Loren D. Fast

In the absence of a CD40 signal, B cells are tolerogenic

When B cells are deprived of signaling through CD40, they exhibit the ability to induce T cell tolerance. The in vivo administration of anti-gp39 and allogeneic B cells diminished the ability of mice to mount an allogeneic response. Tolerance induction was specific for the haplotype expressed on the allogeneic B cells. Selective allospecific unresponsiveness was induced in the CD8 and CD4 compartments by the administration of anti-gp39 and class II-deficient B cells or class I-deficient B cells, respectively. As predicted by studies with anti-gp39 treatment, diminished allospecific responsiveness was induced by the administration of B cells to mice genetically deficient in gp39. Taken together, these data are consistent with the premise that deprivation of CD40 signaling engenders B cells with enhanced tolerogenicity. These studies provide insights into the tolerogenic capacity of resting B cells and outlines a practical approach to exploit this function.

Nonengraftment Haploidentical Cellular Immunotherapy for Refractory Malignancies: Tumor Responses without Chimerism

Allogeneic bone marrow transplantation relies on immunosuppression, which controls graft-versus-host disease (GVHD) and allows engraftment at the expense of diminished graft versus-tumor (GVT) activity. Advances in hematologic transplantation have prompted the development of effective, less-toxic regimens that attempt to balance GVH and GVT immunoreactions. We analyzed the safety and efficacy of haploidentical transplantation in a Phase I/II nonimmunosuppressive, nonmyeloablative setting. A total of 41 patients with relapsed refractory cancer received 100 cGy of total body irradiation (TBI), along with an infusion of 1 x 10(6) to 2 x 10(8) CD3+ cells/kg; 29 patients received the highest dose. A postinfusional cellular graft rejection syndrome resembling engraftment syndrome was noted at the 2 highest CD3+ infusion cohorts. There were 26 patients with hematologic malignancies with 14 responses, 9 of which were major. Two of 6 patients with lymphoma remained free of disease at 76 months and 82 months, respectively; there were 5 durable complete responses and 4 partial responses in 13 patients with acute myelogenous leukemia (AML). All responses occurred outside of donor chimerism. TBI at 100 cGy followed by HLA-haploidentical immunotherapy is a biologically active therapy for patients with refractory AML and lymphoma. Possible mechanisms contributing to its effectiveness include initial GVT kill, breaking of host tolerance to tumor through cross-reactive alloreactive responses, persistent nondetectable microchimerism, or some combination of these.

Developments in the prevention of transfusion‐associated graft‐versus‐host disease

The transfusion of blood products can result in a variety of consequences, including transfer of pathogens and the induction of immune responses, such as the almost invariably fatal transfusion‐associated graft‐versus‐host disease (TA‐GVHD). Exposure of blood products to γ‐irradiation is currently the standard of care for the prevention of TA‐GVHD. Regulatory, technical and clinical challenges associated with the use of γ‐irradiators are driving efforts to develop alternatives. Initially, pathogen reduction methods were developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and leucocytes. These methods have been found to be as effective as γ‐irradiation in preventing T lymphocyte proliferation and TA‐GVHD responses. Additional benefits of pathogen reduction protocols potentially include inhibition of antigen presentation, cytokine production and activation of lymphocytes.

Control of immune responses induced by the transfer of allogeneic white blood cells during transfusion

From the Division of Hematology/Oncology, Rhode Island Hospital, Providence, Rhode Island. Address reprint requests to: Loren Fast, PhD, Division of Hematology/Oncology, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903; e-mail: Loren_Fast@brown.edu. doi: 10.1111/j.1537-2995.2005.00530.x TRANSFUSION 2005;45:44S-50S. Control of immune responses induced by the transfer of allogeneic white blood cells during transfusion

Use of cellular immunotherapy for treatment of refractory acute myeloid leukemia

A previous clinical trial carried out by our group in which high numbers of haploidentical donor lymphoid cells containing CD3+ cells (1 - 2 x 108 CD3+ cells/kg) were infused into recipients with refractory hematological malignancy found that 14/27 patients gave a response, 9 or which were major. These recipients rapidly developed a cytokine storm which was resolved, if needed, by administrating steroids. No persisting donor cells could be detected at 2 weeks following infusion. After gaining FDA and IRB approval, this cellular immunotherapy trial was restarted. One of the goals of the trial was to define the effector mechanisms that were playing a role in anti-leukemic responses. The results from the first recipient enrolled indicated that right after the infusion, 11.3% of peripheral blood mononuclear cells were donor cells as defined by donor-specific anti-HLA antibodies. Donor cell levels had decreased to 0.95% on day 7. Increased expression of granzymes and perforin by both donor and recipient cells especially 2 - 3 days after infusion indicated that these cells exhibited increased cytolytic activity. In addition, both donor and recipient CD4+ cells were found to express increased levels of CD134, and a large fraction of CD8+ cells expressed CD279 (PD-1). The role of cytolytic cells will be assessed further as additional patients are enrolled and the expression of activating or inhibitory receptors will be confirmed. These results could suggest additional therapeutic approaches to be tested for enhancing the anti-leukemic responses following cellular immunotherapy.

Abstract B22: Antitumor responses driven by alloreactivity.

Previous work done by our group had found that infusion of G-CSF mobilized haploidentical donor white blood cells at a dose of 1 – 2 x 108 CD3+ cells/kg into refractory cancer patients after 100 cGy total body irradiation resulted in a number of complete and partial responses in those patients with hematological malignancies. These responses occurred despite elimination of the donor cells by 2 weeks after infusion. The effector mechanism(s) responsible for these anti-cancer responses were not determined although it was noted that there was a cytokine storm appearing within a day following infusion of the cells. Alloreactive responses could be driving the anti-cancer responses using a variety of mechanisms. One possible mechanism is that the alloreactive response overrides inhibitory mechanisms present in the cancer patient allowing the existing cancer reactive effector cells to become activated and lyse cancer cells. Alternatively the alloreactive cytolytic T cells could be cross-reacting with the antigens expressed on the tumor cells. To test these possibilities, peripheral blood mononuclear cells (PBMNC) or the CD3+ T cells isolated from the PBMNC of newly diagnosed leukemic patients were mixed with mitomycin C treated fully allogeneic stimulator PBMNC (from 1 or 2 different normal controls chosen at random) or the CD3- cells from the leukemic patients. Their ability to proliferate in response to the allogeneic stimulation was tested on day five and their ability to lyse autologous leukemia cells after stimulation was tested on day seven. Ten total patients (8 AML, 1 CML, and 1 CMML) were enrolled in the study. The average white blood cell count was 57,640 cells/uL (range 7,100 – 336,500 cells/uL) with 4 -80% peripheral blasts. The results indicated that in 16/20 combinations, leukemic T cells were able to proliferate in response to allogeneic stimulators, however anti-cancer cytolytic activity was only detected in 9/19 evaluable combinations. These results demonstrated that alloreactive responses are able to induce anti-leukemic cytolytic responses by T cells from a subset of leukemic patients. One explanation for the inability of some combinations to generate anti-leukemic cytolytic activity could be the presence of inhibitory mechanisms which could not be overcome by the alloreactive responses. Immunophenotypic analysis of the leukemic T cells did reveal an increased number of T regulatory cells and increases in the expression of inhibitory receptors on the lymphocytes from some of the patients. Alternatively, it may be that the leukemic T cells had not been stimulated with the appropriate allogeneic antigens. An allogeneic stimulator pool comprised of cells from ten different normal donors was prepared and will be used to test whether increasing the number of allogeneic disparities in culture would enhance the ability of the leukemic T cells to generate anti-leukemic responses. These studies have suggested that cancer-reactive cells can be generated from leukemic patient T cells using alloreactive responses. To further test the role of these effector cells and identify additional effector cells, blood samples will be obtained from patients enrolled in the clinical protocol which is in the process of being restarted, having received FDA approval. Clearer definition and characterization of the anti-cancer effector cells should permit alterations to the protocol that would lead to enhanced anti-cancer responses in an increased number of refractory cancer patients. Citation Format: Loren D. Fast, John Reagan, Martha Nevola, Peter Quesenberry. Antitumor responses driven by alloreactivity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B22.

Abstract 1252: Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia.

Introduction: In the treatment of acute leukemia, much of the therapeutic effect of allogeneic stem cell transplantation is centered on engraftment of donor cells into the host, with a resultant graft versus leukemia effect. Work previously done by our group and others has shown the infusion of haploidentical stem cells in patients with acute leukemia results in a cytokine storm and exhibits an anti-tumor effect in the absence of engraftment. Whether the underlying clinical response was due to this cytokine storm or secondary to host or donor effector cell interactions was not determined. We present in vitro data collected from leukemia patients in which patient CD3+ cells are stimulated with randomly selected unrelated normal donor peripheral blood mononuclear cells (PBMC). The stimulated patient CD3+ cells are then collected and tested for their ability to lyse leukemia cells from the patient. Methods: New leukemia patients were identified through standard pathologic diagnostic criteria. Patient samples were collected and separated into CD3+ and CD3- fractions. Patient CD3+ cells were then placed in a mixed lymphocyte culture and stimulated with mitomycin C treated unrelated normal donor PBMC. Cell proliferation was measured on Day 5. On Day 7, the stimulated patient CD3+ cells were collected and then cultured with Chromium 51 labeled CD3- cells, the bulk of which were leukemic blast cells. Cytolytic activity was measured through Cr51 release assays. Cell lysis was recorded as lytic units/million cells (LU/106), which is inversely related to the effector to target ratio at which 30% cell lysis occurs. Patient demographic data including sex and age in addition to leukemia type, cytogenetics, and molecular markers were recorded. Results: 10 total patients were enrolled in the study; 8 AML, 1 CML, and 1 CMML. 9 patients underwent ex vivo CD3+ stimulation with 2 separate donor PBMC samples while 1 sample underwent only 1 donor stimulation for 19 total assays. The average white blood cell count was 57,640 cells/uL (range 7,100-336,500 cells/ul) with 4-80% peripheral blasts. 7 out of 19 (37%) assays showed leukemia cell cytolytic activity greater than 1 lytic unit. All 7 successful assays were derived from patients with AML (Table 1). Conclusions: Patient CD3+ cells stimulated with random unrelated donor PBMC are able to generate a cytolytic anti-leukemia response in a donor dependant fashion. This cytolytic activity appears to be unrelated to host lymphocyte proliferation upon donor antigenic stimulation. Interestingly, of the seven assays with cytolytic activity greater than one lytic unit, four were performed in the two patients with known FLT3 ITD positive leukemia, which has a notoriously poor prognosis. Further studies to elucidate a mechanism of action for cellular therapy are warranted. Citation Format: John L. Reagan, Loren D. Fast, Martha Nevola, Peter J. Quesenberry. Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2013-1252