CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system.

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.

Cytotoxicity of anti-CD64-ricin a chain immunotoxin against human acute myeloid leukemia cells in vitro and in SCID mice.

Blast cells from patients with acute myeloid leukemia (AML) commonly express CD64, the high-affinity receptor for immunoglobulin G (FcgammaRI). An immunotoxin (MDX-44) was constructed by coupling humanized anti-CD64 monoclonal antibody (mAb) H22 via a bivalent linker to deglycosylated ricin A-chain (RA). Human leukemia cell lines were incubated with MDX-44 or H22/free RA. The effect of MDX-44 on the proliferation of leukemia cells was assessed by [(3)H]thymidine incorporation. In the presence of interferon-gamma (IFN-gamma), MDX-44 significantly inhibited the proliferation of CD64(+) HL-60, NB4, and U937 cells in 72-h cultures in a dose-dependent manner. The mechanism of action appeared to be the induction of apoptosis, as measured by propidium iodide staining and flow cytometry analysis. However, CD64(-) KG-1a and Daudi cells were not affected by MDX-44/IFN-gamma. Incubating HL-60 cells with MDX-44/IFN-gamma resulted in a 99% decrease in colony-forming units, whereas colony-forming cells in normal bone marrow were not significantly suppressed by such treatment. Cells from 60% of AML patients (6/10) were inhibited by MDX-44/IFN-gamma, and the inhibition was correlated with CD64 expression on these cells (r = 0.65). In a human AML model in NOD/SCID mice, MDX-44/IFN-gamma inhibited 95-98% of peritoneal exudate AML cell proliferation and 85-90% of solid leukemia masses. The effect of MDX-44 on AML cells was dependent on activation of cells by IFN-gamma. MDX-44/IFN-gamma may have value in the therapy of AML cells expressing cell-surface CD64.

Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen.

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.

Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term.

Multilineage precursor cells from 14-day B6 (C57B1/6J) mouse fetal liver and adult bone marrow that repopulate both the lymphoid and myeloid systems were compared by competitive repopulation. Cells were assayed in normally functioning populations, and enrichment, tissue culture, and induced marking were avoided since these manipulations might affect cell function. Fetal or adult donor cells were mixed with marked adult competitor cells and transplanted into irradiated recipients whose blood was tested at short (25-33-day) or long (105-245-day) time periods after transplantation. Proportions of lymphocytes, granulocytes, and platelets descended from donor precursors were measured by GPI (glucosephosphate isomerase) isozyme genetic markers in congenic mice, and represent the repopulating abilities of these precursors relative to the standard competitor. For short-term repopulation 25-33 days after transplantation, fetal and adult donor cells were similar; in three studies, fetal liver contributed 0.8, 1.1, and 1.4 times as much as adult marrow per 10(5) cells transplanted. However, when long-term (105-245-day) repopulation was tested in the same recipients, fetal liver contributed 3.5, 5.0, and 7.1 times as much as adult marrow. Ratios of long-term/short-term repopulating abilities in fetal liver relative to standard adult marrow competitors were 2.5, 8.9, and 4.7, while in marrow controls, these ratios remained approximately one (1.14 and 0.80). Thus, 14-day fetal liver contains several times more long-term repopulating cells relative to short-term repopulating cells than does adult marrow. Ratios of long-term/short-term fetal cells were unchanged by precursor enrichment. The AA4.1+, Ly-6A/E+, lineage low fraction had a ratio of 4.4, although it repopulated 276 times better than unenriched fetal cells whose ratio was 4.7. There are two hypotheses that explain these data most simply: 1) There may be only a single multilineage precursor, but after transplantation cells seed in different microenvironments that support either long-term or short-term function. 2) Conversely, the difference may be at the stem cell level rather than the microenvironmental level, so that there are tow types of stem cells with multilineage differentiating ability, but only one functions over the long-term. The current report defines new conditions required by each hypothesis. If functional life spans are defined by seeding sites, as in hypothesis 1, fetal cells seed much higher proportions of long-term sites than adult cells. If different types of stem cells function short-term and long-term, as in hypothesis 2, they are not distinguished by markers allowing a 276-fold enrichment to 1367 times the repopulating ability of fresh marrow.

Evaluation of monoclonal antibody-mediated anti-acute myeloid leukemia immunotherapy in a SCID/hu model.

The therapeutic potential of the IgM complement-fixing murine monoclonal antibody (mAb) PM-81 (anti-CD15) against acute myeloid leukemia (AML) was assessed in a SCID/hu leukemia model. Intraperitoneal (i.p.) injection of NB4 leukemia cells resulted in aggressive growth of leukemia cells in the peritoneal cavity of irradiated SCID/CB-17 mice. Flow cytometric analysis of human CD15, 33 and 45 expression, as well as cytologic examination, revealed that leukemia cells disseminated into the peripheral blood and multiple tissues of the mice. The approximately linear relationship between the injected leukemia cells and the subsequent leukemia cell proliferation provided a reliable model for monitoring the therapeutic effects of immunotherapy. Intraperitoneal injection of the mAb PM-81 markedly suppressed leukemia cell growth in this SCID/leukemia model. Most of the untreated mice died within 35-50 days of leukemia cell inoculation. Four weeks after inoculation of NB4 cells, five of nine mAb PM-81 treated mice had no solid tumor growth and six of nine had no detectable peritoneal exudate leukemia cells as determined by flow cytometry. In contrast, 100% of the mice in the untreated or control mAb groups were found to have both solid and peritoneal leukemia growth. In further experiments designed to evaluate the effects of therapy on survival, 50% (4/8) of PM-81 treated mice survived to 150 days, and had no detectable solid or suspension leukemia cells detectable at necropsy. In contrast, the median survival of untreated or negative control antibody-treated mice was 40 days (comparison to PM-81 treated; p = 0.006 and p = 0.03, respectively). The mechanism of leukemia cell suppression is not likely due to complement fixation since we could not demonstrate in vitro any cytotoxicity mediated by SCID mouse plasma. Further study is required to understand the mechanism of the antileukemia effect of PM-81 in this model.

Distinct developmental patterns of short-term and long-term functioning lymphoid and myeloid precursors defined by competitive limiting dilution analysis in vivo.

Functional abilities of individual marrow precursor cells were defined by competitive limiting dilution without enrichment, tissue culture, or induced marking, manipulations that might affect cell functions. We directly measured long-term repopulating abilities in limiting doses (0.25-1.0 x 10(5)) of genetically marked congenic marrow cells. These were mixed with a standard dose of 4 or 5 x 10(5) competitor marrow cells, which contained a predictable distribution of precursor cells and allowed quantitative assays. Percentages of donor type T and B lymphocytes, granulocytes, platelets, and erythrocytes were measured in recipient blood. Applying the maximum likelihood statistic, concentrations (per 10(5)) of precursors repopulating at least one lineage were: 4.7 and 6.0 after 6 wk, 1.6 and 2.7 after 14 to 15 wk, and 1.2 and 1.9 after 30 to 32 wk; concentrations repopulating at least three lineages were 2.3 and 3.4 after 6 wk, 0.9 and 1.7 after 14 to 15 wk, and 0.9 and 1.3 after 32 wk. Almost all precursors functioning after 14 wk repopulated all lineages. At 6 wk, similar levels of donor cells were produced in recipients of both short- and long-term precursors. However, after 14 to 32 wk, contributions by short-term precursors (about two-thirds of the precursors) dropped to zero, while contributions by long-term precursors (about one-quarter of the precursors) expanded severalfold. The latter permanently repopulated all lineages after 30 to 32 wk, functioning as the most primitive stem cells (PSC) in the immune and myeloid systems. Nearly all the variance in long-term repopulated recipients was explained using the Poisson distribution to calculate donor percentages in a model where each donor and competitor PSC contributed equally.

The appearance of Thy-1- donor T cells in the peripheral circulation 3-6 weeks after bone marrow transplantation suggests an extrathymic origin.

Donor and host T cells were distinguished by T cell antigen marker Thy-1 isotype and cytoplasmic isozyme Gpi-1 in this study of bone marrow transplantation between congenic mice. During the first 3-6 weeks after irradiation and marrow transfer, percentages of cells bearing the donor Thy-1 isotype in the periphery are much lower than percentages of T cells bearing the donor Gpi-1 marker. Apparently a population of Thy-1- donor T cells exists for several weeks after bone marrow transplantation. Further study showed that this population of CD3+, Thy-1- donor T cells expressed CD4+ or CD8+ and was found in peripheral blood and spleen but not in the thymus. This finding suggests their extrathymic origin.

Homologous restriction of complement-mediated cell lysis can be markedly enhanced by blocking decay-accelerating factor.

Regulation of complement (C') dependent lysis of cells is attributed to certain membrane proteins. One of these is decay-accelerating factor (DAF), CD55, a 70kD glycosylated protein which functions to protect host cells from damage by autologous C'. We hypothesized that blockade of DAF function by a monoclonal antibody (mAb) could augment C'-dependent lysis mediated by another mAb to a cell surface antigen expressed on leukaemia cells. Thus, we tested the effects of the anti-DAF mAb 1C6 on the ability of both rabbit and human C' to lyse human leukaemia cells through activation by complement-fixing murine mAb. DAF antigen was highly expressed on most leukaemia cell lines and primary acute leukaemia blast cells tested. Murine mAb to CD15 (PM-81) and to gp 120 (AML-1-99), both IgM, also bound to the majority of myeloid and lymphoid leukaemia cells. Using human serum as a source of C', the addition of mAb 1C6 reduced by an additional 85-94% the numbers of clonogenic HL-60 (myeloid leukaemia) cells lysed by mAb PM-81 alone. Similarly, the addition of mAb 1C6 reduced by an additional 87% the numbers of HL-60 colonies eliminated by mAb AML-1-99 alone. Similar results were observed in an experimental purging model using the myeloid leukaemia cell lines HL-60, U937 or NB4 cells as targets. These results show that mAb 1C6 can effectively block the actions of DAF. In the presence of mAb 1C6, the cytotoxic activity mediated by human C' was similar to that of rabbit C'. These results show that increased tumour cell killing can be achieved through DAF blockade. This finding has relevance to clinical trials using C'-fixing mAb for purging bone marrow of occult tumour cells prior to autologous transplantation.

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells.

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.

Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging.

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4-HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4-HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4-HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.

Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations.

Quantitative analyses of primitive hemopoietic stem cell (PHSC) populations are important both for basic biology and for clinical applications. Unfortunately, many conventional assays fail to measure long-term repopulating ability and maximal differentiating ability, the most important characteristics of the PHSC. The competitive repopulation assay described here focuses on this characteristic, assaying the precursors from which most differentiated cells are descended over large fractions of the life span in laboratory mice. Thus long-term repopulating ability and the ability to differentiate into both myeloid and lymphoid lineages are measured directly from 2.5 to 12.5 months after transplantation. This technique also has found high correlations between granulocytes, macrophages, and T and B lymphocytes as early as 3 weeks after transplantation. All or most differentiated cells of these widely disparate types appear to be descended from a common precursor cell, while myeloid-specific or lymphoid-specific precursors produce few or no descendants. However, large increases in variances between 3 and 6 weeks and 12 weeks after transplantation suggest that most of the initially active multilineage precursors are exhausted. Thus the ability to differentiate into widely disparate lineages does not establish long-term repopulating ability.

The same exhaustible multilineage precursor produces both myeloid and lymphoid cells as early as 3-4 weeks after marrow transplantation.

Hemopoietic precursors with the ability to differentiate into wide varieties of cell types are considered primitive, as are precursors with long-term repopulating ability. Here we study the populations of marrow precursors from which both myeloid and lymphoid lineages are descended shortly after transplantation. Surprisingly, few or none of these precursors show long-term repopulating ability. Equal portions of a mixture of marrow cells from C57BL/6J (B6) and congenic B6-Hbbd Gpi-1a mice are transplanted into a group of recipients. Three weeks later, highly significant correlations between percentages of B6 type T cells, B cells, granulocytes, and platelets in each recipient indicate that many lymphoid and myeloid cells are descended from common precursors. After 4-6 weeks, most correlations between lymphoid and myeloid cells improve, indicating that most or all differentiated cells are descended from common precursors. The more differentiated myeloid-specific precursors found in spleen colony-forming cell assays apparently fail to contribute significantly to the differentiated myeloid cell populations tested. By using the binomial model, in which variability of the data among the recipients is inversely related to the number of precursors in the mixture, donor precursor concentrations are estimated as approximately 21 per 10(5) marrow cells after 3 weeks, falling 3-fold to 6.6 per 10(5) after 4-6 weeks. This trend continues, with higher correlations, greater variabilities, and donor precursor concentrations of 1.9 per 10(5) marrow cells after 12-14 weeks and 1.4 per 10(5) after 24 weeks. Strong increases in variances between 3 and 12 weeks after transplantation suggest that most or all of the initially active multilineage precursors are exhausted during this time period. The fact that the ability of a hemopoietic stem cell to differentiate into widely disparate lineages is not associated with long-term repopulating ability requires a change in stem cell definitions, since primitive hemopoietic stem cells have traditionally been defined by both these abilities.

Activity of human placental gamma globulin in blocking immune functions in vitro and in abrogating the xenogeneic, local graft-versus-host reaction.

In order to understand the mechanism of immunosuppression caused by infusion of placental gamma globulin (PGG) in patients with renal allografts, rheumatoid arthritis, and graft-versus-host disease (GVHD) following bone marrow transplantation (BMT), we have examined the effect of PGG in vitro and in a model of the xenogeneic, local graft-versus-host reaction (LGVHR). PGG inhibited lymphocyte proliferation in mixed lymphocyte cultures (MLC) (P less than 0.005) and depressed interleukin-2 (IL-2) levels in such cultures at 72 hours (P less than 0.01). In contrast phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-induced T and B lymphocyte blastogenesis was not affected by such PGG treatment. PGG neither decreased the [3H] TdR pulse incorporation in unstimulated lymphocytes nor affected cell viability. Cell cycle analysis by flow cytometry showed that PGG reduced the percentage of cells in S and G2, M phases during the MLC, but did not alter cell cycling during PWM-stimulated proliferation. An immunosuppressive effect of PGG on the LGVHR was tested in a model of intracutaneous transplantation of PGG-treated human lymphocytes into cyclophosphamide-immunosuppressed rats. Lymphoprep-separated human tonsillar lymphocytes were incubated with RPMI-1640 buffer containing: (1)PGG, 4 mg/ml, (2) human plasma albumin, 4 mg/ml, (3) mitomycin-C, 25 micrograms/ml, or (4) no additive. Cells of each preparation (3 X 10(7) cells in 0.1 ml) were injected intracutaneously into cyclophosphamide-treated male rats at separate abdominal locations. A fifth site received only the buffer solution. Five days after injection of cells, each rat received [125I]UdR (10 muCi) intraperitoneally and was killed after 5 hours.(ABSTRACT TRUNCATED AT 250 WORDS)