Elizabeth J. Read

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (Treg) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking Treg cells develop severe autoimmune disease, and depletion of Treg cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of Treg cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2–responsive Treg cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain Treg cells, the question arises as to the effects of IL-2 therapy on them. We monitored Treg cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4+CD25hi cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the Treg cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent Treg cells in normal hosts, and IL-2–induced Treg cell expansion was further augmented by lymphopenia. On a per-cell basis, Treg cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to Treg cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4+CD25+ Treg cell homeostasis.

Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma.

Lymphoid cells infiltrating into human tumors can be expanded in vitro in medium containing interleukin-2 (IL-2). Adoptive transfer of these tumor-infiltrating lymphocytes (TIL) mediates potent antitumor effects in murine tumor models. Clinical trials to evaluate the efficacy of these cells in patients with advanced cancer are underway. We have investigated whether infused TIL labeled with indium 111 (111In) oxine can traffic and localize to metastatic deposits of tumor. Six patients with metastatic malignant melanoma who had multiple sites of subcutaneous, nodal, and/or visceral disease were the subjects of the study. The patients received cyclophosphamide 36 hours before receiving the intravenous (IV) infusion of TIL followed by IL-2 IV every eight hours. The distribution and localization of the TIL were evaluated using serial whole body gamma camera imaging, serial blood and urine samplings, and serial biopsies of tumor and normal tissue. 111In-labeled TIL localized to lung, liver, and spleen within two hours after the infusion of activity. Activity in the lung diminished within 24 hours. As early as 24 hours after injection of 111In-labeled TIL, localization of TIL to sites of metastatic deposits was demonstrated in all six patients using either imaging studies or biopsy specimens or both. 111In activity in tumor tissue biopsies ranged from three to 40 times greater than activity in normal tissue. A progressive increase in the radioactive counts at sites of tumor deposit was seen. This study shows that labeled TIL can localize preferentially to tumor, and provides information concerning the possible mechanism of the therapeutic effects of TIL.

Granulocyte Colony-Stimulating Factor Mobilizes Functional Endothelial Progenitor Cells in Patients With Coronary Artery Disease

Objective— Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Methods and Results— Sixteen CAD patients had reduced CD34+/CD133+ (0.0224±0.0063% versus 0.121±0.038% mononuclear cells [MNCs], P<0.01) and CD133+/VEGFR-2+ cells, consistent with EPC phenotype (0.00033±0.00015% versus 0.0017±0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2±1/well) compared with 16 healthy subjects at high risk (13±4/well, P<0.05) or 14 at low risk (22±3/well, P<0.001) for CAD. G-CSF 10 &mgr;g/kg per day for 5 days increased CD34+/CD133+ cells from 0.5±0.2/&mgr;L to 59.5±10.6/&mgr;L and CD133+/ VEGFR-2+ cells from 0.007±0.004/&mgr;L to 1.9±0.6/&mgr;L (both P<0.001). Also increased were CD133+ cells that coexpressed the homing receptor CXCR4 (30.4±8.3/&mgr;L, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27±9/well after treatment (P<0.05), with a decline to 9±4/well at 2 weeks (P=0.06). Conclusions— Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture.

Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA.

Aplastic anaemia in adults is usually acquired, but rarely constitutional types of bone marrow failure can occur late in life. We assessed two families with onset of pancytopenia in adults and detected two novel point mutations in the telomerase RNA gene (TERC) in each family. This gene is abnormal in some kindreds with dyskeratosis congenita. Individuals in our families with mutated TERC did not have physical signs of dyskeratosis congenita, and their blood counts were nearly normal, but all had severely shortened telomeres, reduced haemopoietic function, and raised serum erythropoietin and thrombopoietin. Bone marrow failure of variable severity due to dyskeratosis congenita, historically characterised by associated physical anomalies and early pancytopenia, may be present in otherwise phenotypically normal adults, and can masquerade as acquired aplastic anaemia.

Magnetic Resonance Imaging and Confocal Microscopy Studies of Magnetically Labeled Endothelial Progenitor Cells Trafficking to Sites of Tumor Angiogenesis

AC133 cells, a subpopulation of CD34+ hematopoietic stem cells, can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected, magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. Materials and Methods: EPCs labeled with ferumoxide–protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma, and MRI was obtained at different stages of tumor development and size. Results: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However, low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron‐positive cells at the sites corresponding to low signal intensity on MRI. Confocal microcopy showed incorporation into the neovasculature, and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. Conclusion: MRI demonstrated the incorporation of FePro‐labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors.

T cell-depleted bone marrow transplantation and delayed T cell add- back to control acute GVHD and conserve a graft-versus-leukemia effect

Thirty-eight patients with hematological malignancies, received T cell-depleted marrow transplants (BMT) and cyclosporine to prevent acute graft-versus-host disease (aGVHD), followed by delayed add-back of donor lymphocytes to prevent leukemia relapse. In 26 patients scheduled for donor T cell add-back of 2 × 106 cells/kg on day 30 and 5 × 107 cells/kg on day 45 (schedule 1), the overall probability of grade ⩾II aGVHD developing was 31.5%, with a 15.5% probability of aGVHD occurring after T cell add-back. In 12 patients receiving 107 donor T cells/kg on day 30 (schedule 2), the probability of grade ⩾II aGVHD was 100%. The incidence of grade III–IV aGVHD was higher in schedule 2 than in schedule 1 (P = 0.02). Of 24 evaluable patients, 10 (46%) developed chronic GVHD which was limited in eight and extensive in two. Current disease-free survival for 18 patients at standard risk for relapse (chronic myeloid leukemia (CML) in chronic or accelerated phase, acute myeloid leukemia in remission) vs 20 patients with more advanced leukemia or multiple myeloma were respectively 72% vs 12% (P < 0.01) with a 29% vs 69% probability of relapse (P = 0.08). In 12 CML patients surviving more than 3 months, PCR analysis of the BCR/ABL transcript showed that minimal residual disease after T cell add-back was transient except in two patients who developed hematological relapse. Results indicate that the risk of acute GVHD is low following substantial T cell doses, transfused 45 days after transplant, using cyclosporine prophylaxis. Furthermore a graft-versus-leukemia effect was conserved.

Engraftment of Hematopoietic Progenitor Cells Transduced with the Fanconi Anemia Group C Gene (FANCC)

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.

Allogeneic Lymphocytes Induce Tumor Regression of Advanced Metastatic Breast Cancer

PURPOSE Allogeneic T lymphocytes can induce regression of metastatic breast cancer through an immune-mediated graft-versus-tumor (GVT) effect in murine models. To determine if a clinical GVT effect exists against metastatic breast cancer, allogeneic lymphocytes were used as adoptive cellular therapy after a reduced-intensity chemotherapy conditioning regimen and allogeneic hematopoietic stem-cell transplantation (HSCT) from human leukocyte antigen-matched siblings. PATIENTS AND METHODS Sixteen patients with metastatic breast cancer that had progressed after treatment with anthracyclines, taxanes, hormonal agents, and trastuzumab, received allogeneic HSCT. The reduced-intensity transplant conditioning regimen consisted of cyclophosphamide and fludarabine. To distinguish an immunological GVT effect from any antitumor effect of cytotoxic chemotherapy in the transplant-conditioning regimen, allogeneic T lymphocytes were removed from the stem-cell graft and were subsequently administered late postallogeneic HSCT. Allogeneic lymphocytes containing 1 x 10(6), 5 x 10(6), and 10 x 10(6) CD3(+) cells/kg were infused on days +42, +70, and +98 post-allogeneic HSCT, respectively. RESULTS Objective tumor regressions occurred after day +28 post-allogeneic HSCT in six patients and were attributed to allogeneic lymphocyte infusions. Two of these responding patients had disease progression post-allogeneic HSCT before subsequent tumor regression. Tumor regressions occurred concomitantly with the establishment of complete donor T-lymphoid engraftment, were associated with the development of graft-versus-host disease (GVHD), and were abrogated by subsequent systemic immunosuppression for GVHD. CONCLUSION Allogeneic lymphocytes can induce regression of advanced metastatic breast cancer. These results indicate that an immunological GVT effect from allogeneic lymphocytes exists against metastatic breast cancer and provide rationale for further development of allogeneic cellular therapy for this largely incurable disease.

A Pilot Study of Consolidative Immunotherapy in Patients with High-Risk Pediatric Sarcomas

Purpose: Patients with metastatic or recurrent Ewing’s sarcoma family of tumors and alveolar rhabdomyosarcoma have <25% 5-year survival in most studies. This study administered a novel immunotherapy regimen aimed at consolidating remission in these patients. Experimental Design: Fifty-two patients with translocation positive, recurrent, or metastatic Ewing’s sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2. Results: All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2+ patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy. Conclusions: Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

Transplant dose of CD34(+) and CD3(+) cells predicts outcome in patients with haematological malignancies undergoing T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte add-back.

We sought to optimize and standardize stem cell and lymphocyte doses of T cell‐depleted peripheral blood stem cell transplants (T‐PBSCT), using delayed add‐back of donor T cells (DLI) to prevent relapse and enhance donor immune recovery. Fifty‐one patients with haematological malignancies received a T‐PBSCT from an HLA‐identical sibling, followed by DLI of 1 × 107 and 5 × 107 CD3+ cells/kg on d +45 and +100 respectively. Twenty‐four patients were designated as standard risk and twenty‐seven patients with more advanced leukaemia were designated as high risk. Median recipient age was 38 years (range 10–56). Median (range) of CD34+ and CD3+ cell transplant doses were 4·6 (2·3–10·9) × 106/kg and 0·83 (0·38–2) × 105/kg respectively. The cumulative probability of acute GVHD was 39%. No patient died from GVHD or its consequences. The probability of developing chronic GVHD was 54% (18% extensive). The probability of relapse was 12% for the standard‐risk patients and 66% for high‐risk patients. In multivariate analysis, the risk factors for lower disease‐free survival and overall survival were high‐risk disease, CD34+ dose < 4·6 × 106/kg and CD3+ dose < 0·83 × 105/kg. Predictive factors for chronic GVHD were a T‐cell dose at transplant > 0·83 × 105 CD3+ cells/kg. These results further define the impact of CD34 and CD3 cell dose on transplant outcome and show that careful dosing of stem cells and lymphocytes may permit the control and optimization of transplant outcome.