Carolyn A. Keever-Taylor

IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial

Summary:We determined the safety, immune activating effects, and potential efficacy of i.v. infusion of ex vivo interleukin-2 (IL-2) activated natural killer (NK) cells (part I) or IL-2 boluses (part II) during daily s.c. IL-2 administration following hematopoietic recovery from autologous transplantation. In all, 57 patients with relapsed lymphoma (n=29) or metastatic breast cancer (n=28) were enrolled. In part I of the study, 34 patients were enrolled at three dose levels of ex vivo IL-2-activated NK cells. Lymphaphereses were performed on days 28 and 42 of s.c. IL-2 administration. Following overnight ex vivo IL-2 activation of the pheresis product, the cells were reinfused the following day. In part II, 23 patients were enrolled at three dose levels of supplemental i.v. IL-2 bolus infusions, given on days 28 and 35 during s.c. IL-2 administration. Toxicities were generally mild, and no patient required hospitalization. Lytic function was markedly enhanced for fresh peripheral blood mononuclear cells (PBMNCs) obtained 1 day postinfusion of either IL-2-activated cells or IL-2 boluses. IL-2 boluses transiently increased the levels of IL-6, IFN-γ, TNF-α and IL1-β, with increases in IL-6 and IFN-γ being dose dependent. A total of 37 patients (19 patients with lymphoma, 18 with breast cancer) treated with an optimum dose of post-transplant immunotherapy (defined as having received 1.75 × 106 IU/m2/day of s.c. IL-2 plus at least one of the planned ex vivo IL-2-activated cell infusions/IL-2 boluses) could be matched with controls from the Autologous Blood and Marrow Transplant Registry database. The matched-pairs analysis demonstrated no improvement in disease outcomes of survival and relapse. We conclude that IL-2-activated cells/IL-2 boluses can be safely administered, generate PBMNCs with enhanced cytotoxicity against NK-resistant targets, and increase cytokine levels. With this dose and schedule of administration of IL-2, no improvement in patient disease outcomes was noted. Alternative strategies will be needed to exploit the immunotherapeutic potential of IL-2-activated NK cells.

T cell-depleted autologous hematopoietic stem cell transplantation for multiple sclerosis: Report on the first three patients

Multiple sclerosis (MS) is a disease of the central nervous system characterized by immune-mediated destruction of myelin. In patients with progressive deterioration, we have intensified immunosuppression to the point of myeloablation. Subsequently, a new hematopoietic and immune system is generated by infusion of CD34-positive hematopoietic stem cells (HSC). Three patients with clinical MS and a decline of their Kurtzke extended disability status scale (EDSS) by 1.5 points over the 12 months preceding enrollment and a Kurtzke EDSS of 8.0 at the time of enrollment were treated with hematopoietic stem cell (HSC) transplantation using a myeloablative conditioning regimen of cyclophosphamide (120 mg/kg), methylprednisolone (4 g) and total body irradiation (1200 cGy). Reconstitution of hematopoiesis was achieved with CD34-enriched stem cells. The average time of follow-up is 8 months (range 6–10 months). Despite withdrawal of all immunosuppressive medications, functional improvements have occurred in all three patients. We conclude that T cell-depleted hematopoietic stem cell transplantation can be performed safely in patients with severe and debilitating multiple sclerosis. Stem cell transplantation has resulted in modest neurologic improvements for the first time since onset of progressive disease although no significant changes in EDSS or NRS scales are evident at this time.

Comparative Outcomes of Donor Graft CD34+ Selection and Immune Suppressive Therapy As Graft-Versus-Host Disease Prophylaxis for Patients With Acute Myeloid Leukemia in Complete Remission Undergoing HLA-Matched Sibling Allogeneic Hematopoietic Cell Transplantation

PURPOSE T-cell depletion (TCD) reduces the incidence of graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). However, concerns about relapse, graft rejection, and variability in technique have limited the widespread application of this approach. PATIENTS AND METHODS Outcomes of 44 patients receiving HLA-identical sibling TCD grafts using a uniform technique for CD34(+) selection as the sole form of immune suppression were compared with outcomes of 84 patients receiving T-replete grafts and pharmacologic immune suppression therapy (IST). RESULTS Groups were similar, except for fewer men (36% with TCD v 56% with IST) and more frequent use of radiation-containing regimens (100% with TCD v 50% with IST) in the CD34-selected TCD cohort. The proportion of patients with neutrophil engraftment at day 28 was similar (96% with IST and 100% with TCD grafts). The 100-day rates of grade 2 to 4 acute GVHD were 39% and 23% with IST and TCD grafts, respectively (P = .07). Corresponding 2-year rates of chronic GVHD were lower with TCD grafts than IST (19% v 50%, respectively; P < .001). There were no differences in rates of graft rejection, leukemia relapse, treatment-related mortality, and disease-free and overall survival rates. At 1 year, 54% and 12% of patients were still on immunosuppression in the IST and TCD cohorts, respectively. TCD was associated with a higher GVHD-free survival at 2 years compared with IST (41% v 19%, respectively; P = .006). CONCLUSION These results suggest that TCD via CD34 selection might lower long-term morbidity as a result of chronic GVHD without negatively impacting relapse rates in patients with acute myeloid leukemia. Additional prospective studies should be undertaken to definitively address the role of TCD in HCT.

Low Risk of Chronic Graft-versus-Host Disease and Relapse Associated with T Cell–Depleted Peripheral Blood Stem Cell Transplantation for Acute Myelogenous Leukemia in First Remission: Results of the Blood and Marrow Transplant Clinical Trials Network Protocol 0303

Graft-versus-host disease (GVHD) is most effectively prevented by ex vivo T cell depletion (TCD) of the allograft, but its role in the treatment of patients undergoing allogeneic hematopoietic cell transplantation (HCT) for acute myelogenous leukemia (AML) in complete remission (CR) remains unclear. We performed a phase 2 single-arm multicenter study to evaluate the role of TCD in AML patients in CR1 or CR2 up to age 65 years. The primary objective was to achieve a disease-free survival (DFS) rate of >75% at 6 months posttransplantation. A total of 44 patients with AML in CR1 (n = 37) or CR2 (n = 7) with a median age of 48.5 years (range, 21-59 years) received myeloablative chemotherapy and fractionated total body irradiation (1375 cGy) followed by immunomagnetically selected CD34-enriched, T cell‒depleted allografts from HLA-identical siblings. No pharmacologic GVHD prophylaxis was given. All patients engrafted. The incidence of acute GVHD grade II-IV was 22.7%, and the incidence of extensive chronic GVHD was 6.8% at 24 months. The relapse rate for patients in CR1 was 17.4% at 36 months. With a median follow-up of 34 months, DFS for all patients was 82% at 6 months, and DFS for patients in CR1 was 72.8% at 12 months and 58% at 36 months. HCT after myeloablative chemoradiotherapy can be performed in a multicenter setting using a uniform method of TCD, resulting in a low risk of extensive chronic GVHD and relapse for patients with AML in CR1.

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 non‐engraftment and all three patients with grade III/IV GVHD. Other fatalities were due to infections or therapy‐related toxicity. The incidence ≥ 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.

T cell-depleted allogeneic bone marrow transplantation for high-risk non-Hodgkin's lymphoma : clinical and molecular follow-up

The use of allogeneic BMT in patients with relapsed non-Hodgkin lymphoma (NHL) offers the advantage of tumor-free bone marrow and possibly a ‘graft-versus-lymphoma effect’ which may decrease the risk of recurrence. However, allogeneic BMT also poses an increased risk of death due to graft-versus-host disease (GVHD) which can be ameliorated by T cell depletion. We performed a retrospective review of 37 patients who underwent T cell-depleted allogeneic BMT for aggressive and indolent NHL between 1988 and 1996. Polymerase chain reaction (PCR) was used to identify indolent NHL patients with the BCL2/IgH translocation which served as a marker of residual disease. Sixteen of 37 patients (44%) are alive and progression-free with a median follow-up of 4.4 years (range 1–10.3). The incidence of grade 2–4 acute GVHD was 36% and extensive chronic GVHD developed in 12%. Patients with aggressive NHL have an overall PFS of 33% (12–54%); those with chemotherapy-resistant and sensitive disease have PFS of 17% (0–47%), and 40% (15–65%) respectively at 5 years. Patients with indolent histologies have overall PFS of 62% (37–86%); those with chemotherapy-resistant and sensitive disease have PFS of 55% (25–85%) and 80% (45–100%) respectively at 5 years. Eight patients with indolent disease had a BCL2/IgH translocation detectable by PCR. Five of these eight patients remain alive and progression free at a median of 6.5 years after BMT (range 2.1–7.4 years), four of whom remain PCR positive from 1.7 to 2.9 years after transplantation. We conclude that T cell-depleted allogeneic BMT poses a low risk for death due to GVHD, and should be considered for patients with relapsed and refractory indolent NHL.

Effect of T cell subset dose on outcome of T cell-depleted bone marrow transplantation

T cell depletion using the murine monoclonal antibody (moAb) T10B9 is unique in that the T cell receptor (TCR)γδbearing subset is relatively spared compared to the TCRαβ+ subset. We evaluated the probabilities of engraftment, acute and chronic graft-versus-host disease (GVHD), relapse, and survival in 273 recipients of marrow T cell depleted using T10B9. Sixty-two patients received marrow from an HLA-identical sibling, 54 patients received partially matched related donor marrow and 157 patients received unrelated donor marrow. Limiting dilution analysis (LDA) was used to estimate total clonable T cell dose in all patients and a modified LDA using moAb-coated immunomagnetic beads was used to estimate TCRαβ+, CD4+ and CD8+ T cells in a subset of patients. TCRγδ+ cell dose was estimated by flow cytometry. Cox proportional hazards regression models were used to assess the impact of T cell subset dose/kg of body weight on outcome. We found a significant association of TCRγδ+ T cell dose (P = 0.004), but not TCRαβ+ T cell dose or total clonable T cell dose, with the probability of engraftment. TCRαβ+, CD4+, CD8+ and total clonable T cell dose were significantly associated (P < 0.001) with the risks of grade 2–4 acute gvhd in recipients of marrow from related donors but not in recipients of marrow from unrelated donors. neither total clonable t cell dose nor any t cell subset dose was found to be significantly associated with chronic gvhd, relapse or survival. the results confirm preclinical studies showing tcrγδ+ T cells promote engraftment. TCRγδ+ T cells are not associated with an increased risk of acute GVHD while TCRαβT cells are associated with acute GVHD but not engraftment in recipients of marrow grafts T cell depleted using T10B9. These findings support the hypothesis that T cell subsets differentially contribute to alloengraftment and GVHD.

Herpes simplex thymidine kinase gene-transduced donor lymphocyte infusions

OBJECTIVE Donor lymphocytes mediate both a beneficial graft-vs-leukemia/lymphoma (GVL) effect as well as graft-vs-host disease (GVHD), the most dreaded complication of allogeneic hematopoietic stem cell transplantation (HSCT). Transduction of donor lymphocytes with a herpes simplex thymidine kinase (HSVtk) gene prior to infusion confers lethal sensitivity to the anti-herpes drug, ganciclovir (GCV). HSVtk-transduced donor lymphocyte infusions (DLI) have already been used and significant problems have limited the clinical experience to very few patients. To this end, we also report on a study of whether HSVtk-DLI induces GVHD/GVL and if infusion of GCV allows abrogation of GVHD by selective killing of donor lymphocytes. MATERIALS AND METHODS Nine patients with relapsed hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) were infused with HSVtk gene-modified donor lymphocytes. In brief, transgeneic lymphocytes were prepared by 3 days of activation, 1 day of transduction, 6 days of selection with G418, and 2 to 4 weeks of expansion. RESULTS From 5.0 to 199 x 10(6) CD3(+) DLI were infused. There were no toxicities and no correlation between CD3(+) cell dose and either GVHD or GVL was observed. Only one patient who had cutaneous T-cell lymphoma (CTCL) developed GVHD and that same patient is the only patient to have an anti-tumor response. The patient was infused with 23 x 10(6) CD4(+) and 9.7 x 10(6) CD8(+) HSVtk DLI. Following discontinuation of immune suppression and infusion of GCV, GVHD promptly resolved. Although the CTCL relapsed, it has been easily controlled with intermittent topical therapy. One patient with acute myelogenous leukemia (AML) had a remission inversion of undetermined significance. Two patients with AML, one patient with lymphoma, and four patients with chronic myelogenous leukemia (CML) did not respond. CONCLUSION HSVtk-DLI may provide an anti-tumor effect in vivo and may induce GVHD that is abrogated by GCV treatment. While technical aspects to improve response need to be perfected, HSVtk-DLI infusion to induce a transient GVL/GVHD may become an effective future therapy to minimize complications of allogeneic HSCT.

Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen

Invasive aspergillosis (IA) is a major cause of infection‐related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We have prepared overlapping pentadecapeptides (11‐aa overlap with previous peptide) spanning the entire 427‐aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1‐type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4+ T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA‐B*3501+ donors were found to be strongly cytotoxic to autologous Asp f16‐peptide pool‐ and Aspergillus culture extract‐pulsed targets after 4–5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)‐γ production were mediated exclusively by CD8+ T cells in response to pool‐pulsed targets. Interleukin (IL)‐4 production was not detected. CTL activity was restricted by HLA‐B*3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool‐pulsed B*3503+ BLCL but not B*3502+ or B*3508+ BLCL presented peptide to donor no. 1. B*3503+ BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA‐Class I‐restricted, Aspergillus‐specific T cells that may be capable of conferring immunity to IA.

Association of donor-derived host-reactive cytolytic and helper T cells with outcome following alternative donor T cell-depleted bone marrow transplantation.

Recipients of marrow from alternative donors (unrelated or HLA-mismatched related donors) have a higher incidence of post-transplant complications compared to recipients of marrow from HLA-identical siblings. HLA disparity undetected by routine typing techniques has been suggested as one cause for the increased complications observed. Limiting dilution analysis (LDA) of donor-derived, host-reactive T cell precursor frequency prior to transplant has been proposed as a surrogate indicator of underlying HLA disparity which might be used to predict transplant outcome and aid in donor selection. We compared results of LDA of host-reactive IL-2 producing helper T lymphocytes (HTLp) and/or cytolytic T lymphocytes (CTLp) in 77 alternative marrow donor/recipient pairs with transplant outcome using univariate and multivariate analysis. All donor grafts were depleted ex vivo of mature T cells. Median patient age was 15 years (1–53). Donor selection was based on serologic typing for HLA class I and high resolution oligotyping for HLA-DRB1-DRB5, and HLA-DQB1. HLA-A and HLA-B locus antigens were retrospectively defined by one dimensional isoelectric focusing (IEF). Cox proportional hazards regression models were used to assess the impact of frequency and estimated cell dose of CTLp and HTLp on outcome. The CTLp assay was most sensitive to HLA-A and HLA-B locus disparity detected by serology or IEF. The HTLp assay detected class I disparity but was most strongly reactive in the presence of HLA-DRB1 disparity. Univariate analysis indicated a significant association of CTLp frequency and dose with severe (grades 3–4) acute graft-versus-host disease (GVHD), and of CTLp dose with chronic GVHD. Both assays were associated with survival and neither assay was associated with relapse. After adjustment for other significant covariables including known HLA disparity, the association of CTLp with acute GVHD was lost, however, CTLp frequency and CTLp dose remained associated with survival and HTLp frequency was associated with chronic GVHD. These data support the hypothesis that post-BMT complications may be influenced not only by T cell dose but by the alloreactive potential of the cells infused. LDA of alloreactive potential was useful in detecting disparity and in predicting survival or chronic GVHD in recipients of alternative donor TCD marrow grafts.