Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression.

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5-11.4 years, while three required reinstitution of IS after 5-8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction.

Physics of a rapid CD4 lymphocyte count with colloidal gold.

The inherent surface charges and small diameters that confer colloidal stability to gold particle conjugates (immunogold) are detrimental to rapid cell surface labeling and distinct cluster definition in flow cytometric light scatter assays. Although the inherent immunogold surface charge prevents self aggregation when stored in liquid suspension, it also slows binding to cells to timeframes of hours and inhibits cell surface coverage. Although the small diameter of immunogold particles prevents settling when in liquid suspension, small particles have small light scattering cross sections and weak light scatter signals. We report a new, small particle lyophilized immunogold reagent that maintains activity after 42°C storage for a year and can be rapidly dissolved into stable liquid suspension for use in labelling cells with larger particle aggregates that have enhanced scattering cross section. Labeling requires less than 1 min at 20°C, which is ∼30 times faster than customary fluorescent antibody labeling. The labeling step involves neutralizing the surface charge of immunogold and creating specifically bound aggregates of gold on the cell surface. This process provides distinct side-scatter cluster separation with blue laser light at 488 nm, which is further improved by using red laser light at 640 nm. Similar comparisons using LED light sources showed less improvement with red light, thereby indicating that coherent light scatter is of significance in enhancing side-scatter cluster separation. The physical principles elucidated here for this technique are compatible with most flow cytometers; however, future studies of its clinical efficacy should be of primary interest in point-of-care applications where robust reagents and rapid results are important.

Mechanisms of donor-specific tolerance in recipients of haploidentical combined bone marrow/kidney transplantation.

We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

Acute renal endothelial injury during marrow recovery in a cohort of combined kidney and bone marrow allografts.

An idiopathic capillary leak syndrome ('engraftment syndrome') often occurs in recipients of hematopoietic cells, manifested clinically by transient azotemia and sometimes fever and fluid retention. Here, we report the renal pathology in 10 recipients of combined bone marrow and kidney allografts. Nine developed graft dysfunction on day 10-16 and renal biopsies showed marked acute tubular injury, with interstitial edema, hemorrhage and capillary congestion, with little or no interstitial infiltrate (≤10%) and marked glomerular and peritubular capillary (PTC) endothelial injury and loss by electron microscopy. Two had transient arterial endothelial inflammation; and 2 had C4d deposition. The cells in capillaries were primarily CD68(+) MPO(+) mononuclear cells and CD3(+) CD8(+) T cells, the latter with a high proliferative index (Ki67(+) ). B cells (CD20(+) ) and CD4(+) T cells were not detectable, and NK cells were rare. XY FISH showed that CD45(+) cells in PTCs were of recipient origin. Optimal treatment remains to be defined; two recovered without additional therapy, six were treated with anti-rejection regimens. Except for one patient, who later developed thrombotic microangiopathy and one with acute humoral rejection, all fully recovered within 2-4 weeks. Graft endothelium is the primary target of this process, attributable to as yet obscure mechanisms, arising during leukocyte recovery.

Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice.

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

Identification of pre-T cells in human peripheral blood. Extrathymic differentiation of CD7+CD3- cells into CD3+ gamma/delta+ or alpha/beta+ T cells.

CD7+CD3- cells purified (greater than 99.99%) by FACS from the peripheral blood of healthy adults include precursors for mature T cells that have the capacity to differentiate into TCR-1+ or TCR-2+ CD3+ cells. Extrathymic differentiation was demonstrable from all eight healthy donors in the presence of a high concentration of IL-2, mitogenic levels of PHA, and irradiated blood mononuclear feeder cells, after a lag of approximately 40 d in vitro. The extrathymic T (ET) cells were predominantly TCR-1+, although TCR-2+ cells were also derived. ET TCR-1+ cells were CD4-CD8-, CD4-CD8DIM+, and CD4+CD8-, and were distinguished from natural T TCR-2+ cells by a variety of cell surface markers. The ET cells had phenotypes generally displayed by normal mature T cells, although the CD5DIM+ on ET cells was more typical of thymocytes. Acquisition of CD3 on purified CD7+CD3- cells was not due to antigenic modulation or growth of contaminants, and ET cells could be demonstrated at the clonal level. Studies in athymic mice and bone marrow recipients support the view that extrathymic maturation does occur in vivo. Whether the CD7+CD3- cell population was unexposed to the thymus, or exposed but not processed, is unknown. In any case, unusual or "forbidden" autoreactive specificities are predicted since ET cells differentiate without thymic selection of the TCR.

Clinical outcomes of late rather than early full-donor chimerism in patients with advanced lymphomas receiving nonmyeloablative allogeneic hematopoietic SCT.

Allogeneic hematopoietic SCT (HSCT) can ideally provide long-term remission in advanced lymphoma patients by capturing a graft-vs-tumor (GVT) effect. On the basis of a murine model, we attempted to optimize a GVT effect through nonmyeloablative therapy and HLA-matched related donor HSCT with intentional induction of mixed chimerism followed by prophylactic donor lymphocyte infusion. A total of 26 advanced lymphoma patients were separated into an early and late full-donor chimerism (FDC) group using a median of 45 days post-HSCT as the defining point for FDC. Upon generating these groups, analysis by Student's t-test demonstrated that they were statistically distinct in time to develop FDC (P<0.01). There was a trend toward improved CR rates in the late group relative to the early group (62 vs 31%; P=0.12). A trend toward improved progression-free survival at 5 years was also observed in the late compared to the early group by Kaplan-Meier analysis (38 vs 8%; P=0.081). However, this did not correlate to a significant overall survival benefit. In conclusion, these data support the observation from our mouse models that the most potent GVT effect occurs in mixed chimeras with late chimerism conversion.

Comparison of outcomes after transplantation of peripheral blood stem cells versus bone marrow following an identical nonmyeloablative conditioning regimen.

This is the first study to examine the outcomes in 54 patients with hematologic malignancies who received an HLA-matched related donor bone marrow (BM, n = 42) or GCSF-mobilized peripheral blood stem cells (PBSC, n = 12) following identical nonmyeloablative conditioning with the intention of induction of mixed chimerism (MC) followed by prophylactic donor leukocyte infusion (pDLI) to convert MC to full donor chimerism (FDC) and capture a graft-versus-tumor effect without clinical graft-versus-host disease (GVHD). Neutrophil and platelet recovery were faster and transfusion requirement was less in PBSC recipients (P < 0.05). A total of 48% of BMT recipients achieved FDC with a median conversion time of 84 days, including 13 following pDLI. In contrast, 83% (P = 0.04) in the PBSC group had spontaneous FDC at a median of 14 days, precluding the administration of pDLI. There was no significant difference in the incidences of acute or chronic GVHD, though the rates of chronic GVHD were considerably higher in PBSC group than in the BM group (6/7, 86% vs 10/24, 42%). CD4 and CD8 T-cell recovery was faster in PBSC recipients. In PBSC recipients, a higher number of CD34+ cells was associated with increased rates of severe, grade III-IV acute GVHD.

Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants.

Purging of tumor cells and selection of stem cells are key technologies for enabling stem cell transplantation and stem cell gene therapy. Here we report a strategy for cell selection based on physical properties of the cells. Exposing cells to an external pulsed electric field (PEF) increases the natural potential difference across the cell membrane until a critical threshold is reached and pore formation occurs, resulting in fatal perturbation of cell physiology. Attaining this threshold is a function of the applied field intensity and cell size, with larger cells porated at lower field intensities than smaller cells. Since hematopoietic stem cells are smaller than other hematopoietic cells and tumor cells, we found that exposure of peripheral blood mononuclear cells (PBMCs) to PEFs caused stepwise elimination of monocytes without affecting the function of smaller lymphocyte populations. Mobilized peripheral blood exposed to PEFs was enriched for CD34+/CD38- cells and stem cell function was preserved. Furthermore, PEF treatment was able to selectively purge blood preparations of tumor cells and eradicate transplantable tumor.

Cell proliferation kinetics in human tumor xenografts measured with iododeoxyuridine labeling and flow cytometry: a study of heterogeneity and a comparison between different methods of calculation and other proliferation measurements.

The influence of overall treatment time in the results of fractionated radiation treatment was initially established in experimental tumors and, subsequently, in the clinic. The availability of techniques (antibodies against halogenated thymidine analogues and flow cytometry) which permit determinations of the duration of the synthesis phase, the labeling index, and the tumor potential doubling time (Tpot) in a short period of time and requiring only a small biopsy of tumor tissue, has expanded interest in the relationship between tumor cell proliferation and response to irradiation. A valuable tool in the study of this relationship are human tumor xenografts. Previous studies have shown a substantial intratumoral heterogeneity in the determinations of Tpot. Different methods of calculation of the kinetic parameters have been published. We have conducted a heterogeneity analysis and an evaluation of the different calculation methods in order to define the validity of Tpot as a proliferation rate measurement in human tumor xenografts. Results show the intertumoral variability in Tpot [between different types of human tumor xenografts systems (coefficient of variation = 88.2%)] to be greater than mean intratumoral variation (coefficient of variation = 30.8%); this suggests that this variation is potentially adequate to serve as a predictor of response. The diverse calculation methods provided significantly different absolute values but not different tumor ranking, probably because the time interval between labeling and sampling was maintained, for all the samples, between 6 and 8 h. Our study has found significant differences between the labeling index and the S-phase fraction determined with the DNA profile in 9 out of 10 tumor types. No correlation was found between the DNA index of the tumors in this series and their proliferation rate.

Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay.

Most drug discovery efforts have focused on finding new DNA-damaging agents to kill tumor cells preferentially. An alternative approach is to find ways to increase tumor-specific killing by modifying tumor-specific responses to that damage. In this report, we ask whether cells lacking the G1-S arrest in response to X-rays are more sensitive to X-ray damage when treated with agents that override G2-M arrest. Mouse embryonic fibroblasts genetically matched to be (+) or (-) p53 and rat embryonic fibroblasts (+) or (-) for wild-type p53 function were irradiated with and without caffeine, a known checkpoint inhibitor. At low doses (500 microM), caffeine caused selective radiosensitization in the p53(-) cells. At this low dose (where no effect was seen in p53(+) cells), the p53(-) cells showed a 50% reduction in the size of the G2-M arrest. At higher doses (2 mM caffeine), where sensitization was seen in both p53(+) and p53(-) cells, the radiosensitization and the G2-M override were more pronounced in the p53(-) cells. The greater caffeine-induced radiosensitization in p53(-) cells suggests that p53, already shown to control the G1-S checkpoint, may also influence aspects of G2-M arrest. These data indicate an opportunity for therapeutic gain by combining DNA-damaging agents with compounds that disrupt G2-M arrest in tumors lacking functional p53.

Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS.

Six human ovarian cancer cell lines and samples of ascites cells isolated from 27 patients with stage III or IV ovarian papillary serous cystadenocarcinoma were studied individually to test whether recombinant human Mullerian inhibiting substance (rhMIS) acts via its receptor. To do these experiments, we scaled up production of rhMIS and labeled it successfully with biotin for binding studies, cloned the human MIS type II receptor for mRNA detection, and raised antibodies to an extracellular domain peptide for protein detection. These probes were first tested on the human ovarian cancer cell lines and then applied to primary ovarian ascites cells. rhMIS inhibited colony growth of five of six cell lines that expressed the human MIS type II receptor mRNA by Northern analysis while not inhibiting receptor-negative COS cells. Flow cytometry performed on MIS-sensitive ovarian cancer cell lines demonstrated specific and saturable binding of rhMIS (Kd = 10.2 nM). Ascites cells from 15 of 27 or 56% of patients tested bound biotinylated MIS (MIS-biotin) and, of the 11 that grew in soft agarose, 9 of 11 or 82% showed statistically significant inhibition of colony formation. Of the 15 patients who bound biotinylated MIS, mRNA was available for analysis from 9, and 8 of 9 expressed MIS type II receptor mRNA by reverse transcription-PCR, showing a statistically significant correlation, compared with binding, by chi2 analysis (P = 0.025). Solid ovarian cancers were positive for the MIS type II receptor protein by immunohistochemical staining, which colocalized with staining for antibody to CA-125 (OC-125). Thus, the detection of the MIS type I receptor by flow cytometry may be a useful predictor of therapeutic response to MIS and may be a modality to rapidly choose patients with late-stage ovarian cancer for treatment with MIS.

Flow-cytometric and ultrastructural analysis of alveolar macrophage maturation.

Alveolar macrophages (AM) from adult and newborn rats were studied by flow cytometry and ultrastructural morphometry. We observed that the laser scatter and autofluorescent properties of newborn macrophages were different from those of adult cells. Relative to the adult AM, the forward-angle laser scatter obtained with the newborn AM was reduced; this optical measurement appeared to correlate with the smaller mean size, as determined by ultrastructural and electronic volume measurements. The diminished right-angle laser scatter (90 degrees angle) correlated with the presence of fewer small, irregularly shaped lysosomal structures in the newborn AM, compared with AM from adult animals. AM from 1-2-day-old rats displayed large vacuoles containing multilamellar structures, which proved to be less effective at scattering light. Cells from newborn rats were less autofluorescent, a finding that appeared to correlate best with the numbers of secondary lysosomes. Flow cytometry may be used to discern structural alterations that occur during the maturation of AM. These changes correlate well with quantitative ultrastructural analyses of these cells.

Accessory cells with immunophenotypic and functional features of monocyte-derived dendritic cells are recruited to the lung during pulmonary inflammation.

Pulmonary macrophages (Mphi) increase in tissue and bronchoalveolar lavage (BAL) fluid during inflammation caused by bleomycin (BLM). This study demonstrates that increasing numbers of exudate Mphi in BLM lung injury exhibit dendritic cell (DC) features. After the intratracheal administration of BLM (0.075 U), adherent mononuclear cells from the bronchoalveolar lavage fluid (BAMC) of C57BL/6 mice were characterized for morphology, immunophenotype, and accessory cell activities. At day 7 post-BLM, 48% of CD11b+ BAMC displayed features of DC differentiation, as judged by dendritic morphology, expression of class II MHC, 33D1, Factor XIIIa, CD80, and CD86 antigens, and the ability to support a primary allogeneic lymphocyte response (MLR). After BLM treatment, CD11b+ peripheral blood monocytes also showed increased expression of 33D1, Factor XIIIa, CD86, and the ability to stimulate an MLR. We conclude that inflammatory DC with immunophenotypic features of monocyte-derived DC increase in the peripheral blood and lung after an inflammatory stimulus.

A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry.

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.

Activin inhibits basal and androgen-stimulated proliferation and induces apoptosis in the human prostatic cancer cell line, LNCaP.

LNCaP cells, derived from an androgen-sensitive cell line widely employed as an in vitro model of human prostate cancer, have been shown to express activin receptors. Activin is a local regulator of cellular growth, appears to play a key role in mesoderm induction and differentiation during development, and has been implicated in gonadal tumorigenesis. Follistatin, a monomeric glycoprotein that specifically binds and neutralizes activin, is often coexpressed with activin and, thus, modulates the autocrine/paracrine biological activity of this potent growth factor. We tested the hypothesis that LNCaP growth is modulated by the activin/follistatin system. Recombinant human activin A inhibited [3H]thymidine incorporation in a dose-dependent fashion with an ED50 of approximately 0.43 +/- 0.3 nM. Activin (0.1-3 nM) also inhibited dihydrotestosterone (DHT)-stimulated [3H]thymidine incorporation in LNCaP cells. Similarly, recombinant human inhibin A inhibited LNCaP proliferation, but was only 1/100th as potent as activin. Furthermore, activin (3 nM) induced a 3-fold increase in the extent of labeling of low mol wt DNA fragments typical of apoptosis. Activin-induced apoptosis was also indicated by an increase in the number of cells with reduced DNA content, as measured by flow cytometry of activin-treated cells. Both activin-mediated inhibition of cell proliferation and induction of apoptosis could be completely blocked by recombinant human follistatin. Based upon these results using an in vitro model, we speculate that activin functions locally to oppose androgen-driven cell proliferation and, thus, is a key factor controlling prostate growth. Reduced activin biosynthesis, increased follistatin secretion, or signaling defects in the activin receptor system should be further investigated in future studies as potential mechanisms underlying enhanced androgen-independent growth of human prostate cancer cells.

Treatment of plasma cell dyscrasias by antibody-mediated immunotherapy.

The use of serotherapy to treat patients with plasma cell dyscrasias (PCDs) has been sought by us and others. Candidate antigens that have been targeted or proposed for targeting in PCDs include the immunoglobulin idiotype, CD19, CD38, CD54, CD126, HM1.24, and Muc-1 core protein. Unfortunately, many of these antigens are not ideal for use in serotherapy since they are not selectively expressed, are either shed or secreted, or have not been fully characterized. Serotherapy with an anti-CD19 monoclonal antibody (B4) conjugated to a blocked ricin toxin had no significant activity in patients with multiple myeloma (MM). Circulating CD20+ clonotypic B cells have been detected in the circulation of most MM and Waldenstrom's macroglobulinemia (WM) patients. Plasma cells from most WM patients express CD20, but most MM patient plasma cells either lack CD20 or express it weakly. In view of recent successes with anti-CD20-directed serotherapy in other B-cell malignancies, we initiated a phase II trial to study the anti-CD20 monoclonal antibody rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) in patients with MM. We describe two PCD patients (one with WM and one with MM) who responded to therapy. By flow cytometric analysis, CD20+ plasma cells and B cells present in the bone marrow and peripheral blood of a patient with MM disappeared with response to rituximab therapy. However, residual CD20- tumor cells remained in the bone marrow following rituximab therapy, and after 6 months this patient progressed with CD20- myeloma cells. As a potential strategy to overcome this limitation, we demonstrated that interferon-gamma at pharmacologically achievable levels induced CD20 expression on these CD20- plasma cells, consistent with our recent findings that interferon-gamma is a potent inducer of CD20 expression on MM patient plasma cells and B cells. We also characterize a response to rituximab with a decrease in paraprotein and resolution of anemia in a patient with WM whose response to rituximab is ongoing after 19+ months. This preliminary experience supports the potential use of serotherapy targeting CD20 in PCDs. Our studies further suggest that interferon-gamma may enhance CD20 expression on MM plasma cells, thereby increasing their susceptibility to anti-CD20 monoclonal antibody therapies.

The growth and cell kinetics of a secondary tumor after radiation or surgical treatment of the primary tumor.

Early transplants of a spontaneous mouse fibrosarcoma (FSa II) were used to investigate whether radiation or surgical treatment of a tumor in the leg altered the growth or cell kinetics of a second tumor in the axilla. The volume doubling times and in vivo Bromodeoxyuridine labeling indices of the axillary tumors were the same in the control group and in the groups which received 70 Gy to the leg tumor or normal leg. After surgical removal of the leg tumor, there was small reduction in the time to reach 1500 mm3 (4.0 vs 4.6 days) and an increase in Bromo-deoxyuridine labeling index at Day 3 (18% to 24%) in the axillary tumors. In all groups, there was a fall in labeling index with time reflecting increasing tumor size.