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Plasma biomarkers associated with respiratory failure (RF) following hematopoietic cell transplantation (HCT) have not been identified. Therefore, we aimed to validate early (7 and 14 days post-HCT) risk biomarkers for RF. Using tandem mass spectrometry, we compared plasma obtained at day 14 post-HCT from 15 patients with RF and 15 patients without RF. Six candidate proteins, from this discovery cohort or identified in the literature, were measured by enzyme-linked immunosorbent assay in day-7 and day-14 post-HCT samples from the training (n = 213) and validation (n = 119) cohorts. Cox proportional-hazard analyses with biomarkers dichotomized by Youden’s index, as well as landmark analyses to determine the association between biomarkers and RF, were performed. Of the 6 markers, Stimulation-2 (ST2), WAP 4-disulfide core domain protein 2 (WFDC2), interleukin-6 (IL-6), and tumor necrosis factor receptor 1 (TNFR1), measured at day 14 post-HCT, had the most significant association with an increased risk for RF in the training cohort (ST2: hazard ratio [HR], 4.5, P = .004; WFDC2: HR, 4.2, P = .010; IL-6: HR, 6.9, P < .001; and TFNR1: HR, 6.1, P < .001) and in the validation cohort (ST2: HR, 23.2, P = .013; WFDC2: HR, 18.2, P = .019; IL-6: HR, 12.2, P = .014; and TFNR1: HR, 16.1, P = .001) after adjusting for the conditioning regimen. Using cause-specific landmark analyses, including days 7 and 14, high plasma levels of ST2, WFDC2, IL-6, and TNFR1 were associated with an increased HR for RF in the training and validation cohorts. These biomarkers were also predictive of mortality from RF. ST2, WFDC2, IL-6 and TNFR1 levels measured early posttransplantation improve risk stratification for RF and its related mortality.

Plasma biomarkers associated with respiratory failure (RF) following hematopoietic cell transplantation (HCT) have not been identified. Therefore, we aimed to validate early (7 and 14 days post-HCT) risk biomarkers for RF. Using tandem mass spectrometry, we compared plasma obtained at day 14 post-HCT from 15 patients with RF and 15 patients without RF. Six candidate proteins, from this discovery cohort or identified in the literature, were measured by enzyme-linked immunosorbent assay in day-7 and day-14 post-HCT samples from the training (n = 213) and validation (n = 119) cohorts. Cox proportional-hazard analyses with biomarkers dichotomized by Youden's index, as well as landmark analyses to determine the association between biomarkers and RF, were performed. Of the 6 markers, Stimulation-2 (ST2), WAP 4-disulfide core domain protein 2 (WFDC2), interleukin-6 (IL-6), and tumor necrosis factor receptor 1 (TNFR1), measured at day 14 post-HCT, had the most significant association with an increased risk for RF in the training cohort (ST2: hazard ratio [HR], 4.5, P= .004; WFDC2: HR, 4.2, P= .010; IL-6: HR, 6.9, P< .001; and TFNR1: HR, 6.1, P< .001) and in the validation cohort (ST2: HR, 23.2, P= .013; WFDC2: HR, 18.2, P= .019; IL-6: HR, 12.2, P= .014; and TFNR1: HR, 16.1, P= .001) after adjusting for the conditioning regimen. Using cause-specific landmark analyses, including days 7 and 14, high plasma levels of ST2, WFDC2, IL-6, and TNFR1 were associated with an increased HR for RF in the training and validation cohorts. These biomarkers were also predictive of mortality from RF. ST2, WFDC2, IL-6 and TNFR1 levels measured early posttransplantation improve risk stratification for RF and its related mortality.

•Adoptive immunotherapy using natural killer (NK) cells uses 3 different strategies to target cancer.•Activating receptors recognize ligands upregulated on the surface of tumor cells.•Inhibitory receptors recognize self major histocompatibility complex (MHC) that are typically downregulated in tumor cells.•Fc receptors recognize antibodies that bind to surface targets on tumor cells.•Tumor cells may evade NK cells by upregulating MHC (increasing inhibition) or downregulating antibody targets or activating receptor ligands.•Strategies to overcome immune evasion include modifying NK cells with chimeric antigen receptors, redirecting their activity toward a defined target.•Tumor cells may suppress NK cells through a variety of mechanisms.•Strategies to overcome immune suppression include modifying NK cells to resist inhibitory cytokines.

Assessment of prognostic biomarkers of nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT) in the pediatric age group is lacking. To address this need, we conducted a prospective cohort study with 415 patients at 6 centers: 170 were children age 10 years or younger and 245 were patients older than age 10 years (both children and adults were accrued from 2013 to 2018). The following 4 plasma biomarkers were assessed pre-HCT and at days +7, +14, and +21 post-HCT: stimulation-2 (ST2), tumor necrosis factor receptor 1 (TNFR1), regenerating islet–derived protein 3α (REG3α), and interleukin-6 (IL-6). We performed landmark analyses for NRM, dichotomizing the cohort at age 10 years or younger and using each biomarker median as a cutoff for high- and low-risk groups. Post-HCT biomarker analysis showed that ST2 (>26 ng/mL), TNFR1 (>3441 pg/mL), and REG3α (>25 ng/mL) are associated with NRM in children age 10 years or younger (ST2: hazard ratio [HR], 9.13; 95% confidence interval [CI], 2.74-30.38; P = .0003; TNFR1: HR, 4.29; 95% CI, 1.48-12.48; P = .0073; REG3α: HR, 7.28; 95% CI, 2.05-25.93; P = .0022); and in children and adults older than age 10 years (ST2: HR, 2.60; 95% CI, 1.15-5.86; P = .021; TNFR1: HR, 2.09; 95% CI, 0.96-4.58; P = .06; and REG3α: HR, 2.57; 95% CI, 1.19-5.55; P = .016). When pre-HCT biomarkers were included, only ST2 remained significant in both cohorts. After adjustment for significant covariates (race/ethnicity, malignant disease, graft, and graft-versus-host-disease prophylaxis), ST2 remained associated with NRM only in recipients age 10 years or younger (HR, 4.82; 95% CI, 1.89-14.66; P = .0056). Assays of ST2, TNFR1, and REG3α in the first 3 weeks after HCT have prognostic value for NRM in both children and adults. The presence of ST2 before HCT is a prognostic biomarker for NRM in children age 10 years or younger allowing for additional stratification. This trial was registered at www.clinicaltrials.gov as #NCT02194439.

Assessment of prognostic biomarkers of nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT) in the pediatric age group is lacking. To address this need, we conducted a prospective cohort study with 415 patients at 6 centers: 170 were children age 10 years or younger and 245 were patients older than age 10 years (both children and adults were accrued from 2013 to 2018). The following 4 plasma biomarkers were assessed pre-HCT and at days +7, +14, and +21 post-HCT: stimulation-2 (ST2), tumor necrosis factor receptor 1 (TNFR1), regenerating islet–derived protein 3α (REG3α), and interleukin-6 (IL-6). We performed landmark analyses for NRM, dichotomizing the cohort at age 10 years or younger and using each biomarker median as a cutoff for high- and low-risk groups. Post-HCT biomarker analysis showed that ST2 (>26 ng/mL), TNFR1 (>3441 pg/mL), and REG3α (>25 ng/mL) are associated with NRM in children age 10 years or younger (ST2: hazard ratio [HR], 9.13; 95% confidence interval [CI], 2.74-30.38; P= .0003; TNFR1: HR, 4.29; 95% CI, 1.48-12.48; P= .0073; REG3α: HR, 7.28; 95% CI, 2.05-25.93; P = .0022); and in children and adults older than age 10 years (ST2: HR, 2.60; 95% CI, 1.15-5.86; P= .021; TNFR1: HR, 2.09; 95% CI, 0.96-4.58; P= .06; and REG3α: HR, 2.57; 95% CI, 1.19-5.55; P = .016). When pre-HCT biomarkers were included, only ST2 remained significant in both cohorts. After adjustment for significant covariates (race/ethnicity, malignant disease, graft, and graft-versus-host-disease prophylaxis), ST2 remained associated with NRM only in recipients age 10 years or younger (HR, 4.82; 95% CI, 1.89-14.66; P= .0056). Assays of ST2, TNFR1, and REG3α in the first 3 weeks after HCT have prognostic value for NRM in both children and adults. The presence of ST2 before HCT is a prognostic biomarker for NRM in children age 10 years or younger allowing for additional stratification. This trial was registered at www.clinicaltrials.govas #NCT02194439.

•Adoptive immunotherapy using natural killer (NK) cells uses 3 different strategies to target cancer.•Activating receptors recognize ligands upregulated on the surface of tumor cells.•Inhibitory receptors recognize self major histocompatibility complex (MHC) that are typically downregulated in tumor cells.•Fc receptors recognize antibodies that bind to surface targets on tumor cells.•Tumor cells may evade NK cells by upregulating MHC (increasing inhibition) or downregulating antibody targets or activating receptor ligands.•Strategies to overcome immune evasion include modifying NK cells with chimeric antigen receptors, redirecting their activity toward a defined target.•Tumor cells may suppress NK cells through a variety of mechanisms.•Strategies to overcome immune suppression include modifying NK cells to resist inhibitory cytokines.

The Berlin Patient represents the first and only functional HIV cure achieved by hematopoietic stem cell transplant (HSCT). In subsequent efforts to replicate this result, HIV rebounded post-HSCT after withdrawal of antiretroviral therapy. Providing HIV-specific immunity through adoptive T cell therapy may prevent HIV rebound post-HSCT by eliminating newly infected cells before they can seed systemic infection. Adoptive T cell therapy has demonstrated success in boosting Epstein-Barr virus and cytomegalovirus-specific immunity post-HSCT, controlling viral reactivation. However, T cell immunotherapies to boost HIV-specific immunity have been limited by single-epitope specificity and minimal persistence or efficacy in vivo. To improve this strategy, we sought to generate allogeneic HIV-specific T cells from human leukocyte antigen (HLA)-A02+ HIV-negative adult or cord blood donors. We focused on HLA-A02+ donors due to well-characterized epitope restrictions observed in HIV+ populations. We show that multi-antigen HIV-specific T cells can be generated from naive T cells of both cord blood and adults using a reproducible good manufacturing practice (GMP)-grade protocol. This product lysed antigen-pulsed targets and suppressed active HIV in vitro. Interestingly, these cells displayed broad epitope recognition despite lacking recognition of the common HLA-A02-restricted HIV epitope Gag SL9. This first demonstration of functional multi-antigen HIV-specific T cells has implications for improving treatment of HIV through allogeneic HSCT.

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control. This study is registered at clinicaltrials.gov as #NCT00840853.

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control. This study is registered at clinicaltrials.govas #NCT00840853.

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8+ and CD4+ T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8+and CD4+T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.govas NCT00078533.

Immunotherapy with antigen-specific T cells is a promising, targeted therapeutic option for patients with cancer as well as for immunocompromised patients with virus infections. In this review, we characterize and compare current manufacturing protocols for the generation of T cells specific to viral and non-viral tumor associated antigens. Specifically, we discuss: (i) the different methodologies to expand virus-specific T cell (VST) and non-viral tumor-associated antigen specific T cell (TAA-T) products, (ii) the immunological principles involved when developing such manufacturing protocols and (iii) proposed standardized methodologies for the generation of polyclonal, polyfunctional antigen-specific T cells irrespective of donor source. Ex vivo-expanded cells have been safely administered to treat numerous patients with virus-associated malignancies, hematologic malignancies, and solid tumors. Hence, we have performed a comprehensive review of the clinical trial results evaluating the safety, feasibility, and efficacy of these products in the clinic. In summary, this review seeks to provide new insights regarding antigen-specific T cell technology to benefit a rapidly expanding T cell therapy field.

Background: Patients with hematologic malignancies relapsing after allogeneic blood or marrow transplantation (BMT) have limited response to conventional salvage therapies with an expected 1-year overall survival (OS) of <20%. Current strategies to treat relapsed hematopoietic cancer after BMT, such as chemotherapy and donor lymphocyte infusions (DLI) to enhance the immunologic graft-versus-leukemia (GVL) effect, have low efficacy and are associated with graft-versus-host disease (GVHD). An alternative strategy to boost the GVL effect is to infuse donor-derived T-cells targeting tumor-associated antigen (TAA) peptides as circulating TAA-specific T-cells are associated with maintenance of remission post-BMT. In this phase-I study, we evaluated the safety and clinical outcomes following administration of a novel T-cell therapeutic targeting three TAAs, WT1, PRAME and survivin in patients with acute leukemia who relapsed or were at high-risk of relapse after allogeneic BMT.

Cruz: Mana Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Catamaran Bio: Consultancy, Patents & Royalties, Research Funding. Hanley: Mana Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellevolve: Consultancy; Maxcyte: Membership on an entity's Board of Directors or advisory committees. Bollard: SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Catamaran Bio: Membership on an entity's Board of Directors or advisory committees; Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees; ROCHE: Consultancy, Honoraria; Repertoire Immune Medicines: Current equity holder in publicly-traded company; Neximmune: Current equity holder in publicly-traded company; Mana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Background/Objectives:Relapse of hematologic malignancies after hematopoietic cell transplantation (HCT) confers high mortality because of progressive disease and graft-vs.-host disease (GVHD). Hence, we hypothesized that tumor-associated antigen-specific lymphocytes (TAA-L) would be specific, diminish immune escape, and could safely decrease cancer burden.

Adoptive T cell therapy has been successful in boosting viral-specific immunity post-HSCT, preventing viral rebound of CMV and EBV. However, the therapeutic use of T cells to boost HIV-specific T cell immunity in HIV+ patients has been met with limited success. Despite multiple attempts to eradicate HIV with allogeneic HSCT, there is only one case of functional HIV cure post-HSCT, the Berlin Patient. Hence, we hypothesized that broadly HIV-specific CD8 and CD4 T-cells (HXTCs) could be expanded from patients on ARVs, as well as HIV negative adult (dHXTCs) and cord blood donors (cbHXTCs), employing a non-HLA restricted approach for the treatment of HIV+ individuals after autologous or allogeneic HSCT.

Savoldo: Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

No relevant conflicts of interest to declare.

Younes: Celgene: Speakers Bureau; Syndax: Research Funding; SBIO: Research Funding; Genentech: Research Funding; Sanofi-Aventis: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau, Travel Expenses; Gilead: Honoraria; Pharmacyclic: Honoraria.

Abstract 1658

Abstract 4083

No relevant conflicts of interest to declare.

Abstract 3212

Allogeneic stem cell transplantation is the treatment of choice for patients with high-risk hematologic malignancies. Umbilical cord blood (CB) has emerged as an important source of stem cells for allotransplant patients lacking human leukocyte antigen (HLA)-matched donors–a significant problem for minorities. T cells in UCB grafts are, however, virus-naïve, leading to higher mortality rates due to infections with CMV, EBV, adenovirus (Ad) and other viruses. Peripheral Blood Stem Cell Transplant (PBSCT) from CMV-seronegative (CMVneg) donors to CMV-seropositive (CMVpos) recipients produces a similarly high incidence of CMV infection since donor T cells are naïve to this virus. Adoptive immunotherapy with peripheral blood (PB)-derived CMV/Ad-specific CTL generated from CMVpos donors effectively prevents CMV clinical disease after PBSCT, but this option has not been feasible when the donor cells are naive, irrespective of whether they are sourced from CB or CMV-PBS, since CTL generation from these donors has been unsuccessful. We have now overcome this problem and can routinely generate CMV, Ad and EBV-specific CTLs from CB and CMV specific CTLs from seronegative PBSC. We used an Ad5f35vector carrying the CMVpp65 transgene to transduce CB-derived or PB-derived dendritic cells and stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 stimulations with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector. The 9 CB-derived CTL lines we made in this way contained a mean of 87% (range 81-94) CD8+ and 26% (range 12-40) CD4+T cells, and exhibited significant cytotoxicity in 51Cr release assays against CMVpp65, Adhexon, and LCL targets. In IFN-γ ELISPOT assays there was a mean of 209 (range 45-694), 74 (range 0-128), and 157 (range 23-291) SFC following incubation with CMVpp65, Adhexon peptides and LCL, respectively. In addition, we generated CMVpp65, Adhexon, and LCL-specific responses from the peripheral blood of 4 CMVneg adult donors, which produced a mean of 92 (range 50-126), 163 (range 69-293), and 62 (range 37-86) SFC to CMVpp65 respectively. Neither CB- nor CMVneg-derived CTL responded to irrelevant peptides. Of note, the virus-specific T cells that we expanded from both CB and CMVneg donors derived only from T cells with a naive phenotype (CD45RA+/CCR7+). Moreover, both CB and CMVneg-derived CTL recognized ‘unconventional‘ CMV pp65 epitopes, as identified by overlapping pp65 peptide pools and confirmed by IFN-γ ELISPOT as well as multimer analysis (Table 1). In HLA-A2+ subjects, these naive-derived CTLs did not recognize conventional HLA-A2-associated CMV pp65 epitopes such as NLV, suggesting an inherent difference between naïve and memory T cell responses to CMV. In summary, virus-specific responses T cell responses can be obtained even from CB and virus-naive adult donors and may allow prevention and treatment of viral disease in the recipients of these allografts.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 504

Abstract 4089

Abstract 4083

We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that: is least sensitive to chemotherapy and has tumor repopulating potential. In this study we characterized SP cells in ten human lymphoma cell lines. Five of the 11 lymphoma cell lines tested (HDLM2, L428, Ramos, Toledo, Karpas) had a distinct SP ranging from 0.8–2% of total cells. We subsequently found SP cells with tumor associated markers in biopsy samples from patients with Hodgkin’s Lymphoma, T-cell Lymphoma, and Follicular lymphoma. Culture of the sorted cell lines show that SP but not the non-SP cells produced progeny that were both SP and non-SP cells. Gene expression analysis of the SP showed increased expression of ABCG2 and other ABC transporters that mediate both the transport of Hoechst dye and selected chemotherapeutic agents out of the cell. We evaluated whether lymphoma cell lines with an SP are resistant to the chemotherapeutic drug gemcitabine. The viability of lymphoma lines containing SP cells was significantly higher (mean 59.8%; range 37.4–87.8%%) than the viability of lines without an SP (mean 9.3% range 3.0%–12.2%) when cultured with 10nM gemcitabine for 3 days. The SP component of the lymphoma cells became enriched 10 fold when cultured with gemcitabine. Furthermore, when equal numbers of SP and non-SP cells were grown in separate fractions with gemcitabine, there was reduced viability of the non-SP (2,247 +/−294 mean counts per minute (cpm)) by thymidine assay. In contrast, the viability of the SP was preserved (13,200 +/− 7500 mean cpm). There was no difference in the viability between the untreated SP and Non SP, so that the gemcitabine rather than the Hoechst dye accounts for the differences in viability. Although lymphoma SP cells are resistant to chemotherapeutic agents, they also express higher levels of tumor associated antigens that are known targets for cytotoxic T cells. Hence RT-PCR and immunohistochemistry both show that SP cells are HLA positive and have increased expression of MAGE-4, SSX2, Survivin and NY-ESO transcripts and protein compared to non-SP cells from the same tumor. In IFNγ ELISPOT assays, we have shown that T cells secrete IFNγ in response to SP targets. Hence, lymphoma cell lines and primary lymphoma tissue contain chemotherapy-resistant SP cells which express tumor associated antigens and may be targeted by specific T cells.

Umbilical cord blood (UCB) transplantation is a promising alternative source of hematopoietic stem cells for patients lacking HLA-matched donors. Nearly 60% of UCB transplants to date have been performed on minority individuals for whom an unrelated donor was not available; moreover, the naïve phenotype of UCB cells may be responsible for the lower incidence and reduced severity of GvHD in these patients. The relatively low cell numbers and naïvety of T lymphocyte populations in UCB grafts has, however, lead to delayed immune reconstitution and higher mortality due to infection. Reactivations of latent viruses such as cytomegalovirus (CMV) are particularly problematic, as is overt infection from adenovirus (Adv). Previous studies have shown that prophylactic adoptive immunotherapy with peripheral blood-derived CTL directed against CMV and Adv can effectively prevent the clinical manifestations of these viruses after hematopoietic stem cell transplant raising the possibility that a similar approach could be developed after UCB transplant. We hypothesized that virus-specific CTL could be generated from UCB for clinical use to restore anti-viral immunity and reduce viral infection post UCB transplant. Bi-virus specific CTL were generated from frozen UCB mononuclear cells using a clinical-grade recombinant adenovirus type5 vector pseudotyped with a type35 fiber carrying a transgene for CMVpp65 as a source of Adv and CMV antigens. UCB-derived dendritic cells were transduced with this Ad5f35pp65 vector as the initial source of antigen presenting cells to stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 rounds of weekly stimulation with autologous UCB-derived EBV-lymphoblastoid cell lines (LCL) transduced with the same vector in the presence of IL-15 and IL-2. UCB from donors of varied HLA types were selected. 40×106 UCB mononuclear cells (available in the 20% fraction of frozen UCB units) were thawed and used in the manufacturing process. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 64–94%) CD8+ve T-cells and 27% (range 12–40%) CD4+ve T-cells. Flow cytometric analysis of memory markers after 3 weeks expansion revealed a predominance of CD45RA− CD62L− T-cells (69±18%; range 25–93%) with a smaller population of CD45RA− CD62L+ T-cells (10±5%; range 1–23%). Evaluable UCB CTL lines showed specific cytolytic activity in 51Cr release assays against targets loaded with CMV and Adv antigens. The observed cytotoxicity was specific because unloaded targets and MHC-mismatched targets were not killed. IFNγ ELISPOT assays on CTL lines demonstrated a mean of 209 (range 45–694) and 74 (range 0–188) spot forming cells/1×105 T-cells following incubation with CMV-pp65 and Adv-hexon/penton peptides respectively. No significant response to CMV-IE1 peptides was demonstrated. The expanded UCB CTL had a broad Vβ repertoire and were specific for multiple viral epitopes. In addition, the virus-specific T cells were shown to be expanded only from T-cells with a naïve phenotype (CD45RA+/CCR7+). These results demonstrate that, despite the generally naïve nature of UCB lymphocytes, bi-virus-specific responses can be expanded in vitro and could potentially be used clinically in UCBT patients who develop infectious complications prior to immune reconstitution.

We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that:

Umbilical cord blood (UCB) transplantation is a promising alternative source of hematopoietic stem cells for patients lacking HLA-matched donors. Nearly 60% of UCB transplants to date have been performed on minority individuals for whom an unrelated donor was not available; moreover, the naïve phenotype of UCB cells may be responsible for the lower incidence and reduced severity of GvHD in these patients. The relatively low cell numbers and naïvety of T lymphocyte populations in UCB grafts has, however, lead to delayed immune reconstitution and higher mortality due to infection. Reactivations of latent viruses such as cytomegalovirus (CMV) are particularly problematic, as is overt infection from adenovirus (Adv). Previous studies have shown that prophylactic adoptive immunotherapy with peripheral blood-derived CTL directed against CMV and Adv can effectively prevent the clinical manifestations of these viruses after hematopoietic stem cell transplant raising the possibility that a similar approach could be developed after UCB transplant. We hypothesized that virus-specific CTL could be generated from UCB for clinical use to restore anti-viral immunity and reduce viral infection post UCB transplant. Bi-virus specific CTL were generated from frozen UCB mononuclear cells using a clinical-grade recombinant adenovirus type5 vector pseudotyped with a type35 fiber carrying a transgene for CMVpp65 as a source of Adv and CMV antigens. UCB-derived dendritic cells were transduced with this Ad5f35pp65 vector as the initial source of antigen presenting cells to stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 rounds of weekly stimulation with autologous UCB-derived EBV-lymphoblastoid cell lines (LCL) transduced with the same vector in the presence of IL-15 and IL-2. UCB from donors of varied HLA types were selected. 40×106UCB mononuclear cells (available in the 20% fraction of frozen UCB units) were thawed and used in the manufacturing process. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 64–94%) CD8+ve T-cells and 27% (range 12–40%) CD4+ve T-cells. Flow cytometric analysis of memory markers after 3 weeks expansion revealed a predominance of CD45RA− CD62L− T-cells (69±18%; range 25–93%) with a smaller population of CD45RA− CD62L+ T-cells (10±5%; range 1–23%). Evaluable UCB CTL lines showed specific cytolytic activity in 51Cr release assays against targets loaded with CMV and Adv antigens. The observed cytotoxicity was specific because unloaded targets and MHC-mismatched targets were not killed. IFNγ ELISPOT assays on CTL lines demonstrated a mean of 209 (range 45–694) and 74 (range 0–188) spot forming cells/1×105T-cells following incubation with CMV-pp65 and Adv-hexon/penton peptides respectively. No significant response to CMV-IE1 peptides was demonstrated. The expanded UCB CTL had a broad Vβ repertoire and were specific for multiple viral epitopes. In addition, the virus-specific T cells were shown to be expanded only from T-cells with a naïve phenotype (CD45RA+/CCR7+). These results demonstrate that, despite the generally naïve nature of UCB lymphocytes, bi-virus-specific responses can be expanded in vitroand could potentially be used clinically in UCBT patients who develop infectious complications prior to immune reconstitution.