Reversal of in situ T-cell exhaustion during effective human antileukemia responses to donor lymphocyte infusion.

Increasing evidence across malignancies suggests that infiltrating T cells at the site of disease are crucial to tumor control. We hypothesized that marrow-infiltrating immune populations play a critical role in response to donor lymphocyte infusion (DLI), an established and potentially curative immune therapy whose precise mechanism remains unknown. We therefore analyzed marrow-infiltrating immune populations in 29 patients (22 responders, 7 nonresponders) with relapsed chronic myelogenous leukemia who received CD4(+) DLI in the pre-tyrosine kinase inhibitor era. Immunohistochemical analysis of pretreatment marrow revealed that the presence of >4% marrow-infiltrating CD8(+) (but not CD4(+)) T cells predicted DLI response, even in the setting of high leukemia burden. Furthermore, mRNA expression profiling of marrow-infiltrating T cells of a subset of responders compared with nonresponders revealed enrichment of T-cell exhaustion-specific genes in pretreatment T cells of DLI responders and significant downregulation of gene components in the same pathway in responders in conjunction with clinical response. Our data demonstrate that response to DLI is associated with quantity of preexisting marrow CD8(+) T cells and local reversal of T-cell exhaustion. Our studies implicate T-cell exhaustion as a therapeutic target of DLI and support the potential use of novel anti-PD1/PDL1 agents in lieu of DLI.

Biologic activity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic stem cell transplantation.

Through an immune-mediated graft-versus-leukemia effect, allogeneic hematopoietic stem cell transplantation (HSCT) affords durable clinical benefits for many patients with hematologic malignancies. Nonetheless, subjects with high-risk acute myeloid leukemia or advanced myelodysplasia often relapse, underscoring the need to intensify tumor immunity within this cohort. In preclinical models, allogeneic HSCT followed by vaccination with irradiated tumor cells engineered to secrete GM-CSF generates a potent antitumor effect without exacerbating the toxicities of graft-versus-host disease (GVHD). To test whether this strategy might be similarly active in humans, we conducted a Phase I clinical trial in which high-risk acute myeloid leukemia or myelodysplasia patients were immunized with irradiated, autologous, GM-CSF-secreting tumor cells early after allogeneic, nonmyeloablative HSCT. Despite the administration of a calcineurin inhibitor as prophylaxis against GVHD, vaccination elicited local and systemic reactions that were qualitatively similar to those previously observed in nontransplanted, immunized solid-tumor patients. While the frequencies of acute and chronic GVHD were not increased, 9 of 10 subjects who completed vaccination achieved durable complete remissions, with a median follow-up of 26 months (range 12-43 months). Six long-term responders showed marked decreases in the levels of soluble NKG2D ligands, and 3 demonstrated normalization of cytotoxic lymphocyte NKG2D expression as a function of treatment. Together, these results establish the safety and immunogenicity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic HSCT, and raise the possibility that this combinatorial immunotherapy might potentiate graft-versus-leukemia in patients.

Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. The administration of fully human anti-CTLA-4 blocking monoclonal antibodies to advanced-cancer patients increases immune-mediated tumor destruction in some subjects. Nonetheless, patients that respond also frequently manifest serious inflammatory pathologies, raising the possibility that the therapeutic and toxic effects of CTLA-4 blockade might be linked. Here we show that periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX) generate clinically meaningful antitumor immunity without grade 3 or 4 toxicity in a majority of metastatic melanoma patients. The application of this sequential immunotherapy to advanced ovarian carcinoma patients also revealed that tumor destruction and severe inflammatory pathology could be dissociated, although further refinements are required to increase clinical responses and to minimize toxicity in this population. The extent of therapy-induced tumor necrosis was linearly related to the natural logarithm of the ratio of intratumoral CD8(+) effector T cells to FoxP3(+) regulatory T cells (Tregs) in posttreatment biopsies. Together, these findings help clarify the immunologic and clinical effects of CTLA-4 antibody blockade in previously vaccinated patients and raise the possibility that selective targeting of antitumor Tregs may constitute a complementary strategy for combination therapy.

Combined CD4+ donor lymphocyte infusion and low-dose recombinant IL-2 expand FOXP3+ regulatory T cells following allogeneic hematopoietic stem cell transplantation.

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4(+) lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4(+)DLI alone (P = .001). FOXP3 expression correlated with absolute CD4(+)CD25(+) cell counts. Moreover, expanded CD4(+)CD25(+) T cells displayed normal suppressive function and treatment with CD4(+)DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4(+) cellular therapy may provide a mechanism to expand Treg in vivo.

Low dose interleukin-2 following intensification therapy with high dose cytarabine for acute myelogenous leukemia in first complete remission.

The most important problem in the therapy of patients with acute myeloid leukemia (AML) is relapse after intensive therapy. We sought to determine if interleukin-2 (low-dose with intermittent boluses) administration could be feasibly administered after standard therapy to potentiate anti-tumor immunity in a fashion analogous to the post-allogeneic stem cell transplant "graft-vs-leukemic" effect. Adults with de novo AML received daunorubicin and cytosine arabinoside induction therapy. Patients achieving complete remission received high dose ara-C (HIDAC) for three courses followed by low dose rIL-2 (Amgen), administered by continuous infusion (450,000 U/m(2)/day) for 10 weeks with intermittent boluses (500,000/U/m(2) over 2 hr) given in weekly intervals starting on Week 4. Of the 32 enrolled patients, 27 achieved CR; 8/11 who received rIL-2 completed therapy. 6/11 are long term survivors (median follow-up, 139 months). rIL-2 was well tolerated and associated with a 5-fold increase in circulating NK-lymphocytes and a 3-fold increase in circulating T-cells. Mononuclear cells from patients receiving rIL-2 exhibited enhanced cytolytic activity in vitro against cryopreserved autologous leukemia cells. This study supports further investigation of immunotherapy in the post-intensive chemotherapy setting in the management of patients with AML.

Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: A Phase I Clinical Trial.

Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB-IV melanoma.Experimental Design: Residual iNKT cells [<0.05% of patient peripheral blood mononuclear cell (PBMC)] were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL2 in vitro to obtain up to approximately 109 cells.Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1-2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters.Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510-9. ©2017 AACR.

Expansion of tumor-specific CD8+ T cell clones in patients with relapsed myeloma after donor lymphocyte infusion.

Donor lymphocyte infusions (DLI) provide effective therapy for patients with multiple myeloma who have relapsed after allogeneic bone marrow transplantation. However, the immunological mechanisms of the graft-versus-myeloma (GVM) effect have not been defined, and the target antigens of this response have not been identified. Molecular analysis of CDR3 Vbeta repertoire after CD4+ DLI demonstrated previously that the development of GVM and graft-versus-host-disease (GVHD) were associated with the clonal expansion of distinct T-cell populations in patient peripheral blood. In the current study, we undertook a molecular and functional characterization of GVM- and GVHD-associated T-cell clones. T-cell clones associated with GVM were detectable by clone-specific PCR at a low level in peripheral blood before DLI and expanded approximately 10-fold after DLI. In contrast, T-cell clones associated with GVHD were not detectable before DLI or before the development of clinical GVHD. Two T-cell clones associated with GVM were isolated and expanded in vitro, allowing their phenotypic and functional characterization. Both GVM clones were derived from donor cells and had a CD3+CD8+CD4- phenotype. One GVM clone specifically recognized patient myeloma cells in an HLA class I-restricted manner, but was not reactive with patient normal bone marrow cells or patient EBV transformed B cells. Taken together, these findings suggest that the GVM response is mediated by donor-derived CD8+ T-cell clones with antimyeloma specificity that may be present before DLI. In contrast, T-cell clones associated with GVHD are expanded de novo after DLI.

CML28 is a broadly immunogenic antigen, which is overexpressed in tumor cells.

Donor lymphocyte infusion (DLI) reliably induces durable remission in 75-80% of patients with relapsed chronic myelogenous leukemia (CML) after allogeneic hematopoietic stem cell transplantation. To identify immunological targets of the graft-versus-leukemia response (GVL) after DLI, we used CML post-DLI responder sera to screen a CML cDNA expression library. One of the antigens identified in this screen is a M(r) 28,000 protein, termed CML28. CML28 is identical to hRrp46p, a component of the human exosome, a multiprotein complex involved in the 3' processing of RNA. Components of the human exosome include known autoantigens, such as PMScl-100, an autoantibody target in patients with polymyositis, scleroderma, or polymyositis-scleroderma overlap syndrome. Recombinant CML28-GST fusion protein was purified, and used in Western blot and ELISA to demonstrate the development of a high-titer CML28-specific IgG antibody response in a patient with relapsed CML who responded to DLI. Northern blotting demonstrated that CML28 is highly expressed in a variety of hematopoietic and epithelial tumor cell lines, but not in normal hematopoietic tissues or other normal tissue, with the exception of testis. Purified recombinant CML28 was used to generate a CML28-specific murine monoclonal antibody. Western blotting with CML28 monoclonal antibody against whole-cell lysates derived from blood and marrow of normal donors and patients with leukemia revealed high expression of this antigen in tumor but not in normal samples. Because CML28 was highly expressed in epithelial tumor cell lines, anti-CML28 responses were also examined in patients with solid tumors. By ELISA, we found specific serological responses in 10-33% of patients with lung cancer, melanoma, and prostate cancer. Our studies suggest that immunogenicity of CML28 is likely because of overexpression of this antigen in tumor cells. Moreover, given its expression and immunogenicity in a wide variety of malignancies, CML28 merits additional evaluation as a target for antigen-specific immunotherapy.

Infusion of CD4+ donor lymphocytes induces the expansion of CD8+ donor T cells with cytolytic activity directed against recipient hematopoietic cells.

PURPOSE: Donor lymphocyte infusions (DLIs) provide effectivetherapy for patients with relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation. Previous studies have suggested that depletion of CD8+ T cells from the infused donor lymphocytes can reduce the incidence of graft-versus-host disease associated with DLI without reducing antileukemia activity. In this situation however, the immune effector cells responsible for tumor rejection have not been identified. The goal of this study was to characterize these effector populations. EXPERIMENTAL DESIGN: We studied three representative patients with relapsed chronic myeloid leukemia who achieved complete molecular remission after receiving CD8+ T-cell-depleted DLI from HLA-identical sibling donors. Effector T cells were characterized in patient samples after in vitro stimulation and functional assessment. T-cell clones relevant to the immune response were then isolated and further characterized. RESULTS: Analysis of peripheral blood samples collected after DLI indicated the presence of a high frequency of circulating host-reactive cytolytic CD8+ T cells secreting IFN-gamma. These HLA class I-restricted CTLs were specific for recipient minor histocompatibility antigens (mHAs) because they did not recognize target cells of donor origin. One CTL clone was further expanded in vitro and shown to recognize a broadly expressed mHA presented by HLA-B5701. Using a molecular approach, we demonstrated that this clone was expanded in peripheral blood and marrow after DLI. It was not detected before DLI. CONCLUSIONS: These results indicate that CD4+ DLI elicits a potent allogeneic response mediated by mHA-specific CD8+ T cells.

Angiogenic cytokines are antibody targets during graft-versus-leukemia reactions.

PURPOSE: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). EXPERIMENTAL DESIGN: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. RESULTS: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. CONCLUSIONS: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias.

Biologic Activity of Autologous, Granulocyte-Macrophage Colony-Stimulating Factor Secreting Alveolar Soft-Part Sarcoma and Clear Cell Sarcoma Vaccines.

PURPOSE: Alveolar soft-part sarcoma (ASPS) and clear cell sarcoma (CCS) are rare mesenchymal malignancies driven by chromosomal translocations that activate members of the microphthalmia transcription factor (MITF) family. However, in contrast to malignant melanoma, little is known about their immunogenicity. To learn more about the host response to ASPS and CCS, we conducted a phase I clinical trial of vaccination with irradiated, autologous sarcoma cells engineered by adenoviral-mediated gene transfer to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). EXPERIMENTAL DESIGN: Metastatic tumors from ASPS and CCS patients were resected, processed to single-cell suspensions, transduced with a replication-defective adenoviral vector encoding GM-CSF, and irradiated. Immunizations were administered subcutaneously and intradermally weekly three times and then every other week. RESULTS: Vaccines were successfully manufactured for 11 of the 12 enrolled patients. Eleven subjects received from three to 13 immunizations. Toxicities were restricted to grade 1-2 skin reactions at inoculation sites. Vaccination elicited local dendritic cell infiltrates and stimulated T cell-mediated delayed-type hypersensitivity reactions to irradiated, autologous tumor cells. Antibody responses to tissue-type plasminogen activator (tTPA) and angiopoietins-1/2 were detected. Tumor biopsies showed programmed death-1 (PD-1)-positive CD8(+) T cells in association with PD ligand-1 (PD-L1)-expressing sarcoma cells. No tumor regressions were observed. CONCLUSIONS: Vaccination with irradiated, GM-CSF-secreting autologous sarcoma cell vaccines is feasible, safe, and biologically active. Concurrent targeting of angiogenic cytokines and antagonism of the PD-1-negative regulatory pathway might intensify immune-mediated tumor destruction.

Autologous CLL cell vaccination early after transplant induces leukemia-specific T cells.

BACKGROUND: Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. METHODS: We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. RESULTS: At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. CONCLUSION: Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. TRIAL REGISTRATION: Clinicaltrials.gov NCT00442130. FUNDING: NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Detecting T-cell reactivity to whole cell vaccines: Proof of concept analysis of T-cell response to K562 cell antigens in CML patients.

BCR-ABL(+) K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (DC)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous DCs. These DCs were used to both stimulate and detect antigen-specific CD8(+) T-cell reactivity. As proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anti-CML antigen T-cell reactivity in CML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 CML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM-CSF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown.

Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid-immunoglobulin complexes in humans.

Donor lymphocyte infusion (DLI), whereby donor mononuclear cells are infused into patients, is one of the few effective immunotherapeutic strategies that generate long-lasting tumor remissions. We previously demonstrated that chronic myelogenous leukemia (CML) patients treated with DLI develop high-titer plasma antibodies specific for CML-associated antigens, the majority of which have been reported to bind nucleic acids These observations led us to predict that circulating antibody-antigen complexes in DLI-responsive patients carry nucleic acids that can engage innate immune sensors. Consistent with this, we report here that post-DLI plasma from 5 CML patients that responded to DLI treatment induced massive upregulation of MIP-1α, IP-10, and IFN-α in normal blood mononuclear cells. Importantly, this was not observed with plasma obtained before DLI and from DLI nonresponders and imatinib-treated patients. This endogenous immunostimulatory activity required nucleic acid and protein for its adjuvant effect and activated antigen-presenting cells through the RNA and DNA sensors TLR8 and TLR9. Presence of the immunoglobulin Fc receptor CD32 enhanced cellular responses, suggesting that immunoglobulins associate with this activity. Finally, a TLR-induced expression signature was detectable in post-DLI but not pre-DLI blood, consistent with an active circulating TLR8/9-stimulating factor. We have therefore demonstrated that effective tumor immunity correlates with the presence of endogenous nucleic acid-immunoglobulin complexes in patient plasma, thus providing a putative mechanism for the induction of potent antigen-specific immunity against malignant cells.

Graft-versus-leukemia antigen CML66 elicits coordinated B-cell and T-cell immunity after donor lymphocyte infusion.

PURPOSE: The target antigens of graft-versus-leukemia that are tumor associated are incompletely characterized. EXPERIMENTAL DESIGN: We examined responses developing against CML66, an immunogenic antigen preferentially expressed in myeloid progenitor cells identified from a patient with chronic myelogenous leukemia who attained long-lived remission following CD4+ donor lymphocyte infusion (DLI). RESULTS: From this patient, CML66-reactive CD8+ T-cell clones were detected against an endogenously presented HLA-B*4403-restricted epitope (HDVDALLW). Neither CML66-specific antibody nor T-cell responses were detectable in peripheral blood before DLI. However, by 1 month after DLI, CD8+ T cells were present in peripheral blood and at 10-fold higher frequency in marrow. Subsequently, plasma antibody to CML66 developed in association with disease remission. Donor-derived CML66-reactive T cells were detected at low levels in vivo in marrow before DLI by ELISpot and by a nested PCR-based assay to detect clonotypic T-cell receptor sequences but not in blood of the patient pre-DLI nor of the graft donor. CONCLUSIONS: CD4+ DLI results in rapid expansion of preexisting marrow-resident leukemia-specific donor CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in blood. Our single-antigen analysis thus shows that durable posttransplant tumor immunity is directed in part against nonpolymorphic overexpressed leukemia antigens that elicit coordinated cellular and humoral immunity.

Efficacious immune therapy in chronic myelogenous leukemia (CML) recognizes antigens that are expressed on CML progenitor cells.

Curative effects of graft-versus-leukemia-based therapies such as donor lymphocyte infusion (DLI) for chronic myelogenous leukemia (CML) may result from immunologic ablation of self-renewing CML progenitor cells. Patients who achieved durable remissions after DLI developed a significant B-cell lymphocytosis after treatment, which did not occur in patients who were unresponsive to DLI. In this study, we identified antigen targets of this B-cell response by probing two immunoproteomic platforms with plasma immunoglobulins from seven CML patients with clinically apparent graft-versus-leukemia responses after DLI. In total, 62 antigens elicited greater reactivity from post-DLI versus pre-DLI plasma. Microarray analysis revealed that >70% of the antigens were expressed in CML CD34(+) cells, suggesting that expression in malignant progenitor cells is a feature common to antibody targets of DLI. We confirmed elevated expression of three target antigens (RAB38, TBCE, and DUSP12) in CML that together consistently elicited antibody responses in 18 of 21 of an additional cohort of CML patients with therapeutic responses, but not in normal donors and rarely in non-CML patients. In summary, immunologic targets of curative DLI responses include multiple antigens on CML progenitor cells, identifying them as potential immunogens for vaccination and/or monitoring of immunotherapeutics designed to eliminate myeloid leukemia stem cells.

Serologic markers of effective tumor immunity against chronic lymphocytic leukemia include nonmutated B-cell antigens.

Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens-C6orf130, MDS032, and ZFYVE19-were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity.

IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo.

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

Graft-versus-tumor response in patients with multiple myeloma is associated with antibody response to BCMA, a plasma-cell membrane receptor.

Donor lymphocyte infusions (DLIs) induce effective graft-versus-tumor responses in patients with multiple myeloma who relapse after allogeneic hematopoietic stem-cell transplantation. The graft-versus-myeloma response is presumably mediated primarily by donor T cells, but recent studies have also demonstrated the presence of antibodies specific for a variety of myeloma-associated antigens in patients who achieve complete remission after DLI. One of the B-cell antigens identified in these studies was B-cell maturation antigen (BCMA), a transmembrane receptor of the tumor necrosis factor (TNF) superfamily that is selectively expressed by mature B cells. The present studies were undertaken to characterize the functional significance of antibodies to BCMA in vivo. Using transfected cells expressing BCMA, antibodies in patient serum were found to react with the cell-surface domain of BCMA. Post-DLI patient serum was able to induce complement-mediated lysis and antibody-dependent cellular cytotoxicity (ADCC) of transfected cells and primary myeloma cells expressing BCMA. BCMA antibodies were only found in post-DLI responders and not in other allogeneic transplant patients or healthy donors. These results demonstrate that BCMA is a target of donor B-cell immunity in patients with myeloma who respond to DLI. Antibody responses to cell-surface BCMA may contribute directly to tumor rejection in vivo.