Induction of antiviral cytotoxic T cells by plasmacytoid dendritic cells for adoptive immunotherapy of posttransplant diseases.

Virus-associated hematologic malignancies (EBV lymphoproliferative disease) and opportunistic infections (CMV) represent a major cause of hematopoietic stem cell and solid organ transplantation failure. Adoptive transfer of antigen-specific T lymphocytes appears to be a major and successful immunotherapeutic strategy, but improvements are needed to reliably produce high numbers of virus-specific T cells with appropriate requirements for adoptive immunotherapy that would allow extensive clinical use. Since plasmacytoid dendritic cells (pDCs) are crucial in launching antiviral responses, we investigated their capacity to elicit functional antiviral T-cell responses for adoptive cellular immunotherapy using a unique pDC line and antigens derived from Influenza, CMV and EBV viruses. Stimulation of peripheral blood mononuclear cells from HLA-A*0201(+) donors by HLA-A0201 matched pDCs pulsed with viral-derived peptides triggered high levels of multi-specific and functional cytotoxic T-cell responses (up to 99% tetramer(+) CD8 T cells) in vitro. Furthermore, the central/effector memory cytotoxic T cells elicited by the pDCs strongly display antiviral activity upon adoptive transfer into a humanized mouse model that mimics a virus-induced malignancy. We provide a simple and potent method to generate virus-specific CTL with the required properties for adoptive cellular immunotherapy of post-transplant diseases.

Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes.

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.

Dual T cell receptor beta chain expression on human T lymphocytes.

Allelic exclusion of lymphocyte antigen receptor chains has been hypothesized as a mechanism developed by the immune system to ensure efficient lymphocyte repertoire selection and tight control of lymphocyte specificity. It was effectively shown to be operative for both the immunoglobulin (Ig) and the T cell receptor (TCR) beta chain genes. Our present observations suggest that close to 1% of human T lymphocytes escape this allelic control, and express two surface TCR beta chains with distinct superantigenic reactivities. Since this high frequency of dual beta chain expressors did not result in any dramatic immune dysregulations, these results question the need for a mechanism ensuring clonal monospecificity through allelic exclusion.

Surface expression of two distinct functional antigen receptors on human gamma delta T cells.

Lymphocytes recognize antigens with highly variable heterodimeric surface receptors. Although four distinct antigen receptors could in principle be produced by any lymphocyte, only one functional combination of receptor chains has thus far been found expressed on their surface. Examination of human gamma delta T cells revealed a population that violated this rule by expressing on their surface two distinct functional gamma delta T cell receptors (TCRs) that used different TCR gamma gene alleles. Thus, current models for T cell clonal selection may need modification, and a possible escape mechanism for autoreactive TCRs is suggested.

Acute graft versus host disease due to T lymphocytes recognizing a single HLA-DPB1*0501 mismatch.

Analysis of a large number of unrelated bone marrow transplantations (BMT) has shown that HLA-DP incompatibility did not detectably influence the risk for acute graft-versus-host disease (aGVHD). Accordingly, it was proposed that HLA-DP determinants did not function as transplantation antigens in the same way as HLA-A, -B, or -DR. We have previously shown that HLA-DP (as well as HLA-A, -B, -DQ, or -DR)-specific T cells could be isolated from skin biopsies of patients who developed an aGVHD after semiallogeneic BMT. Nevertheless, whether a single HLA-DP mismatched allele could induce a detectable allo-specific reaction in vivo after BMT remained to be established. To directly address this issue we studied one patient who presented aGVHD after receiving purified CD34+ bone marrow (BM) cells from an unrelated donor with a single HLA-DP mismatch in the GVHD direction. To characterize the immunological events associated with GVHD, we analyzed the peripheral T cell repertoire, the T cell receptor Vbeta diversity, and the specificity of T cells invading a skin biopsy at the onset of GVHD. Our results demonstrated that a large fraction of skin-infiltrating lymphocytes, which expressed diverse T cell receptors, were reactive against this single HLA-DPB1 *0501 mismatch and consequently that a single HLA-DP mismatch between BM donor and recipient can activate a strong T cell response in vivo.

Specificity of T cells invading the skin during acute graft-vs.-host disease after semiallogeneic bone marrow transplantation.

The mechanisms responsible for skin lesions during acute graft-vs.-host disease (aGVHD) after allogeneic bone marrow transplantation (BMT) are poorly understood. The exact role of various effector cell populations and "major" (particularly HLA-DP) or "minor" antigens as target molecules is not known. To investigate the nature of cells responsible for tissue injury, we cultured T cells from skin biopsy first with interleukin 2 (IL-2) alone and then in polyclonal activation conditions to avoid in vitro antigenic sensitization before specificity testing. We applied this method to two biopsies performed during aGVHD after semiallogeneic BMT and obtained cytotoxic T cells against four graft mismatches: CD8+ T cells against HLA-A2.2 and HLA-B27 and CD4+ T cells against HLA-DP101 and HLA-DP401. This demonstrates that T cells with documented specificity can be obtained from an aGVHD lesion without antigenic selection. Moreover, these data directly implicate DP as a potential target antigen for aGVHD.

[Adoptive transfer of T lymphocytes]. / Transfert adoptif de lymphocytes T.

Within a few years, the success of treatments based on the use of T-cells armed with a chimeric T-receptor for the CD19 molecule (CAR-T CD19) has revolutionized the perception of adoptive transfer approaches. The levels of responses observed in acute leukemias, of the order of 70-90 % are indeed unprecedented. The medical and financial enthusiasm aroused by these results has led to the current situation where more than 300 clinical trials are under way, against some thirty different antigens. This enthusiasm, well justified by the first successes, must however be tempered by the difficulties associated with the use of these cells. Indeed, the management of patients is made very complex both for medical reasons, because the toxicities associated with these treatments are important, and for technical reasons, because the preparation of T lymphocytes for therapeutic use requires dedicated structures. During this same period, knowledge of the mechanisms of regulation of T lymphocytes and the possibilities offered by synthetic biology and techniques of genome engineering have progressed considerably. Combined, they allow envisaging a true "programming" of the T lymphocytes, intended to improve the efficiency of the treatments and the safety of the patients. Medical and industrial perspectives and the role of these approaches in the arsenal of cancer therapies will depend largely on two conditions: the emergence of a robust demonstration of their effectiveness in solid tumors, and the establishment of an acceptable production and distribution model 1.

Isolation of human lymphocyte antigens class I-restricted cytotoxic T lymphocytes against autologous myeloma cells.

Peripheral blood T cells from a patient with multiple myeloma in complete remission were selected in vitro against an autologous myeloma cell line (SBN-1), using a protocol designed for the selection of relatively rare precursor cytotoxic T cells (pCTL). Delayed addition (2 weeks) of interleukin 2 induced T-cell proliferation, and a bulk culture (T-cell line) was obtained 2 days later. This T-cell line displayed cytotoxicity against SBN-1. A CD8+ CD4- cytotoxic T-cell clone (CT5) was then obtained that recognized SBN-1 but not autologous EBV+ B-lymphoblastoid cells, autologous T PHA-blasts, or Daudi, Raji, K562, and 11 allogeneic myeloma cell lines. Moreover, CT5 cytotoxic activity against SBN-1 was blocked by monoclonal antibodies recognizing human lymphocyte antigen class I molecules. This seems to be the first demonstration of myeloma-specific pCTL in peripheral blood T cells of patients with multiple myeloma.

Decline, with age, in the proportion of mouse T cells that express IL-2 receptors after mitogen stimulation.

Receptors for IL-2 can be demonstrated on the majority of mouse splenic T cells within 48 h after stimulation with Concanavalin A. We show here that aging leads to a decline in the frequency of cells able to generate detectable levels of IL-2 receptor, as measured by cytofluorimetry using antibodies to the receptor. The age-associated deficit cannot be overcome either by increasing the mitogen dose, or by supplementing the cultures with exogenous IL-2. At least a part of the decline, with age, in T cell-mediated immunity may represent an inability of individual T cells to produce sufficient IL-2 receptor to enable them to reach the IL-2 dependent phases of their first cell cycle.

Preparative two-step purification of human IL-2 by HPLC and hydrophobic affinity chromatography.

Interleukin 2 (IL-2) has been purified by a protocol using gel filtration high performance liquid chromatography (HPLC) and hydrophobic affinity chromatography with blue-trisacryl M. Peripheral blood lymphocytes or tonsillar lymphocytes were stimulated with phytohemagglutinin (PHA). Serum free conditioned medium (CM) containing IL-2, other lymphokines and residual PHA molecules was analyzed after 3 variations of ammonium sulfate (AS) precipitation: (1) precipitation of CM with 50% AS yielded a precipitate containing most of the residual PHA but also a fraction of IL-2. (2) Precipitation with direct 80% AS of crude CM yielded both IL-2 and residual PHA. (3) A double step procedure (50% AS followed by 80% AS) yielded a precipitate containing IL-2 but free of residual lectin. HPLC purification of these various AS-precipitated materials or of lyophilized crude CM yielded 2 peaks with mitogenic activity as assayed with the CTLL2 murine clone or IL-2-dependent human Con A-stimulated lymphoblasts. IFN was easily separated from IL-2 and PHA, but BCGF still copurified with IL-2. Peak I (25 kDa) was enriched 400-fold for IL-2 while peak II (68 kDa) contained the residual PHA. The IL-2-containing fractions eluted from HPLC were further purified by blue-trisacryl M chromatography. The IL-2 eluted with 0.4 M NaCl. The entire protocol (HPLC followed by blue-trisacryl) led routinely to 8000-fold IL-2 enrichment. Preparative HPLC directly applied to lyophilized crude (CM) enriched IL-2 activity 400-fold with yield averaging 60% of the IL-2 input. The final material was free from interferon and IL-1, but BCGF still copurified with IL-2. The 2-step purified material (HPLC and blue-trisacryl) gave 2 bands in SDS-PAGE both of which contained IL-2.

Structural analysis of gamma delta TCR using a novel set of TCR gamma and delta chain-specific monoclonal antibodies generated against soluble gamma delta TCR. Evidence for a specific conformation adopted by the J delta 2 region and for a V delta 1 polymorphism.

We recently showed that secretion of non-chimeric disulfide-linked human gamma delta TCR ('soluble' TCR, sTCR) comprising V gamma 9 and V delta 2 regions could be achieved by simply introducing translational termination codons upstream from the sequences encoding TCR transmembrane region. Here we extended these findings by demonstrating efficient secretion and heterodimerization of gamma delta sTCR comprising V gamma 8, V delta 1 and V delta 3 regions, obtained via the same strategy. After immunization against immunoaffinity-purified soluble TCR, several hundreds of TCR-specific monoclonal antibodies (mAb) were generated, which fell in at least seven groups. One set of mAb was directed against a V gamma 8-specific epitope. Strikingly, despite the high degree of sequence homology between V gamma 8 and other V gamma I domains, none of these mAb were crossreactive with other members of the V gamma I family. Three other sets of mAbs were shown to recognize delta chains comprising V delta 1, V delta 2 and V delta 3 regions respectively, regardless of their junctional sequence or of the gamma chain to which they were paired. Among the V delta 1-specific mAb, some specifically recognized V delta 1D delta J delta C delta chains while others reacted with both V delta 1 D delta J delta C delta and V delta 1J alpha C alpha chains, which suggested V domain conformational alterations induced by the C region. Moreover, reactivity of one V delta 1-specific mAb (#R6.11) was affected by a polymorphic residue located on the predicted CDR4 loop of the V delta region. Two delta chain-specific mAb (#178 and #515) showed a highly unusual reactivity, which was negatively affected by particular V delta and J delta sequences: (i) mAb #515 and #178 recognized all TCR delta chains except those comprising V delta 1 or V delta 2 regions, respectively and (ii) within TCR delta chains carrying 'permissive' V delta regions, none of those comprising the J delta 2 region were recognized by #515 and/or #178 mAbs, which suggested a highly specific conformation adopted by this particular J delta sequence. Apart from its usefulness in TCR structural studies, this novel set of mAb represents an important tool for the characterization and isolation of gamma delta T cells expressing particular combinations of V gamma/V delta regions and for analysis of V alpha/V delta usage by alpha beta T cells. Moreover, since our present data strongly suggest that gamma delta TCR are easier to obtain in a soluble form than alpha beta TCR, an efficient strategy for the generation of V alpha region-specific mAb might be to immunize with chimeric gamma delta sTCR comprising particular V alpha regions.

T cell colony-forming frequency of mononucleated cells extracted from rejected human kidney transplants. Functional and phenotypic studies of the colonies.

One line of investigation of cellular events leading to rejection of an allograft has been to collect the cells infiltrating the rejected allograft and to subject them to in vitro functional and cell marker analysis. Outlined in this article is a description of the limiting dilution analysis technique applied to mechanically disrupted cells obtained from three different rejected human kidney allografts and a phenotypic cell surface marker, as well as a functional study of the progeny of such cells at a clonal level. Of the harvested cells, 65% were T11+, 58% were OKT8+, and 14% were OKT4+. The frequencies of colony forming cells (CFC) were assessed in liquid medium supplemented with lectin-free purified IL-2 (selecting in vivo activated cells) or with IL-2 plus PHA. The CFC frequencies ranged from 1/777 to 1/120 cells plated and from 1/250 to 1/90 cells plated in, respectively, IL-2 and IL-2 plus PHA. Only colonies with a probability of monoclonality more than 80% were further expanded in the presence of lectin-free IL-2. Among 31 colonies grown in the presence of IL-2 alone, all colony-cells were OKT11+, 4/31 were T4+T8-, 24/31 T4-T8+--and, finally, 3/31 were T4+T8+. On the other hand, 122 colonies grown in the presence of IL-2 plus PHA were also all OKT11+, but 47/122 were T4+T8-, and 62/122 were T4-T8+. In addition, expression of DR molecules was highly variable from colony to colony. Significant antidonor cytotoxicity was recorded in 24 colonies, most of them expressing T8 molecules at their surface. Moreover cytotoxic antidonor colonies seemed to recognize an antigen determinant different from the known HLA incompatibilities between donor and recipient. In three long-term-cultured colonies, we noticed a shift in surface marker: from the expression of T8 to either the coexpression of T4 or the loss of the T8 to the sole expression of the T4 molecules. This methodology is a step forward in the elaboration of techniques for determining the relationships between the surface marker identity and the immune function of cells activated in vivo, which are found in a rejected human kidney.

Generation of helper and cytotoxic CD4+T cell clones specific for the minor histocompatibility antigen H-Y, after in vitro priming of human T cells by HLA-identical monocyte-derived dendritic cells.

BACKGROUND: There is now convincing evidence that minor histocompatibility antigens (mHag) may play a significant role in the pathogenesis of graft-versus-host disease after HLA-identical bone marrow transplantation. Indeed, in this clinical situation, T cells specific for mHag have been isolated. Here, we addressed whether one can generate mHag-specific T cells in vitro, without any in vivo immunization, among healthy blood donors. METHODS: We used monocyte-derived dendritic cells (Mo-DCs) as antigen presenting cells to induce primary responses between healthy HLA-identical siblings, in mixed lymphocyte dendritic cell reactions (MLDCRs). RESULTS: We show that CD4+ T-cell clones, specific for the mHag H-Y, can be generated in vitro. These clones were derived from a gender-mismatched positive MLDCR pair of HLA-identical siblings and were restricted by the HLA DQB1*0502 molecule. In addition, these CD4+ T clones were also able to lyse allogeneic targets with the same pattern of restriction and specificity than helper function. Finally, acute myeloid leukemia (AML) blast cells were susceptible to lysis by these clones. CONCLUSIONS: Altogether, these results predict that Mo-DCs could help to generate class II-associated, mHag-specific, T-cell lines or clones in vitro, between healthy blood donors, without any need of transplantation-mediated immunization.

Polyclonal rabbit gamma globulins against a human cytotoxic CD4 T cell clone. II. Use in prevention of rejection in kidney transplantation: a pilot study.

Antiblast globulins (GAB) were prepared by immunization of rabbits with activated T lymphocytes (AT) derived from a rejected kidney allograft. AT consisted of a CD4+ (CD3+, CD2+ TCR alpha+ beta+) clone cytotoxic for HLA DR8-positive targets. The immunizing cells were adapted to industrial growth conditions by repetitive stimulations with an EBV-transformed line from the kidney donor and recombinant IL-2. In the pilot study, GAB (1.0-1.5 mg/kg/day) was given in 12-hr infusions, in association with prednisone (Pred) 1 mg/kg/day and azathioprine (Aza) 2 mg/kg/day, as prophylactic treatment of rejection in 12 kidney-transplanted patients during the first 2 weeks postgrafting. GAB dosage was further adapted according to the level of circulating E-rosette-forming T cells (ERFT). Cyclosporine A (8 mg/kg/day) was given at day 14 as a monotherapy after Pred and Aza were progressively tapered. No patient died, but one kidney was lost from surgical complication. No rejection occurred under GAB treatment; 41% of patients had at least one episode in the first 3 months and 16% from 3 to 9 months. GAB side effects were minor (skin rash: 2, low grade fever: 4) except for one acute serum sickness. Platelet and white blood cell counts were unchanged, but there was a significant decrease in hemoglobin during the 2 weeks of GAB infusions. Few infectious episodes occurred (3 bacterial, 2 viral). GAB monitoring showed a dramatic drop in T11+, T3+, T4+, and T8+ circulating T cells (less than 10% of normal values between days 3 and 14), whereas EFRT cells had a delayed and somewhat lower decrease (less than 10% after day 6 only). Consequently, mean GAB doses had to be raised to 1.3 mg/kg/day at day 4 and 1.6 at days 8 and 14. This pilot study suggests that this new bioreagent should be of major interest in the prophylaxis and treatment of rejection in allograft recipients. A controlled study is in progress.

Polyclonal rabbit gamma globulins against a human cytotoxic CD4+ T cell clone. I. Clone characteristics and antiblast globulin preparation.

Antihuman lymphocyte rabbit (or horse) gamma globulins used in recipients of organ transplantation are prepared against thymocytes or immortalized cell lines, the only two sources so far allowing enough antigen preparation. These cells lack, however, the surface determinants characteristic of alloreactive blasts involved in the rejection process. We have derived long-term cultures of a panel of alloreactive (untransformed) clones from a rejected kidney. Among them, clone 1E7 has been chosen as a cytotoxic CD4+ (CD2+ CD3+ TCR alpha beta+) clone proliferating against HLA-DR8 targets. This clone (clonality assessed on T cell receptor genomic rearrangements) has been grown using weekly stimulations with the kidney donor-derived EBV cell line and recombinant IL-2. Clone cultures have been adapted to mass production after optimization of culture conditions satisfying pharmaceutical requirements. This procedure warranted a reproducible source of antigen since the functional and phenotypic characteristics of the immunizing 1E7 cells remained identical through the life span of the culture. In addition, the study of the total growth capacity of 1E7 cells showed consistent expansion until the 40th cell cycle, ensuring a progeny that will satisfy the large-scale requirement for a clinical trial. Rabbits were injected with 100 x 10(6) 1E7 cells (21, 14, and 7 days before bleeding). Sera were depleted of agglutinin by red blood cell absorption and globulin antiblast (GAB) prepared by SO4Na precipitation and ion exchange chromatography; 50% complement-mediated target cell lysis and 50% inhibition of E rosette formation and alloproliferation were obtained at GAB dilutions of about 1:250-1:500. Prescreened on cynomolgus monkeys, GAB could significantly prolong skin grafts when given prophylactically. Finally GAB have been used in human recipients of kidney grafts for prophylaxis of early rejection. Results of this pilot study are given in a separate report in this issue. In conclusion we have, for the first time, set up a large-scale preparation of polyclonal globulin against a normal human alloreactive clone, and this new reagent should present several advantages over classic antilymphocyte or antithymocyte sera because it contains specificities against activation antigens and has less crossreactivity variation among batches.

Evidence that early acute renal failure may be mediated by CD3- CD16+ cells in a kidney graft recipient with large granular lymphocyte proliferation.

We report here on a patient with a large granular lymphocyte proliferative disease who received a third kidney allograft. This patient presented a lymphocytosis (culminating at approximately 30,000/mm3) with a large proportion (approximately 70%) of CD3- WT31- CD2+ CD16+ lymphocytes. Five days after a kidney graft and during prophylactic treatment by Ortho pan OKT3, he presented an acute graft failure with an apparent interruption of graft blood flow as assessed by the Tc99 scan pattern and an arteriogram. The biopsy showed an abnormal accumulation of intravascular CD3- CD16+ cells bound to endothelial cells with thrombilike patterns in small and middle-sized arteries, whereas CD3+ mononucleated cells infiltrate was restricted to interstitium as observed in his previous graft, performed before the appearance of the lymphoproliferative disorder. The syndrome resolved spontaneously. The role of OKT3-mediated release of cytokines able to upregulate endothelial cell adhesion molecules in triggering this phenomenon is discussed.

Characterization of skin-infiltrating T lymphocytes during acute graft-versus-host disease.

A panel of 34 clones was established from a cell line derived from the skin biopsy of a patient (genotype: A1, A2, B7, B8, DR3, DR6) undergoing acute graft-versus-host disease after semiallogeneic bone marrow transplantation with his mother's bone marrow (genotype: A1, A1, B7, B8, DR3, DR6). The T-cell line obtained presented the following phenotype: CD3+, CD4+, CD8-, CD16-, WT31+, T-cell receptor delta 1-, 4B4+, 2H4-, CD25+, DR+. This CD4+ T-cell line was poorly cytotoxic against the target cells tested, including the mother's phytohemagglutinin blasts as a negative control (autologous T cells), the father's phytohemagglutinin blasts bearing the mismatch haplotype, K562, U937, SVK14 (a keratinocyte cell line), and a panel of B-lymphoblastoid cell lines bearing HLA-A2, the known mismatch antigen. All but 1 of the 34 clones obtained were of CD4+ phenotype, and none was CD16+. Only the sole CD8+ clone showed significant cytotoxicity against the father's phytohemagglutinin blast; however, this cytotoxic activity was associated with the highest score for nonspecific killing against both K562 and U937. This work demonstrates the feasibility of obtaining a large panel of clones from a graft-versus-host disease target organ to constitute the basic cellular material for in vitro study of the graft-versus-host process.

Alteration of the T cell repertoire after bone marrow transplantation.

We investigated T cell receptor (TCR) alpha/beta and gamma/delta repertoire reconstitution after autologous and allogeneic bone marrow transplantation (BMT) in humans using 13 monoclonal antibodies directed at constant and variable regions of the TCR. The TCR gamma/delta repertoire was studied kinetically during the first month and then 1 year post-BMT whereas alpha/beta peripheral blood T lymphocytes (PBTL) were studied within the first 3 months and 1 year post-BMT. Through these two studies, we found 7 of 26 patients with over-represented TCR gamma/delta subsets during the early period post-BMT. Moreover, during this period the V gamma 9V delta 2 frequency among gamma/delta T cells was found to be higher than among normal donors. Study on TCR alpha/beta T cells also revealed abnormally expanded V-specific subset (5 of 10 patients within 3 months following BMT) demonstrating that repertoire alteration early after BMT is a general phenomenon concerning potentially all T cell subsets. More surprisingly, the alpha/beta T cell repertoire was also found to be altered late after BMT (7 of 15 patients after 1 year post-BMT presented one or more overepresented alpha/beta TCR subset). These alterations of TCR combinatorial diversity should be taken into account in understanding the immunological status of patients after BMT.

Recognition of leukemic blasts by HLA-DPB1-specific cytotoxic T cell clones: a perspective for adjuvant immunotherapy post-bone marrow transplantation.

There is increasing evidence that the immune response plays a role in the prevention of leukemic relapses after allogeneic bone marrow transplantation (BMT). Producing this effect (referred to as the graft-versus-leukemia reaction or GVL) is a current goal of clinical transplantation. At present, all protocols rely on the injection of donor T cells with unknown specificities. In keeping with this approach, we recently proposed the use of a single allogeneic T cell clone transfected with the HSv-tk gene to target an HLA-DPB1 mismatch in the GVH direction. For this strategy to be successful, HLA-DP antigens must be expressed on leukemic cells, which should be recognised by the HLA-DP-specific T cell clone and subsequently destroyed. In the present study, differential expression of HLA-DR, -DQ and -DP was tested by fluorescence using monoclonal antibodies on a panel of 46 acute myeloid leukemias (AML), 28 acute lymphoblastic leukemias (ALL) and 31 chronic lymphocytic leukemias of B cell origin (B-CLL). The vast majority of leukemic cells expressed HLA-DP antigens although with considerable variability. HLA-DPB1 genotyped leukemic cells were used as target cells for an HLA-DPB1*0401-specific T cell clone. Specific recognition of leukemic blasts was demonstrated for 11 out of 11 B-CLL, 11 out of 19 AML and nine out of 16 ALL. These data show that most leukemic blasts are accessible to direct lysis by allogeneic HLA-DP-specific T cells.