Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis.

BACKGROUND: CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells. METHODS: CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity. RESULTS: In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin. DISCUSSION: The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.

Donor natural killer (NK1.1+) cells do not play a role in the suppression of GVHD or in the mediation of GVL reactions after DLI.

Donor regulatory T cells (CD3+ alphabetaT-cell receptor [TCR]+) derived from the repopulating host thymus have been shown to be primarily responsible for suppression of GVHD following DLI therapy in murine BMT models. However, natural killer (NK) T cells also have regulatory properties, and a role for NK T cells in suppression of GVH reactivity has not been completely excluded. NK cells may also contribute to the graft-versus-leukemia (GVL) effect associated with DLI therapy. In this study, we used a murine BMT model (C57BL/6 into AKR) to study whether depletion of donor NK cells had any impact on the suppression of GVH reactivity after DLI or on the DLI-induced GVL effect against acute T-cell leukemia. Depletion of donor NK cells was accomplished in vivo by giving DLI-treated bone marrow chimeras multiple injections of anti-NK1.1 monoclonal antibody (MoAb). The chimeras treated with anti-NK1.1 MoAb had significantly fewer splenic NK1.1 cells than nontreated chimeras, and splenocytes from anti-NK1.1-treated mice were deficient in the ability to generate lymphokine-activated lytic activity. Results presented here showed that NK-cell depletion had no effect on the suppression of GVH reactivity after DLI. When DLI-treated chimeras were challenged with an acute T-cell leukemia, NK-cell depletion had no discernible effect on GVL reactivity. These preclinical data suggest that donor NK cells do not have a significant role in the suppression of GVHD after DLI or in the mediation of GVL reactivity induced by DLI.

Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following donor leukocyte infusion therapy.

In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR beta-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed alphabetaTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+alphabetaTCR+ cells consisted of two subpopulations: a CD4+CD8- subpopulation and a CD4-CD8- subpopulation. Together, these results show that thymus-derived, Thy1+alphabetaTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.

Photochemical treatment with S-59 psoralen and ultraviolet A light to control the fate of naive or primed T lymphocytes in vivo after allogeneic bone marrow transplantation.

Donor leukocyte infusions after allogeneic bone marrow transplantation can provide a curative graft-vs-leukemia (GVL) effect, but there is a significant risk of graft-vs-host (GVH) disease. A simple and effective method for controlling the fate of naive or primed T-lymphocytes in vivo without eliminating their beneficial properties is needed. In this report, photochemical treatment (PCT) ex vivo with a synthetic psoralen (S-59) and UVA light was evaluated as a pharmacological approach to limiting the proliferation and GVH potential of naive and primed donor T cells in vivo. S-59 rapidly intercalates into and cross-links DNA on UVA illumination. The effects of PCT on T cells were found to be both S-59 and UVA dose dependent. With selected PCT regimens, treated T cells still expressed activation markers (CD25 and CD69) and secreted IL-2 on activation, but they showed limited proliferative capacity in vitro and in vivo. Clonal expansion of CTL in MLR was reduced after PCT, but short term lytic activity of primed CTL was not affected. In a murine model of MHC-mismatched bone marrow transplantation, the addition of PCT-treated T cells to T-depleted bone marrow facilitated donor engraftment and complete chimerism without causing acute or chronic graft-vs-host disease. Allospecific GVL reactivity was reduced but not eliminated after PCT treatment. In an MHC-matched model using host-presensitized donor T cells, PCT significantly reduced GVH-associated mortality without eliminating GVL reactivity. Thus, PCT ex vivo offers a simple, rapid, and inexpensive method by which to control the fate of naive and primed T cells in vivo.

Graft-vs.-host and graft-vs.-leukemia reactions after delayed infusions of donor T-subsets.

Infusions of donor leukocytes have been given to allogeneic bone marrow recipients after transplant to treat leukemia relapse. Treatment with these delayed infusions of donor cells has been called delayed or donor leukocyte infusion (DLI). While graft-vs.-host disease (GVHD) has typically been less severe than expected after DLI, it still remains a significant risk factor. Recently, we used a full major histocompatibility complex (MHC)-mismatched model (C57BL/6 into AKR) to determine how increased immunogenetic disparity affects GVH and graft-vs.-leukemia (GVL) reactions after DLI. In contrast to an MHC-matched model (B10.BR into AKR), GVHD was still observed when MHC-mismatched donor T cells were infused 3 weeks posttransplant. Limiting dilution analysis was used to determine the frequency of alloreactive cytotoxic T lymphocytes (CTL) and interleukin (IL)-2-secreting T helper cells in the spleens of MHC-mismatched recipients 7 days after DLI treatment. GVHD correlated with elevated frequencies of alloreactive T-helper cells. One strategy for reducing the severity of GVHD after DLI is the selective administration of CD4 or CD8 T-subsets. Delayed infusion of purified T-subsets 3 weeks posttransplant resulted in significantly less GVHD than infusion of a mixture of the two subsets. No GVH-associated mortality was observed after DLI with purified donor CD4+ T cells. In GVL studies, MHC-mismatched CD8+ T cells were the most potent antitumor effectors against an acute T cell leukemia. The GVL effect of MHC-mismatched T-subsets was compared with that of MHC-matched subsets. When naive MHC-matched cells were given as DLI, depletion of either T-subset eliminated the GVL effect. CD8+ T cells from MHC-matched donors primed against host alloantigens, however, mediated a CD4 (T-helper)-independent GVL reaction. Together, these results suggest that administration of T-subsets can significantly reduce GVHD after DLI without loss of the beneficial GVL effect.

Sca1(+)/Mac1(+) nitric oxide-producing cells in the spleens of recipients early following bone marrow transplant suppress T cell responses in vitro.

Spleen cells collected from allogeneic chimeras early after bone marrow transplantation (BMT) consistently showed suppressed proliferative responses to interleukin-2 in vitro and failed to proliferate in mixed lymphocyte reaction (MLR) assays. However, isolation of Thy 1.2(+) T cells from the heterogeneous spleen cell suspension prior to culture resulted in heightened proliferation, suggesting the presence of cells capable of suppressing T cell responses in vitro. When separated into subpopulations by negative and positive selection with specific monoclonal antibodies, a non-T, non-B population with immunosuppressive properties was identified. The suppressive cells were found in the spleens of both allogeneic and syngeneic chimeras, but not normal donor mice. Suppressor activity was transient and typically declined by 3 weeks post-BMT. The cells suppressed the response of alloactivated T cells isolated from BMT chimeras as well as naive donor T cells in MLR assays in a dose-dependent manner. To explore the mechanism(s) involved in the suppression, the effects of interferon-gamma (IFN-gamma)-specific mAb and the nitric oxide (NO) synthase inhibitor NG-methyl-l-arginine were examined. The results support a role for both IFN-gamma and NO in the suppressive activity. Separation of cells based on Mac-1 expression indicated that there were both Mac-1-enriched and Mac-1-depleted cells capable of producing NO, but that the Mac-1-depleted cells were the most potent suppressors in MLR assays. The Mac-1-depleted cells still contained a residual population of Mac-1(dim) cells which showed increased levels of Mac-1 expression after overnight culture. Intracellular staining with an inducible nitric oxide synthase (iNOS)-specific mAb indicated that the NO-producing cells expressed the cell surface markers Mac-1 and Sca-1. When iNOS knockout transgenic mice were used as transplant donors, in vitro suppression of T cell responses was reduced but not eliminated, suggesting that other mechanism(s) could contribute to the suppression. Collectively, these results demonstrate that Sca-1(+)/Mac-1(+) cells capable of producing NO are present in the spleens of recipients early after BMT and suggest that these cells may have immunoregulatory roles in vivo.

Glycosphingolipids as novel targets for T-cell suppression by the B subunit of recombinant heat-labile enterotoxin.

Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived from Escherichia coli that binds to ganglioside GM1, a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8+ T cells bound twice as much as CD4+ T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8+ T cells were more susceptible to rLTB than either CD4+ T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4+ T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8+ T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37 degrees C. Binding to ganglioside GM1 was essential for suppression, since rLTB/G33D, a mutant which does not bind GM1, failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8+ T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.

The graft-versus-leukemia effect of post-transplant donor leukocyte infusion.

Tumor relapse remains a major obstacle to the success of allogeneic bone marrow transplantation (BMT) as a treatment for leukemia. Due to limited treatment options, the outlook for most patients that relapse following allogeneic BMT has been poor. The infusion of normal immunocompetent leukocytes from the original marrow donor has become a promising new option for treating/preventing leukemia relapse in allogeneic BMT recipients. This form of treatment has often been referred to as donor leukocyte infusion (DLI) therapy. Our laboratory is using murine models of allogeneic BMT to address important unresolved issues regarding DLI therapy in an effort to make the treatment more effective. These include identification of the antileukemic effector cells, augmentation of the antileukemic effect, and understanding why graft-versus-host-disease (GVHD) is less severe than anticipated. This article reviews our work in murine models of DLI and introduces our current working hypotheses concerning DLI therapy.

Human adenovirus-specific CD8+ T-cell responses are not inhibited by E3-19K in the presence of gamma interferon.

Adenovirus has considerable potential as a gene therapy vector, but recent animal data suggest that transduced cells are destroyed by adenovirus-specific cytotoxic T-lymphocyte (CTL) responses. Therefore, it will be important to develop strategies to evade adenovirus-specific CTL responses in humans. As a first step, an assay was developed to detect and characterize human CTLs directed against adenovirus. Adenovirus-specific CTL responses were demonstrated to be present in four of five healthy adults by in vitro stimulation of peripheral blood mononuclear cells with autologous fibroblasts infected with the adenovirus type 2 (Ad2) E3 deletion mutant Ad2+ND1. Killing by adenovirus-specific CTLs was major histocompatibility complex class I restricted and was documented to be mediated by CD8+ T cells. Wild-type-Ad2-infected cells were poor CTL targets compared with cells infected with the E3 deletion mutant because of the expression of E3-19K, an early viral glycoprotein which prevents transport of major histocompatibility complex class I antigens out of the endoplasmic reticulum to the cell surface. However, preincubation of targets with gamma interferon resulted in enhanced killing of wild-type-Ad2-infected cells, to levels comparable to those obtained with Ad2+ ND1-infected cells. Radioimmunoprecipitation analysis revealed that gamma interferon not only increased the synthesis of class I antigens but also allowed excess molecules to escape from the endoplasmic reticulum. It is concluded that E3-19K expression in adenovirus-infected cells inhibits human CTL recognition in vitro but that gamma interferon may help overcome the E3-19K effect during acute infection in vivo.

Unrelated donor bone marrow transplantation to treat severe aplastic anaemia in children and young adults.

Alternative donor bone marrow transplantation (BMT) to treat severe aplastic anaemia (SAA) in children and young adults has been complicated by high rates of graft rejection and severe graft-versus-host disease (GVHD). We hypothesized that increased immunosuppression combined with T-cell depletion of the marrow graft would enable successful use of unrelated donor BMT in this disease. Preconditioning consisted of cytosine arabinoside, cyclophosphamide, and total body irradiation (TBI). T-cell depletion was with the anti-CD3 antibody T10B9. GVHD prophylaxis consisted of cyclosporine A. 28 previously transfused patients were transplanted. Nine donor/recipient pairs were HLA matched. As of 1 January 1996, 15/28 (54%) patients are alive, transfusion independent and well with a range of follow-up of 13 months to 8 years (median 2.75 years). Fatalities include all three patients with nonengraftment and all three patients with grade III/IV GVHD. Other fatalities were due to infections or therapy-related toxicity. The incidence >or= grade II acute GVHD was 28%. These data show that in children with SAA who have failed immunosuppression, unrelated donor BMT offers a reasonable hope of long-term survival.

Hematologic and immunologic evaluation of recombinant human interleukin-6 in patients with advanced malignant disease: evidence for monocyte activation.

Eighteen advanced cancer patients received weekday subcutaneous injections of recombinant interleukin-6 (rIL-6) for 4 weeks at escalating doses. Patients were evaluated for hematologic and immune system effects. Hematologic monitoring included WBC, differential, Hgb and Hct, platelet counts, and assessment of marrow and peripheral blood progenitors. Immunologic monitoring included evaluation of acute-phase reactants (APRs), immunophenotyping, serum cytokine levels, cytokine-induced proteins, and cytokine messenger RNA (mRNA). The maximal tolerated dose (MTD) was 8.0 micrograms/kg/day, with neurocortical toxicity as the major limiting factor. All patients became anemic, and most had fever and chills. APRs were increased throughout treatment. WBCs increased transiently on day 2; granulocytes and monocytes increased again through day 26, whereas lymphocytes decreased to baseline or lower levels. Platelets responded by day 12 and increased through day 26 at the MTD with no effect on colony-forming unit-megakaryocyte (CFU-Mk). Peripheral WBC and RBC progenitors were not affected but decreased in the marrow. T-cell percentages declined with little effect on absolute numbers; T-cell activation was seen. CD45RO+ T cells decreased, but there was no significant effect on CD8+ CD28+ T cells. Neither B cells nor natural killer (NK) cells were affected. However, evidence of monocyte effects included upregulation of CD71, induction of the cytokine-induced proteins 2-5A synthetase and neopterin, and increases in tumor necrosis factor-alpha (TNF-alpha) mRNA. Serum cytokines were undetected, and mRNA for IL-1 beta, IL-2, and interferon-gamma (IFN-gamma) was not induced; however, mRNA for IL-4 and IL-10 did increase suggesting activation of Th2-like T cells. One mixed tumor response was seen. We conclude that IL-6 alone has systemic activity on the immune system, as well as the hematopoietic system, which at the MTD, primarily involves induction of APR, activation and expansion of monocytes, and activation of Th2-like T cells.

Immunoregulatory properties of ISG15, an interferon-induced cytokine.

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.

Delayed infusion of immunocompetent donor cells after bone marrow transplantation breaks graft-host tolerance allows for persistent antileukemic reactivity without severe graft-versus-host disease.

The development of graft-host tolerance after bone marrow transplantation (BMT) is crucial to avoid the problems of graft-versus-host disease (GVHD) and graft rejection. GVHD can be eliminated by depleting mature donor T cells from the BM inoculum, thereby facilitating the development of graft-host tolerance. However, T-cell depletion often results in an increased incidence of graft rejection and an increased frequency of leukemia relapse. Thus, although graft-host tolerance is a desirable outcome, it can pose a significant threat to leukemia-bearing hosts. Using a major histocompatability complex (MHC)-matched allogeneic model of BMT (B10.BR into AKR), we found that irradiated recipients given donor BM alone displayed mixed T-cell chimerism and did not develop GVHD. Graft-host tolerance developed by 8 weeks after BMT in these chimeras, and they were susceptible to low-dose leukemia challenge. When sufficient numbers of donor spleen cells, as a source of T-cells, were added to the BM graft, AKR hosts developed severe and lethal GVHD. Antihost reactive donor T cells persisted in chimeras undergoing GVHD, indicating that graft-host tolerance did not develop. When administration of the spleen cells was delayed for 7 to 21 days after BMT, there was significantly less mortality because of GVHD. Day 21 was the optimal time for infusion of cells without development of GVHD. Graft-host tolerance was broken by the delayed infusion of donor cells, as indicated by the persistence of antihost-reactive donor T cells in these chimeras in T-cell receptor cross-linking and mixed lymphocyte reaction assays. Importantly, the persistence of antihost-reactive donor T cells correlated with along-term antileukemic effect that was still present at 100 days after transplant. Multiple infusions of immunocompetent donor cells could be administered without increasing the risk for GVHD if delayed until 21 days post-BMT. Delayed infusions of donor spleen cells also resulted in a long-term antileukemic effect in the absence of GVHD in an MHC-haplotype-mismatched model of BMT (SJL into [SJL x AKR]F1). Although delayed infusion of normal donor cells did not induce GVHD, spleen cells from donors previously sensitized to host alloantigens induced GVHD when infused 21 days after BMT. Thus, the ability of previously activated cells to induce GVHD was not inhibited in the same manner as naive cells. Results from limiting dilution analysis assays indicated that alloactivated interleukin-2-secreting CD4+ T cells were preferentially inhibited over cytolytic T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of human proliferative T cell responses to adenovirus.

Although cellular immune responses are likely important for recovery from acute adenovirus infection, they have not been studied in humans. As a first step, a sensitive assay for the detection of adenovirus-specific proliferative T cell responses was developed. Peripheral blood mononuclear cells from 29 of 30 healthy adults exhibited specific proliferative responses to adenovirus type 2 antigen. Adenovirus-specific proliferation was found to be mediated by CD4+ T cells. Specific proliferative responses were detected to purified Ad2 virions and by Ad2-infected cell lysates, indicating that adenovirus structural proteins are important targets. In addition, specific proliferative responses to the uncommon adenovirus type 35 were found in persons without serologic evidence of prior Ad35 infection. These data suggest that adenovirus-specific CD4+ T cells recognize conserved antigens among different serotypes and that a majority of persons develop long-lived CD4+ T cell responses to adenovirus.

Unrelated bone marrow donor transplants for children with leukemia or myelodysplasia.

Allogeneic bone marrow transplantation is the treatment of choice for many childhood leukemias. The donor of choice-an HLA matched sibling-is only available about 30% of the time. Unrelated donors are an alternative choice. In this report, we describe the results of unrelated donor bone marrow transplants (BMT) in 50 children with leukemia (25 acute lymphoblastic leukemia [ALL], 3 acute myeloid leukemia [AML], 3 juvenile chronic myelogenous leukemia [JCML], 10 chronic myeloid leukemia [CML]) or myelodysplastic syndrome (MDS; 9). The median age of the 31 male and 19 female patients was 9 years (range 2 to 18). Only 13 patients were serologically matched at HLA-A, B, DR, and DQ with their donors; 6 of these were reactive in mixed lymphocyte culture. The other 37 patients were mismatched for one (36 patients) or more (1 patient) HLA antigens. Pretransplant conditioning included cytosine arabinoside, cyclophosphamide, fractionated total body irradiation (TBI) (with lung, liver, and more recently, kidney shielding), and methylprednisolone. High-risk patients also received busulfan. Graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion with IgM monoclonal antibody T10B9 plus complement and posttransplant cyclosporine-A. Forty-nine patients (98%) engrafted. Median times to greater than 500 polymorphonuclear leukocytes (PMN)/microL and greater than 25,000 platelets/microL were 18 and 20 days, respectively. Acute GVHD > or = grade II occurred in 16 patients (33%); 13 (81%) of these died. Chronic GVHD developed in 30 of 40 patients at risk, but was extensive in only 5. Event-free survival (EFS) for all patients was 44% +/- 7% (median follow-up was 49 months), and overall survival was 50 +/- 7%. Patients with low-risk disease (ALL or AML in first or second remission and CML in chronic phase) had a better EFS than children with high-risk disease (60% v 34%, P = .07). There was no significant difference in EFS between patients who were serologically matched with their donors (46%) and those who were partially mismatched (43%) (P = .97). These data compare favorably with published reports for children transplanted with HLA-matched sibling donors and should encourage earlier consideration of unrelated donor BMT in children with leukemia or myelodysplasia.

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation.

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.

A subcutaneous capillary filtrate collector for measurement of blood chemistries.

The capillary filtrate collector (CFC) contains 30,000 molecular weight cut-off, hollow fiber ultrafiltration membranes that are placed below the skin. A transcutaneous tube leads to an evacuated glass tube that provides a vacuum to pull ultrafiltrate at 40-60 microliters/hr from blood, through the fibers, and past a sampling port to the glass tube. Long-term (1-6 months) animal and clinical studies have shown that the ultrafiltrate concentration of chemicals such as glucose and a variety of drugs is exactly the same as that of the blood plasma water when the ultrafiltrate is created. In this study, the device was placed in six home monitored diabetics and four in-center hemodialysis diabetic patients. Over the following month, blood glucose concentrations were compared to CFC glucose concentrations. In spite of difficulties in diluting and assaying small samples of filtrate, there was a good correlation between blood and CFC glucose levels. A flow-through enzymatic glucose sensor has been tested and shown to accurately measure glucose in CFC filtrate. When placed in the transcutaneous tubing near the skin, this should allow a small external device to continuously monitor glucose levels in brittle or out of control diabetes with high accuracy and little risk, discomfort, or cost.

Delayed infusion of normal donor cells after MHC-matched bone marrow transplantation provides an antileukemia reaction without graft-versus-host disease.

When allogeneic BMT is used for the treatment of leukemia, depletion of T cells from the donor BM to avoid GVHD may be accompanied by persistence of host cells and post-transplant relapse. In this report, a murine model of MHC-compatible BMT was used to show that delayed infusion of immunocompetent donor cells early after T cell-deficient BMT eliminated residual host cells and provided an antileukemic effect without causing lethal GVHD. AKR (H-2k) recipient mice were pre-conditioned with 9 Gy total body irradiation (LD50) and transplanted with 10(7) BM cells from MHC-matched B10.BR donors. These mice did not develop GVHD and became stable, long-term mixed (donor-host) T cell chimeras. In this model, mixed or incomplete donor T cell chimerism was associated with decreased GVL reactivity. AKR hosts that were transplanted with B10.BR bone marrow admixed with 3 x 10(7) B10.BR spleen cells (as a source of T cells) became complete donor T cell chimeras, but they developed severe and lethal GVHD. However, when the infusion of donor spleen cells was delayed until 21 days after BMT, few mice exhibited any clinical signs of GVHD, and > 95% of the mice became long-term survivors. The infused spleen cells eliminated residual host T cells by 21 days after infusion, and most chimeras were able to resist a supralethal challenge with AKR leukemia/lymphoma cells. Thus, post-transplant adoptive immunotherapy with normal mononuclear cells from the marrow donor may be an effective way to eliminate residual disease or treat leukemia relapse after BMT without causing significant GVHD.