

a

DDL ˆ donor-derived lymphocytes; ML ˆ mononuclear leukocytes; PLT-LR ˆ leukocyte-reduced platelet concentrate; RBC-LR ˆ leukocyte-reduced red blood cell concentrate; PLT ˆ platelet concentrate; RBC ˆ red blood cell concentrate; LLC ˆ log lymphocyte content (relative to the lymphocyte content of leukocyte-reduced blood components). b DDL collected as a therapeutic blood component contains an ML dose typically 10 000-fold greater than the number of ML which persist as residual cellular contaminant in leukocyte-reduced blood components.

594 J.-H. Lee and H. G. Klein

considerations, including: (1) instrument availability, (2) donor availability, (3) adequacy of the donor venous access, and (4) operator haemapheresis skill. As in any cytapheresis procedure, the purity and cell yield of the DDL component depend on donor variables (peripheral lymphocyte count, haematocrit, adequacy of venous access), instrument characteristics (eciency of cell interface detection and cell separation), as well as procedural variables (volume of blood processed, blood ¯ow rate, operational skill). The resulting DDL units are transfused, with or without additional laboratory processing, as they become available after brief storage at room temperature. The extent of laboratory cell processing depends on the attempt to reduce GvHD. Cryopreserving the DDL component may allow ¯exibility in designing the optimal treatment schedule. Advanced-phase CML and other haematological malignancies Antigenically less di€erentiated, more rapidly proliferating leukaemias have been dicult to control using DLI. Chronic-phase CML has responded more readily to DLI than has more advanced CML (75 versus 19%), and a response rate of 20% seen in acute myelogenous leukaemia (AML) is comparable to that of accelerated phase CML (Table 2). An analysis of the collective experience has been complicated by many clinical variables, most notably by the role of chemotherapy administered prior to DLI. To date, DLI therapy has been used to treat a variety of disorders in addition to CML and AML: acute lymphocytic leukaemia (ALL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), myelodysplastic syndrome (MDS), polycythaemia vera (PV), juvenile CML (JCML), and chronic lymphocytic leukaemia (CLL). Lymphoid leukaemias have responded less frequently than have myeloid disorders, including myelodysplastic syndromes. Clinical response rates of 10% in ALL (three of 31 evaluable patients) and 20% in AML (15 of 76 evaluable patients)33±36 may be an early indication that DLI is less often e€ective against cells of its own lineage. Despite its lymphoid origin, MM may respond to DLI at a rate comparable to that in AML, and a T cell dose of greater than 1  108/kg has been suggested as being predictive of a clinical graft-versus-myeloma e€ect.78±80 As in CML, favourable clinical predictors of GvL in acute leukaemias have included DLI-associated GvHD and a long duration of remission following initial AHT. A few reports have suggested that IFN-a, which appears not to a€ect the response rate in CML, may be more e€ective in acute leukaemias.33,81 Among other diseases treated to date, DLI has been successful in MDS and PV, but no responses have been reported in NHL, HD, JCML or CLL.33±35,82,83 Interestingly, it has been suggested that in rapidly proliferating disease, male recipients of lymphocytes from female donors may respond more favourably than those receiving DLI under other donor-recipient gender combinations.84 Other leukocyte transfusion therapies Besides leukaemias, the adoptive transfer of donor immunity through lymphocyte transfusion has been under investigation for the treatment of other disorders in which immune dysfunction plays a pathogenetic role. In fact, adoptive immunotherapy as a therapeutic concept arose with the lymphokine-activated killer (LAK) phenomenon de®ned in 198085, in which a heterogeneous population of autologous major histocompatibility complex (MHC) non-restricted lymphocytes gain the ability to lyse tumour cells under the in¯uence of IL-2. Co-culturing lymphocytes and tumour cells in IL-2 results in the generation of MHC class I-restricted T cells possessing a more potent and speci®c antitumour activity referred to as tumour in®ltrating lymphocytes (TIL)86,87,

E€ects of transfused leukocytes 595

or tumour-derived activated cells (TDAC).88 These lymphocyte-based cancer therapies have required signi®cant ex vivo cell manipulation, have been limited largely to the autologous setting, and have depended on the in¯uence of IL-2 used either during exvivo cell culture (LAK cells and TIL) or in conjunction with cell transfusion (LAK cells). Lymphocytes, LAK cells and TIL, in particular, continue to receive investigative attention based on observed clinical response rates of up to 40% in selected patients with advanced malignancies.51±53,86±88 A better understanding of the relationship among antigen presentation, tissue di€erentiation antigens and tumour-speci®c antigens is probably necessary for the further advancement of this therapy.89 The potential use of blood transfusion to mediate immune e€ects has been appreciated for nearly 30 years. In 1973, Opelz et al90 ®rst provided direct clinical evidence in kidney transplantation that allogeneic blood may improve renal allograft survival when transfused during or prior to transplantation. Further studies have shown that: (1) leukocytes play a major role in the immunomodulatory e€ect91, (2) the donor and the transfusion recipient must share at least one HLA-DR antigen while being a haplotype mismatch, and (3) CD4 regulatory T cells are important in mediating the immunomodulatory e€ect.92 Beyond improving graft survival in organ transplantation, leukocyte transfusion has been attempted with limited success in treating selected disorders of immune function, including HIV disease93±96, type I diabetes mellitus97±99, recurrent spontaneous abortion100±102, and autoimmunity. Nelson103 recently proposed that leukocyte microchimerism may be important in the pathophysiology of autoimmunity based on a series of related clinical observations from diverse medical disciplines: (1) the long-term persistence of fetal leukocytes in the mother, (2) the female predilection for autoimmune disorders, particularly at or beyond child-bearing years, (3) the induction of an autoimmune disorder by pregnancy and the modulation of an existing autoimmune disorder during pregnancy, (4) cell engraftment and the long-term persistence of lymphocytes from the mother or a twin sibling as an explanation of autoimmunity in males, (5) the clinical similarity of some autoimmune disorders (e.g. scleroderma) to chronic GvHD post-AHT, and (6) the frequent association of HLA class II molecules with autoimmune disorders. In view of the long-term microchimerism frequently possible by blood transfusion21, Nelson's proposal may be extended to include blood transfusion as a cause for autoimmunity, and conversely, leukocytes as a therapeutic agent against autoimmune disorders. Mechanisms by which microchimerism may trigger or modulate autoimmunity have not been studied, however. DLI for the treatment of relapsed chronic-phase CML following AHT, as described in this chapter, represents the most successful to date of many types of developing leukocyte therapy. CELLULAR VACCINES USING ANTIGEN-PRESENTING CELLS Cellular immunotherapy has relied primarily on cells that e€ect direct cytotoxicity. Successful leukocyte therapies to date, however, include those involving an indirect approach; increasing interest has concentrated on cells with a regulatory function, such as antigen-presenting cells (APC), that mediate a downstream e€ect. Candidate cells include macrophages, dendritic cells and B cells, although other cells may also present antigen, with or without stimulation by cytokines.104 APC have been pulsed with peptide antigens and non-peptide antigens such as glycolipids, and transfected with gene sequences encoding immunostimulatory antigens or co-stimulatory molecules. Such engineered cells have been used to stimulate and amplify T cells to produce `cellular

596 J.-H. Lee and H. G. Klein

vaccines' against tumour antigens or viral infections in vitro.105,106 The ability to isolate antigen-presenting cells, modify them by various molecular techniques, and expand both speci®c clones and polyclonal T cell repertoires promises to revolutionize the approach to cellular therapy. However, the basic science underlying the immune response to tumours is in its infancy and much more will have to be learned before these innovative approaches become e€ective cellular therapeutics. CONCLUSION To date, clinical studies in leukocyte reduction and leukocyte transfusion have progressed along separate tracks as if adverse and therapeutic e€ects of the transfused leukocytes result from separate immunological mechanisms. However, common mechanisms may elicit opposing clinical outcomes in the transfusion recipient, depending on the clinical circumstance and immunological compatibility. TA-GvHD, a typically lethal complication of allogeneic blood transfusion, may transform into predictably life-saving GvL in patients with relapsed chronic-phase CML following AHT in response to DLI. The terms TA-GvHD, DLI-GvHD, and GvL may simply re¯ect di€erent clinical circumstances surrounding the same immunological phenomenon. Further advances in our understanding of cellular immune interactions and blood component preparation technology may lead to new ways of deriving immunological bene®t from transfused blood leukocytes while minimizing their adverse e€ects. REFERENCES 1. Code of Federal Regulations. Washington, DC: US Government Printing Oce. Title 21, Subchapter F, Biologics. 2. Menitove JE (ed.). Standards for Blood Banks and Transfusion Services, 19th edn. Bethesda: American Association of Blood Banks, 1999. 3. Circular of Information of the Use of Human Blood and Blood Components. American Association of Blood Banks, America's Blood Centers, American National Red Cross, 1997. 4. Vengelen-Tyler V (ed.). Technical Manual, 12th edn, p 548. Bethesda: American Association of Blood Banks, 1996. 5. Menitove JE, McElligott MC & Aster RH. Febrile transfusion reactions: what component should be given next? Vox Sanguinis 1982; 42: 318±321. 6. Heddle NM, Klama LN, Grith L et al. A prospective study to identify the risk factors associated with acute reactions to platelet and red cell transfusions. Transfusion 1993; 33: 794±797. 7. Dzik WH. Leukoreduced blood components: laboratory and clinical aspects. In Rossi EC, Simon TL, Moss GS & Gould SA (eds) Principles of Transfusion Medicine, 2nd edn, pp 353±373. Baltimore: Williams & Wilkins, 1996. 8. Dinarello CA & Wol€ SM. Molecular basis of fever in humans. American Journal of Medicine 1982; 72: 799±819. 9. McFarland JG. Platelet immunology and alloimmunization. In Rossi EC, Simon TL, Moss GS & Gould SA (eds) Principles of Transfusion Medicine, 2nd edn, pp 231±244. Baltimore: Williams & Wilkins, 1996. 10. Lee EJ & Schi€er CA. Serial measurement of lymphocytotoxic antibody and response to non-matched platelet transfusions in alloimmunization patients. Blood 1987; 70: 1727±1729. 11. Bowden RA, Slichter SJ, Sayers M et al. A comparison of ®ltered leukocyte-reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusion-associated CMV infection after marrow transplant. Blood 1995; 86: 3598±3603. 12. Dzik S, Blajchman MA, Blumberg N et al. Current research on the immunomodulatory e€ect of allogeneic blood transfusion. Vox Sanguinis 1996; 70: 1987±1994. 13. Ghio M, Contini P, Mazzei C et al. Soluble HLA Class I, HLA Class II, and Fas ligand in blood components: a possible key to explain the immunomodulatory e€ects of allogeneic blood transfusion. Blood 1999; 93: 1770±1777.

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14. Blajchman MA. Transfusion-associated immunomodulation and universal white cell reduction: are we putting the cart before the horse? Transfusion 1999; 39: 665±670. 15. Houbiers JGA, Brand A, van de Watering LMG et al. Randomised controlled trial comparing transfusions of leukocyte depleted or bu€y coat depleted blood in surgery for colorectal cancer. Lancet 1994; 344: 573±578. 16. Busch ORC, Hop WCJ, van Papendrecht MAWH et al. Blood transfusions and prognosis in colorectal cancer. New England Journal of Medicine 1993; 328: 1372±1376. 17. Fast LD. Recipient CD8‡ cells are responsible for the rapid elimination of allogeneic donor lymphoid cells. Journal of Immunology 1996; 157: 4805±4810. 18. Linden JV & Pisciotto PT. Transfusion-associated graft-versus-host disease and blood irradiation. Transfusion Medicine Reviews 1992; 6: 116±123. 19. Wagner FF & Flegel WA. Transfusion-associated graft-versus-host disease: risk due to homozygous HLA haplotypes. Transfusion 1995; 35: 284±291. 20. Anderson KC & Weinstein HJ. Transfusion-associated graft-versus-host disease. New England Journal of Medicine 1990; 323: 315±321. 21. Lee TH, Paglieroni T, Ohoto H et al. Survival of donor leukocyte subpopulations in immunocompetent transfusion recipients: frequent long-term microchimerism in severe trauma patients. Blood 1999; 93: 3127±3139. 22. Bianchi DW, Zickwolf GK, Weil GJ et al. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proceedings of the National Academy of Sciences of the USA 1996; 93: 705±708. 23. Maloney S, Smith A, Furst DE et al. Microchimerism of maternal origin persists into adult life. Journal of Clinical Investigation 1999; 104: 41±47. 24. Truitt RL, LeFever AV, Shich CC-Y et al. Graft-vs-leukemia e€ect. In Burako€ SJ, Deeg HJ, Ferrara J & Atkinson K (eds) Graft-vs-Host Disease: Immunology, Pathophysiology, and Treatment, pp 177±181. New York: Marcel-Dekker, 1990. 25. Gale RP & Butturini A. How do transplants cure chronic myelogenous leukemia? Bone Marrow Transplantation 1992; 9: 83±85. 26. Horowitz MM, Gale RP, Sondel PM et al. Graft-versus leukemia reactions after bone marrow transplantation. Blood 1990; 75: 555±562. 27. Weiden PL, Flournoy N, Sanders JE et al. Anti-leukemic e€ect of graft-versus-host disease contributes to improved survival after allogeneic marrow transplantation. Transplantation Proceedings 1981; 13: 248±251. 28. Weiden PL, Sullivan K, Flournoy N et al. The Seattle Marrow Transplant Team: antileukemic e€ect of chronic graft-versus-host disease. Contribution to improved survival after allogeneic marrow transplantation. New England Journal of Medicine 1981; 304: 1529±1531. 29. Goldman JM, Gale RP, Horowitz MM et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: increased risk for relapse associated with T-cell depletion. Annals of Internal Medicine 1988; 108: 806±807. 30. Marmont A, Horowitz MM, Gale RP et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991; 78: 2120±2130. 31. Ot K, Burns JP, Cunningham I et al. Cytogenetic analysis of chimerism and leukemia relapse in chronic myelogenous leukemia patients ater T cell-depleted bone marrow transplantation. Blood 1990; 75: 1346±1355. 32. Kolb HJ, Mittermueller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462±2465. 33. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia e€ect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995; 86: 2041±2050. 34. Kolb HJ & Holler E. Adoptive immunotherapy with donor lymphocyte transfusions. Current Opinion in Oncology 1997; 9: 139±145. 35. Collins RH, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. Journal of Clinical Oncology 1997; 15: 433±444. 36. Helg C, Starobinski M, Jeannet M & Chapuis B. Donor lymphocyte infusion for the treatment of relapse after allogeneic hematopoietic stem cell transplantation. Leukemia and Lymphoma 1998; 29: 301±313. 37. Baurmann H, Nagel S, Binder T et al. Kinetics of the graft-versus-leukemia response after donor leukocyte infusions for relapsed chronic myeloid leukemia after alllogenic bone marrow transplantation. Blood 1998; 92: 3582±3590. 38. van Rhee F, Lin F, Cullis JO et al. Relapse of chronic myeloid leukemia after allogeneic bone marrow transplant: the case for giving donor leukocyte transfusions before the onset of hematologic relapse. Blood 1994; 83: 3377±3383.

598 J.-H. Lee and H. G. Klein 39. Gardiner N, Lawler M, O'Riordan JM et al. Monitoring of lineage-speci®c chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia. Bone Marrow Transplantation 1998; 21: 711±719. 40. Keil F, Haas O, Fritsch G et al. Donor leukocyte infusion for leukemic relapse after allogeneic marrow transplantation: lack of residual donor hematopoiesis predicts aplasia. Blood 1997; 89: 3113±3117. 41. Keil F, Kalhs P, Haas OA et al. Graft failure after donor leucocyte infusion in relapsed chronic myeloid leukaemia: successful treatment with cyclophosphamide and antithymocyte globulin followed by peripheral blood stem cell infusion. British Journal of Haematology 1996; 94: 120±122. 42. Weiss L, Lubin I, Factorowich I et al. E€ective GvL e€ects independent of GvHD after T cell-depleted allogeneic bone marrow transplantation in a murine model of B cell leukemia/lymphoma. Journal of Immunology 1994; 153: 2562±2567. 43. Glass B, Uharek L, Gassmann W et al. Graft-versus-leukemia activity after bone marrow transplantation does not require graft-versus-host disease. Annals of Hematology 1992; 64: 255±259. 44. Rocha M, Umansky V, Lee K et al. Di€erences between graft-versus-leukemia and graft-versus-host reactivity I: interaction of donor immune T cells with tumor and/or host cells. Blood 1997; 89: 2189±2202. 45. Giralt S, Hester J, Huh Y et al. CD8-depleted donor lymphocyte infusion as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation. Blood 1995; 86: 4337±4343. * 46. Pan L, Delmonte J, Jalonen CK & Ferrara JLM. Pretreatment of donor mice with granulocyte colonystimulating factor polarizes donor T lymphocytes toward type-2 cytokine production and reduces severity of experimental graft-versus-host disease. Blood 1995; 86: 4422±4429. 47. Lotzova E, Savary CA & Keating MJ. Leukemia diseased patients exhibit multiple defects in natural killer cell lytic machinery. Experimental Hematology 1983; 10: 83±95. 48. Lotzova E. Role of interleukin-2 activated MHC-nonrestricted lymphocytes in antileukemia activity and therapy. Leukemia and Lymphoma 1992; 7: 15±28. 49. Soi€er RJ, Murray C, Gonin R & Ritz J. E€ect of low-dose interleukin-2 on disease relapse after T-celldepleted allogeneic bone marrow transplantation. Blood 1994; 84: 964±971. 50. Slavin S, Naparstek E, Nagler A et al. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse after allogeneic bone marrow transplantation. Blood 1996; 87: 2195±2204. 51. Dutcher JP, Creekmore S, Weiss GR et al. A Phase II study of interleukin-2 and lymphokine-activated killer cells in patients with metastatic malignant melanoma. Journal of Clinical Oncology 1989; 7: 477±485. 52. Fisher RI, Coltman CA, Doroshow JH et al. Metastatic renal cancer treated with interleukin-2 and lymphokine-activated killer cells. Annals of Internal Medicine 1988; 108: 518±523. 53. Berendt MJ & North RJ. T-cell-mediated suppression of anti-tumor immunity: an explanation for progressive growth of an immunogenic tumor. Journal of Experimental Medicine 1980; 151: 69±80. 54. Falkenburg JHF, Wafelman AR, Joosten P et al. Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes. Blood 1999; 94: 1201±1208. 55. Smit WM, Reijnbeek M, van Bergen CA et al. Generation of dendritic cells expressing bcr/abl from CD34-positive chronic myeloid leukemia precursor cells. Human Immunology 1997; 53: 216±223. 56. Choudhury A, Gajewski JL, Liang JC et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome positive chronic myelogenous leukemia. Blood 1997; 89: 1133±1142. 57. Smit WM, Rijnbeek M, van Bergen CA et al. T cells recognizing leukemia CD34‡ progenitor cells mediate the antileukemic e€ect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation. Proceedings of the National Academy of Sciences of the USA 1998; 95: 10 152±10 157. 58. den Haan JM, Sherman NE, Blokland E et al. Identi®cation of a graft versus host disease-associated human minor histocompatibility antigen. Science 1995; 268: 1476±1480. 59. den Haan JM, Meadows LM, Wang W et al. The human immunodominant minor histocompatibility antigen HA-1 represents a diallelic gene with a single amino acid polymorphism. Science 1998; 279: 1054±1057. 60. Falkenburg JHF, Smith WM & Willemze R. Cytotoxic T-lymphocyte (CTL) responses against acute or chronic myeloid leukemia. Immunological Reviews 1997; 157: 223±230. 61. Goulmy E. Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy. Immunological Reviews 1997; 157: 125±140. 62. Molldrem J, Dermime S, Parker K et al. Targeted T-cell therapy for human leukemia: cytotoxic T lymphocytes speci®c for a peptide derived from proteinase 3 preferentially lyse human myeloid leukemia cells. Blood 1996; 88: 2450±2457.

E€ects of transfused leukocytes 599 63. Cardoso AA, Schultze JL, Boussiotis VA et al. Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen. Blood 1996; 88: 41±48. 64. Cardoso AA, Seamon MJ, Afonso HM et al. Ex vivo generation of human anti-pre-B leukemia-speci®c autologous cytolytic T cells. Blood 1997; 90: 549±561. 65. Guinan EC, Gribben JG, Boussiotis VA et al. Pivotal role of the B7:CD28 pathway in transplantation tolerance and tumor immunity. Blood 1994; 84: 3261±3262. 66. Papadopoulos EB, Ladanyi M, Emanuel D et al. Infusions of donor leukocytes to treat Epstein±Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. New England Journal of Medicine 1994; 330: 1185±1191. 67. Heslop HE, Brenner NK & Rooney CM. Donor T cells as therapy for EBV lymphoproliferation post bone marrow transplantation. New England Journal of Medicine 1994; 331: 679±680. 68. Caldas C & Ambinder R. Epstein±Barr virus and bone marrow transplantation. Current Opinion in Oncology 1995; 7: 102±106. * 69. Bonini C, Ferrari G, Verzeletti S et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 1997; 276: 1719±1724. 70. Tiberghien P, Reynolds CW, Keller J et al. Ganciclovir treatment of herpes simplex thymidine kinasetransduced primary T lymphocytes: an approach for speci®c in vivo T-cells depletion after bone marrow transplantation. Blood 1994; 84: 1333±1341. 71. Munshi NC, Govindarajan R, Drake R et al. Thymidine kinase (TK) gene-transduced human lymphocytes can be highly puri®ed, remain fully functional, and are killed eciently with ganciclovir. Blood 1997; 89: 1334±1340. 72. Rooney CM, Smith CA, Ng CYC et al. Use of gene-modi®ed virus-speci®c T lymphocytes to control Epstein±Barr-virus-related lymphoproliferation. Lancet 1995; 345: 9±13. 73. Bordignon C, Bonini C, Verzeletti S et al. Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Human Gene Therapy 1995; 6: 813±817. 74. Walter EA, Greenberg PD, Gilbert MJ et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell cllones from the donor. New England Journal of Medicine 1995; 333: 1038±1044. 75. Shouval D & Ilan Y. Immunization against hepatitis B through adoptive transfer of immunity. Intervirology 1995; 38: 41±46. 76. Ho M, Armstrong J, McMahon D et al. A phase I study of adoptive transfer of autologous CD8‡ T lymphocytes in patients with acquired immunode®ciency syndrome (AIDS)-related complex or AIDS. Blood 1993; 81: 2093±2101. 77. Lieberman J, Skolnik PR, Parkerson GR et al. Safety of autologous ex vivo-expanded human immunode®ciency virus (HIV)-speci®c cytotoxic T-lymphocyte infusion in HIV-infected patients. Blood 1997; 90: 2196±2206. 78. Lokhorst HM, Schattenberg A, Cornelissen JJ et al. Donor leukocyte infusions are e€ective in relapsed multiple myeloma after allogeneic bone marrow transplantation. Blood 1997; 90: 4206±4211. 79. Verdonck LF, Lokhorst HM, Dekker AW et al. Graft-versus-myeloma e€ect in two cases. Lancet 1996; 347: 800±801. 80. Tricot G, Vesole DH, Jagannath S et al. Graft-versus-myeloma e€ect: proof of principle. Blood 1996; 87: 1196±1198. 81. Porter DL, Roth MS, Lee SJ et al. Adoptive immunotherapy with donor mononuclear cell infusions to treat relapse of acute leukemia or myelodysplasia after allogeneic bone marrow transplantation. Bone Marrow Transplantation 1996; 18: 975±980. 82. Collins RH, Pineiro LA, Nemunaitis JJ et al. Transfusion of donor bu€y coat cells in the treatment of persistent or recurrent malignancy after allogeneic bone marrow transplantation. Transfusion 1995; 35: 898±899. 83. Barrett AJ & Malkovska V. Graft-versus-leukaemia: understanding and using the alloimmune response to treat haematological malignancies. British Journal of Haematology 1996; 93: 754±761. 84. Goulmy E, Termijteelen A, Bradley BA & van Rood JJ. Y-antigen killing by T-cells of women is restricted by HLA. Nature 1977; 266: 544±545. 85. Lotze MT, Line BR, Mathisen DJ et al. The in vivo distribution of autologous human and murine lymphoid cells grown in T cell growth factor (TCGF): implications for the adoptive immunotherapy of tumors. Journal of Immunology 1980; 125: 1487±1493. 86. Rosenberg SA, Yang JC, Topalian SL et al. Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2. Journal of the American Medical Association 1994; 271: 907±913.

600 J.-H. Lee and H. G. Klein 87. Kohler PC, Hank JA, Exten R et al. Clinical response of a patient with di€use histiocytic lymphoma to adoptive chemoimmunotherapy using cyclophosphamide and alloactivated haploidentical lymphocytes: a case report and phase I trial. Cancer 1985; 55: 552±560. 88. Oldham RK, Lewko WM, Good RW & Sharp E. Cancer biotherapy with interferon, interleukin-2 and tumor-derived activated cells (TDAC). In vivo 1994; 8: 653±664. 89. Ochsenbein AF, Klenerman P, Karrer U et al. Immune surveillance against a solid tumor fails because of immunologic ignorance. Proceedings of the National Academy of Sciences of the USA 1999; 96: 2233±2238. 90. Opelz G, Mickey MR & Terasaki P. E€ect of blood transfusions on subsequent kidney transplants. Transplantation Proceedings 1973; 5: 253±259. 91. Persijn GG, Cohen B, Lansbergen Q & van Rood JJ. Retrospective and prospective studies on the e€ect of blood transfusions in renal transplantation in The Netherlands. Transplantation 1979; 28: 396±401. 92. van Rood JJ & Claas F. Impact of histocompatibility testing, or the Yin-Yang of transplantation. In Tilney NL, Strom TB & Paul LC (eds) Transplantation Biology: Cellular and Molecular Aspects, p 341. Philadelphia: Lippincott-Raven, 1996. 93. Lane HC, Zunich KM, Wilson W et al. Syngeneic bone marrow transplantation and adoptive transfer of peripheral blood lymphocytes in human immunode®ciency virus (HIV) infection. Annals of Internal Medicine 1990; 113: 512±519. 94. Wiviott LD, Walker CM & Levy JA. CD8 ‡ lymphocytes suppress HIV production by autologous CD4‡ cells without eliminating the infected cells from culture. Cellular Immunology 1990; 128: 628±634. 95. Pantaleo G, De Maria A, Koenig S et al. CD8‡ T lymphocytes of patients with AIDS maintain normal broad cytolytic function despite the loss of human immunode®ciency virus-speci®c cytotoxicity. Proceedings of the National Academy of Sciences of the USA 1990; 87: 4818±4822. 96. Whiteside TL, Elder EM, Moody D et al. Generation and characterization of ex vivo propagated autologous CD8‡ cells used for adoptive immunotherapy of patients infected with human immunode®ciency virus. Blood 1993; 81: 2085±2092. 97. Pozzilli P, Ghirlanda G, Manna G et al. White cells transfusion in recent onset type 1 diabetes. Diabetes Research 1986; 3: 273±276. 98. Krug J, Verlohren H-J, Bierwolf B et al. Lymphocyte transfusion in recent onset type I diabetes mellitus: a one-year follow-up of cell-mediated anti-islet cytotoxicity and C-peptide secretion. Journal of Autoimmunity 1990; 3: 601±609. 99. Cavanaugh J, Chopek M, Binimelis J et al. Bu€y coat transfusions in early type 1 diabetes. Diabetes 1987; 36: 1089±1093. 100. Bux J, Westphal E, de Sousa F et al. Alloimmune neonatal neutropenia is a potential side e€ect of immunization with leukocytes in women with recurrent spontaneous abortions. Journal of Reproductive Immunology 1992; 22: 299±302. 101. Smith JB, Cowchock FS, Lata JA & Hankinson BT. The number of cells used for immunotherapy of repeated spontaneous abortion in¯uences pregnancy outcome. Journal of Reproductive Immunology 1992; 22: 217±224. 102. Clark DA, Gunby J & Daya S. The use of allogeneic leukocytes or IVIG for the treatment of patients with recurrent spontaneous abortions. Transfusion Medicine Reviews 1997; 11: 85±94. *103. Nelson JL. Non-host cells in the pathogenesis of autoimmune disease: a new paradigm? Annals of the Rheumatic Diseases 1999; 58: 518±520. 104. Tykocinski ML, Kaplan DR & Medof ME. Antigen-presenting cell engineering. American Journal of Pathology 1996; 148: 1±16. *105. Rosenberg SA, Yang JC, Schwartzentruber DJ et al. Immunologic and therapeutic evaluation of a synthetic tumor associated peptide vaccine for the treatment of patients with metastatic melanoma. Nature Medicine 1998; 4: 321±327. 106. Hodge JW, Abrams S, Schlom J & Kantor JA. Induction of antitumor immunity by recombinant vaccinia viruses expressing B7-1 or B7-2 costimulatory molecules. Cancer Research 1994; 54: 5552±5555.