Nancy Hensel

Successful Treatment of Metastatic Renal Cell Carcinoma With a Nonmyeloablative Allogeneic Peripheral-Blood Progenitor-Cell Transplant: Evidence for a Graft-Versus-Tumor Effect

PURPOSE A 50-year-old man developed progressive pulmonary metastasis resistant to interferon alfa-2b treatment 7 months after he underwent left nephrectomy for stage III renal cell carcinoma. We performed a nonmyeloablative allogeneic peripheral-blood stem-cell transplant in this patient to exploit a possible graft-versus-tumor effect from allogeneic lymphocytes. MATERIALS AND METHODS The conditioning regimen consisted of fludarabine and cyclophosphamide followed by a T-cell replete, granulocyte-colony stimulating-factor-mobilized peripheral-blood stem-cell transplant from his HLA-identical brother. Cyclosporine was administered from days -4 to +45 to prevent graft rejection and acute graft-versus-host disease (GVHD). RESULTS Serial polymerase chain reaction analysis of hematopoietic lineage-specific minisatellites initiallyshowed mixed chimerism in CD14(+) and CD15(+) myeloid cells, CD3(+) T cells, and CD34(+) progenitor cells, with rapid conversion to 100% donor T-cell chimerism by day +60 and 100% donor myeloid cells by day +100. Serial computed tomography scans of the chest showed stable disease at day +30, slight regression of pulmonary lesions at day +63, and complete disappearance of all pulmonary metastatic disease by day +110. Mild transient acute GVHD disease of the skin occurred on day +60 and limited chronic GVHD of the skin occurred by day +200. CONCLUSION The complete regression of metastatic disease, which has now been maintained for more than 1 year, is compatible with a graft-versus-tumor effect.

Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte‐mediated inhibition of CFU‐GM and alterations in T‐cell receptor Vβ profiles

We have demonstrated that 44% of myelodysplastic syndrome (MDS) patients with cytopenia have a haematological response to antithymocyte globulin (ATG). Three ATG responders and two non‐responders with refractory anaemia were further studied for lymphocyte‐mediated inhibition of bone marrow using a standard CFU‐GM assay. In responders, peripheral blood lymphocytes (PBL) added at a 5:1 ratio suppressed CFU‐GM by 54 ± 9% (P = 0.04) and was reversed by ATG treatment. Pre‐treatment marrow depleted of CD3 lymphocytes, increased CFU‐GM by 32% (P = 0.02) in an ATG responder, but not in a non‐responder. CD3 lymphocytes from 6‐month post‐treatment marrow did not inhibit pre‐treatment CFU‐GM, indicating ATG had affected the T cells. Pre‐treatment marrow depleted of CD8 lymphocytes, increased CFU‐GM by 60% (P = 0.01) and 49% (P = 0.03) in two ATG responders, but not in a non‐responder. Inhibition required cell–cell interaction through MHC I. TCR Vβ families, analysed by SSCP, changed from clonal to polyclonal in one ATG responder after 6 months, but clones persisted in a non‐responder. These results indicate patients with refractory anaemia who respond to ATG have CD8 T‐cell clones that mediate MHC‐I‐restricted suppression of CFU‐GM which are replaced by polyclonal T cells that do not suppress CFU‐GM after ATG treatment.

Mitochondrial Metabolism Modulates Differentiation and Teratoma Formation Capacity in Mouse Embryonic Stem Cells

Relatively little is known regarding the role of mitochondrial metabolism in stem cell biology. Here we demonstrate that mouse embryonic stem cells sorted for low and high resting mitochondrial membrane potential (ΔΨmL and ΔΨmH) are indistinguishable morphologically and by the expression of pluripotency markers, whereas markedly differing in metabolic rates. Interestingly, ΔΨmL cells are highly efficient at in vitro mesodermal differentiation yet fail to efficiently form teratomas in vivo, whereas ΔΨmH cells behave in the opposite fashion. We further demonstrate that ΔΨm reflects the degree of overall mammalian target of rapamycin (mTOR) activation and that the mTOR inhibitor rapamycin reduces metabolic rate, augments differentiation, and inhibits tumor formation of the mouse embryonic stem cells with a high metabolic rate. Taken together, our results suggest a coupling between intrinsic metabolic parameters and stem cell fate that might form a basis for novel enrichment strategies and therapeutic options.

Rapid natural killer cell recovery determines outcome after T-cell-depleted HLA-identical stem cell transplantation in patients with myeloid leukemias but not with acute lymphoblastic leukemia

Natural killer (NK) cells are the first lymphocytes to recover after allogeneic stem cell transplantation (SCT) and can exert powerful graft-versus-leukemia (GVL) effects determining transplant outcome. Conditions governing NK cell alloreactivity and the role of NK recovery in sibling SCT are not well defined. NK cells on day 30 post-transplant (NK30) were measured in 54 SCT recipients with leukemia and donor and recipient killer immunoglobulin-like receptor (KIR) genotype determined. In univariate analysis, donor KIR genes 2DL5A, 2DS1, 3DS1 (positive in 46%) and higher numbers of inhibitory donor KIR correlated with higher NK30 counts and were associated with improved transplant outcome. NK30 counts also correlated directly with the transplant CD34 cell dose and inversely with the CD3+ cell dose. In multivariate analysis, the NK30 emerged as the single independent determinant of transplant outcome. Patients with NK30 >150/μl had less relapse (HR 18.3, P=0.039), acute graft-versus-host disease (HR 3.2, P=0.03), non-relapse mortality (HR 10.7, P=0.028) and improved survival (HR 11.4, P=0.03). Results suggest that T cell-depleted SCT might be improved and the GVL effect enhanced by selecting donors with favorable KIR genotype, and by optimizing CD34 and CD3 doses.

Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation

Summary:Donor lymphocyte infusions (DLI) have been the mainstay of treatment for chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation (allo-SCT). Imatinib mesylate (IM) is also effective in these patients. However, advanced phase relapse (APRel) responds poorly with either treatment. To test the possibility that combinations of DLI and IM might be more effective, 37 patients with CML relapsing after allo-SCT between August 1994 and May 2004 were studied. Ten had molecular relapse (MRel), 14 hematological relapse (HRel) and 13 APRel. Thirteen received DLI, 9 IM and 11 DLI+IM. Four patients received DLI+IM but not concurrently. Thirty (81%) patients responded (actuarial survival and current leukemia-free survival of 80.6±6.7% and 69.1±7.7%). Of 30 patients, 26 are in molecular remission (MR), median follow-up of 1226 days (range 249–3257) since relapse. Ten of 11 patients (including four with APRel) treated with DLI+IM achieved MR in 3 months and all are alive in MR. In contrast, only two of 22 treated with either modality (1/13 DLI and 1/9 IM) achieved MR at 3 months, 15 are alive, 11 in MR. Four patients receiving nonconcurrent DLI+IM are also alive in MR. In conclusion, DLI appears to synergize with IM to induce rapid and durable MR.

Specific depletion of alloreactive T cells in HLA‐identical siblings: a method for separating graft‐versus‐host and graft‐versus‐leukaemia reactions

Accumulating evidence indicates that alloreactive donor T cells confer both graft‐versus‐host (GVH) and graft‐versus‐leukaemia (GVL) reactivity following allogeneic bone marrow transplantation. We have developed a method to deplete alloreactive donor T cells with an immunotoxin targeting the α chain of the IL‐2 receptor. In patients with chronic myeloid leukaemia and their HLA‐identical sibling donors, we measured donor helper T‐lymphocyte precursor frequencies (HTLPf) against recipient peripheral blood mononuclear cells (PBMNC; donor versus host), recipient leukaemia cells (donor versus leukaemia) and third‐party PBMNC, before and after the depletion. In seven pairs there was a 4.3‐fold reduction of donor‐versus‐host HTLPf (P = 0.017), without a significant change in the donor frequencies against third party (P = 0.96). In eight further donor–recipient pairs, immunotoxin‐depleted donor versus patient PBMNC HTLPf 4.5‐fold (mean 1/155 000 before and 1/839 000 after depletion, P = 0.007). There was a smaller non‐significant 1.8‐fold reduction in donor‐versus‐leukaemia HTLPf from 1/192 000 to 1/334 000 (P = 0.19). These results suggest that selective T‐cell depletion can significantly deplete donor anti‐host reactivity while conserving antileukaemia reactivity in HLA‐matched donor–recipient pairs.

Immune escape from a graft-versus-leukemia effect may play a role in the relapse of myeloid leukemias following allogeneic bone marrow transplantation

We studied patients relapsing with myeloid leukemias following allogeneic bone marrow transplantation (BMT) for evidence of immune escape by clonal evolution of the leukemia. Relapsed cells from four out of five patients had a reduced ability to stimulate proliferation of lymphocytes from an HLA-mismatched responder. There was decreased susceptibility to lysis by CTL in three and reduced susceptibility to NK-mediated lysis in one. Relapsed leukemias had marked alterations in expression of critical surface molecules involved in immune responsiveness. Three had decreased expression of MHC class I and II, with no change or increase in CD54 (ICAM-1) or CD80 (B7.1). None of these responded to treatment with donor lymphocytes. Three patients showed no change, or increased expression of MHC with no change or decrease in ICAM-1 or B7.1. Two achieved remission – one in response to donor lymphocytes and one following withdrawal of cyclosporine. In one patient transplanted with myelodysplastic syndrome in transformation, interferon-gamma upregulated expression of MHC molecules in relapsed cells and increased their stimulatory capacity and target susceptibility to unmatched responder lymphocytes. These results suggest that immune escape through clonal evolution of the leukemia is a common occurrence in patients who relapse with myelogenous leukemias after BMT.

In vitro induction of myeloid leukemia-specific CD4 and CD8 T cells by CD40 ligand-activated B cells gene modified to express primary granule proteins

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate postallogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-γ or were cytotoxic to the patients CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.

The transfer of adaptive immunity to CMV during hematopoietic stem cell transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the donor

The successful reconstitution of adaptive immunity to human cytomegalovirus (CMV) in hematopoietic stem cell transplantation (HSCT) recipients is central to the reduction of viral reactivation-related morbidity and mortality. Here, we characterized the magnitude, specificity, phenotype, function, and clonotypic composition of CMV-specific T-cell responses in 18 donor-recipient pairs both before and after HSCT. The principal findings were: (1) the specificity of CMV-specific T-cell responses in the recipient after HSCT mirrors that in the donor; (2) the maintenance of these targeting patterns reflects the transfer of epitope-specific T-cell clonotypes from donor to recipient; (3) less differentiated CD27(+)CD57(-) CMV-specific memory T cells are more likely to persist in the recipient after HSCT compared with more terminally differentiated CD27(-) CD57(+) CMV-specific memory T cells; (4) the presence of greater numbers of less differentiated CD8(+) CMV-specific T cells in the donor appears to confer protection against viral reactivation in the recipient after HSCT; and (5) CMV-specific T cells acquire a more differentiated phenotype and a restricted functional profile after HSCT. Overall, these findings define the immunologic factors that influence the successful adoptive transfer of antigen-specific T-cell immunity during HSCT, which enables the identification of recipients at particular risk of CMV reactivation after HSCT.

High avidity myeloid leukemia-associated antigen-specific CD8+ T cells preferentially reside in the bone marrow

The activity of allogeneic CD8(+) T cells specific for leukemia-associated antigens (LAAs) is thought to mediate, at least in part, the curative effects of hematopoietic stem cell transplantation (HSCT) in myeloid malignancies. However, the identity and nature of clinically relevant LAA-specific CD8(+) T-cell populations have proven difficult to define. Here, we used a combination of coreceptor-mutated peptide-major histocompatibility complex class I (pMHCI) tetramers and polychromatic flow cytometry to examine the avidity profiles, phenotypic characteristics, and anatomical distribution of HLA A*0201-restricted CD8(+) T-cell populations specific for LAAs that are over-expressed in myeloid leukemias. Remarkably, LAA-specific CD8(+) T-cell populations, regardless of fine specificity, were confined almost exclusively to the bone marrow; in contrast, CD8(+) T-cell populations specific for the HLA A*0201-restricted cytomegalovirus (CMV) pp65(495-503) epitope were phenotypically distinct and evenly distributed between bone marrow and peripheral blood. Furthermore, bone marrow-resident LAA-specific CD8(+) T cells frequently engaged cognate antigen with high avidity; notably, this was the case in all tested bone marrow samples derived from patients who achieved clinical remission after HSCT. These data suggest that concomitant examination of bone marrow specimens in patients with myeloid leukemias might yield more definitive information in the search for immunologic prognosticators of clinical outcome.