ALBERT

Description: The presence of breast cancer cells in the patient’s bone marrow (BM) at diagnosis is associated with resistance to treatment, disease relapse and poor prognosis. Identification of the factors implicated in the homing, survival and latency of breast cancer cells in the BM should contribute to the design of more efficient therapeutic strategies for breast cancer. There is evidence that breast cancer can recruit endothelial progenitors from the BM. Also, other epithelial tumors seem to preferentially adhere to BM endothelial cells. Therefore, we hypothesized that BM endothelium may play a significant role in the biology of breast cancer cells residing in the BM. Co-cultures in Matrigel showed that breast cancer cells interact with BM endothelium to form heterotypic multicellular networks. Moreover, breast cancer cells migrate towards BM endothelium assembled as capillary-like structures, but not to structures of BM mesenchymal stem cells or BM stroma. This migration was abrogated by pertussis toxin-mediated blockade of chemokine receptor signaling, suggesting the involvement of endothelium-secreted chemokine(s). We then evaluated the impact of breast cancer cells in the survival and proliferation of BM endothelium. All breast cancer lines tested (n=4) promoted the proliferation of BM-derived endothelial cells. This effect is mediated through the engagement of the PI3K/Akt pathway (phosphorylation of Akt at Ser437 and Thr308, and activation of its downstream substrates GSK3β, PRAS-40 and FKHRL1) since its specific blockade abrogated the stimulatory effects of breast cancer on BM endothelium. We next determined whether, reciprocally, BM endothelium impacts on breast cancer cell survival. These experiments were performed in serum-free media to enhance dependency of breast cancer cells from microenvironmental stimuli. In all cases tested, BM endothelium promoted survival/proliferation of breast cancer cells. This stimulation was accompanied by the engagement of the PI3K/Akt pathway in breast cancer cells and, in three of the four lines, the phosphorylation of Erk1/2. These effects were also observed for breast cancer cells that showed constitutive activation of Akt (MCF-7 and ZR-75-1 cells). Specific blockade of PI3K/Akt abrogated the BM endothelium-promoted survival of breast cancer cells, thus demonstrating the critical role of this pathway. These studies show that crosstalk between BM endothelial cells and breast cancer cells may impact on the survival of both cell types. These findings provide new light on the mechanisms that may facilitate the development of a tumor-permissive BM microenvironment in breast cancer, and the creation of breast cancer-supporting BM niches. Importantly, this study implicates BM endothelium as a therapeutic target in breast cancer and suggest that blockade of PI3K/Akt may impact the outcome of patients with metastatic breast cancer.

Description: We report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 ×106/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

Description: Targeted disruption of signaling pathways essential for tumor cell survival and proliferation offer great promise for the design of more efficient and specific therapies. mTOR plays a central role in sensing mitogenic, nutrient and energy signals, and in the integration of different signaling pathways. Moreover, increasing evidence implicates mTOR in tumorigenesis and in the maintenance of transformed phenotypes. Therefore, we hypothesized that the mTOR pathway may play a significant role in T-cell acute lymphoblastic leukemia (ALL). BM and peripheral blood specimens from 15 pediatric patients were analyzed for the phosphorylation status of two downstream substrates of mTOR, S6 Ribosomal Protein and eIF4G. In all cases, primary leukemia T cells showed marked phosphorylation of S6 Ribosomal Protein (Ser235/236) and phosphorylation of the eukaryotic initiation factor eIF4G (Ser1108), showing that the mTOR pathway is constitutively active in T-ALL cells. Since leukemia T cells did not show significant basal phosphorylation of Akt/PKB, this observation suggests that the mTOR activation observed in T-ALL is Akt-independent. Specific blockade of mTOR by Rapamycin (100nM) reduced or abrogated phosphorylation of S6 ribosomal protein. Moreover, the mTOR blockers Rapamycin (10–100nM) and CCI-779 (10–100nM) significantly inhibited cell survival, and IL-7-promoted proliferation (40% to 85% inhibition) and cell cycle progression of T-ALL cells. Since we have shown previously that PI3K/Akt is critically involved in T-ALL responses to exogenous stimuli, we next evaluated whether inhibition of PI3K/Akt signals would synergize or potentiate the effects of mTOR blockade. We observed that the PI3K-inhibitor LY294002 (10μM) synergized with Rapamycin to abrogate IL-7-promoted leukemia T-cell proliferation. Finally, we explored the possibility that mTOR blockade can enhance the cytotoxic effects on T-ALL of the conventional drugs Dexamethasone (100nM) and Doxorubicin (100nM). Rapamycin potentiated the cytotoxic effects of both Dexamethasone (1.3 to 10 fold) and Doxorubicin (1.7 to 7.3 fold), suggesting that the combination of mTOR blockade with chemotherapy can improve the anti-leukemia efficacy of conventional regimens. In conclusion, we observed that the mTOR pathway is constitutively activated in primary leukemia T cells, and that the mTOR blockers Rapamycin and CCI-779 significantly inhibit T-ALL survival and proliferation. In addition, Rapamycin potentiates the inhibitory activity of other signaling inhibitors and the cytotoxic effects of the conventional drugs Dexamethasone and Doxorubicin. This study suggests that the mTOR pathway is a valid target for the treatment of T-ALL and lay the groundwork for the inclusion of mTOR blockade in the current therapeutic regimens.

Description: Appropriate processing and presentation of tumor associated antigens (TAA) by antigen presenting cells (APC) is absolutely required for the development of clinically relevant anti-tumor T cell responses. One common approach which utilizes the exogenous pulsing of synthetic peptides onto APC can sometimes generate ineffective immune responses. This failure may, in part, be the consequence of erroneous conformations of HLA/synthetic pulsed peptides which can differ from conformations formed with HLA/endogenous peptides that are derived from intracellular proteins. Also, endogenously processed peptides sometimes undergo post-translational modifications during transport to the cell surface, a process that does not occur with exogenously loaded peptides. Since our goal is to induce immunity that can recognize TAA that are endogenously presented by tumors, it is logical that the ideal APC would not only express the required immunoaccessory molecules, but would also endogenously process and appropriately present target antigenic peptides. In this report, we employed our artificial APC (aAPC) that expresses HLA-A2, CD80, and CD83 and is capable of priming and supporting the prolonged expansion of peptide specific CD8+ cytotoxic T cells (CTL). We hypothesized that aAPC can endogenously process and present HLA class I peptides and can induce functional T cell immunity. To test this, aAPC was transduced with an EGFP-mini MP1 (aa 55–66) fusion gene containing the sequence for the influenza derived peptide MP58 (aa 58–66). We observed that this HLA-A2 restricted peptide is processed and presented by aAPC by demonstrating that MP58 specific CTL are able to recognize aAPC/mini MP1 target cells. This was completely abrogated by treating aAPC/mini MP1 with proteasome inhibitors, suggesting that MP58 is endogenously processed by the proteasome. This was confirmed by the elevation of EGFP-mini MP1 fusion protein and the accumulation of ubiquitinated forms as detected by flow cytometry and Western blot analysis, respectively. At the protein level, aAPC was shown to express all proteasome subunits examined and to upregulate immunoproeosome subunits with exposure to IFN-γ. We biochemically confirmed the presence of MP58 in the A2 groove on the surface of aAPC/mini MP1, by performing reverse phased HPLC, mass spectrometry and peptide sequencing of peptides directly acid stripped from the cell surface. Since aAPC expresses only one HLA allele, A2, this finding provides strong support that MP58 is processed and presented in the groove of the A2 molecule on aAPC/mini MP1. We next evaluated the density of MP58 presented by HLA-A2 on aAPC/mini MP1. A2 positive CD8+ T cells were stimulated at weekly intervals by either aAPC/mini MP1 or parental aAPC exogenously pulsed with graded concentrations of synthetic MP58. After three stimulations, peptide specificity of generated CTL was examined by tetramer analysis. The comparison of tetramer staining revealed that the density of endogenously processed and presented MP58 corresponded to pulsing aAPC with 100 μg/ml. In order to extend this strategy to a TAA, we transduced aAPC with EGFP-mini MART1 (aa 27–35) mini gene. We have demonstrated that MART1 peptide is processed and presented on the cell surface and have induced the expansion of MART1 peptide specific T cells. These results suggest that our APC can naturally process and present class I restricted peptides, resulting in the efficient priming and expansion of clinically relevant antigen specific CTL.

Description: Abstract 354 Adoptive cell therapy utilizes unique mechanisms of action to prevent the development of infections in immunocompromised patients and treat chemotherapy resistant malignancies. In adoptive cell therapy, the major effector cells appear to be CD8+ T cells, since they are armed with antigen-specific effector functions, i.e. cytotoxicity and cytokine secretion. However, the roles of antigen-specific CD4+ T cells in T cell immunity are also critical. In immunocompromised patients adoptively transferred with CMV-specific CD8+ T cells, long-term in vivo persistence was achieved only when CMV-specific CD4+ T cells were also present in vivo. Recently, adoptive transfer of a NY-ESO-1 specific CD4+ T cell clone was reported to induce a complete response in a patient with metastatic melanoma. These results suggest that adoptive cell therapy for infectious diseases and cancer can be improved by infusing both antigen-specific CD4+ helper T cells as well as CD8+ CTL. Unfortunately, however, few versatile systems are available for producing large numbers of antigen-specific human CD4+ T cells for the purpose of adoptive therapy. K562 is a human erythroleukemic cell line, which lacks the expression of HLA class I and II, invariant chain (Ii), and HLA-DM, but does express adhesion molecules such as ICAM-1 and LFA-3. Given this unique immunologic phenotype, K562 has served as a useful tool in clinical cancer immunotherapy trials. Previously, we reported the generation of a K562-based artificial APC (aAPC), which expresses HLA-A2, CD80, and CD83. aAPC/A2 can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory phenotype, possess potent effector function, and can be maintained in vitro without any feeder cells or cloning. aAPC/A2 is equipped with constitutive proteasome and inducible immunoproteasome machinery and can naturally process and present CD8+ T cell peptides via transduced A2 molecules. We have successfully generated clinical grade aAPC/A2 under cGMP conditions and conducted a clinical trial where patient with advanced melanoma are infused with large numbers of MART1-specific CTL generated ex vivo using aAPC/A2, IL-2 and IL-15. Based on our experience with aAPC/A2 and CD8+ T cells, we have generated a series of novel aAPC (aAPC/DR1, DR3, DR4, DR7, DR10, DR11, DR13, and DR15) to stimulate HLA-DR-restricted antigen-specific CD4+ T cells. K562 has been engineered to express HLA-DRα and β chains as a single HLA allele in conjunction with Ii, HLA-DMα and β chains, CD80 and CD83. CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Following the transduction of Ii, CLIP (class II invariant chain-associated peptide) appeared on the cell surface of aAPC. Furthermore, CLIP expression on aAPC was almost completely abrogated by the introduction of HLA-DM. This result is in accordance with previous studies showing that HLA-DM catalyzes the removal of CLIP from DR thus enabling exogenous peptides to bind to empty DR molecules in late endosomes. In addition to its endogenous pinocytic activity, aAPC was made capable of Fcγ receptor-mediated endocytosis by transduction of CD64. Comparison of naïve aAPC and CD64-transduced aAPC confirmed that Fcγ receptor-mediated endocytosis is more efficient than pinocytosis to take up soluble protein and process and present DR-restricted peptides to CD4+ T cells. Using these standardized and renewable aAPC, we determined novel viral protein-derived DR-restricted CD4+ T cell epitopes and expanded large numbers of viral antigen-specific CD4+ T cells without growing bystander Foxp3+ regulatory T cells. Without any feeder cells or cloning, expansion of CD4+ T cells using aAPC and low dose IL-2 and IL-15 was sustainable up to 150 days. Immunophenotypic analysis using HLA-DR tetramers and specific mAbs revealed that expanded CD4+ T cells were CD45RA−, CD45RO+, CD62L+-, demonstrating a central/effector memory phenotype. Furthermore, intracellular cytokine analysis showed that expanded DR-restricted viral-specific CD4+ T cells secreted IL-2 and IFN-γ but much less IL-4, displaying a Th1-biased phenotype. Taken all together, these results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD4+ helper T cells and CD8+ CTL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2086 CD8+ T cells play a primary role in rejecting pathogens and tumors. Studies of CD8 null mice show that CD8 coreceptor is critical for the development of MHC class I-restricted CD8+ T cells in the thymus. However, while these mice possess low numbers of CTL with limited clonality, they are highly avid and contain acute and chronic infections. In humans, CD8 deficiency leads to no or only mild symptoms of immunodeficiency. These results suggest that the CD8 coreceptor is not absolutely necessary for the generation of antigen-specific CTL and that there exists a compensatory mechanism for the loss of CD8 expression. Common γ chain receptor cytokines (e.g. IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) transmit STAT-mediated signaling in T cells. Importantly, it has been demonstrated that crosstalk between TCR and STAT signaling occurs in CD8+ T cells. For example, weak or partial TCR-initiated signaling via the Ras/MAPK pathway can be complemented by IL-2 induced STAT activation. This mechanism enables common γ chain receptor cytokines to modulate optimal and supplement suboptimal TCR signaling. Recently, we reported the generation of K562-based artificial APC (aAPC) expressing HLA-A2 as a single HLA allele in conjunction with CD80 and CD83 (wt A2-aAPC). This aAPC can prime naïve CD8+ T cells ex vivo and generate antigen-specific CD8+ CTL with a central memory~effector memory phenotype. It has been demonstrated that the D227K/T228A mutations of A2 molecules (mut A2) inhibit the CD8/MHC interaction without affecting TCR/pMHC interactions. In this human ex vivo study, using a modified aAPC which expresses mut A2 molecules (mut A2-aAPC), we tested our hypothesis that CD8 coreceptor-independent T cell stimulation in the presence of complementary adaptive cytokines preferentially stimulates high avidity antigen-specific CTL. When freshly isolated A2+ CD8+ T cells were restimulated against recall antigen with mut A2-aAPC, both antigen specificity measured by tetramer positivity and number of expanded antigen-specific CD8+ T cells were significantly lower compared with wt A2-aAPC. Similar results were obtained when naïve CD8+ T cells were primed ex vivo with wt-A2 aAPC in the presence of CD8 coligation and then restimulated with mut-A2 aAPC in the absence of CD8 binding. When naïve CD8+ T cells were initially primed with mut A2-aAPC in the absence of CD8 binding, subsequent restimulation using wt A2-aAPC in the presence of CD8 coligation was not able to induce the proliferation of antigen-specific CD8+ CTL. As expected, when naïve CD8+ T cells were both primed and restimulated with mut A2-aAPC in the complete lack of CD8 coligation, antigen-specific CD8+ CTL did not grow. However, adding IL-21 overcame this deficiency. When CD8-independent T cell priming and restimulation by mut A2-aAPC was supplemented with IL-21, antigen-specific CD8+ CTL expansion with high functional avidity occurred. Lack of CD8 binding to MHC results in partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of MHC/TCR. While the responses of IL-21R- Jurkat and its Lck-null mutant, J.CaM1.6, to IL-21 were minimal, both showed robust IL-21 responses when stably transduced with IL-21R. Intracellular staining revealed that IL-21 induced robust phosphorylation of STAT3 but not STAT1 upon stimulation in both IL-21R-transduced Jurkat and J.CaM1.6. When IL-21R-transduced Lck-null J.CaM1.6 cells were stimulated in the presence of IL-21, T cell responses were completely abrogated by STAT3 inhibition. In contrast, the MAPK inhibitor only partially blocked the T cell responses. The combination of suboptimal doses of the STAT3 inhibitor and the MAPK inhibitor completely nullified the T cell responses, indicating an additive effect of STAT3 and MAPK inhibition. These results suggest that STAT3 but not STAT1 is critically involved in IL-21 signaling that rescues the defective T cell responses caused by the lack of Lck. Taken all together, this study suggests that CD8 ligation is critical for the expansion of post-thymic peripheral antigen-specific CTL in humans. However, STAT3-mediated IL-21 signaling can complement partial TCR signaling caused by the lack of CD8 association and expand CTL with high functional avidity. Since high functional avidity CTL are optimal effectors for the clearance of pathogens and tumors in vivo, our findings may be important for generating high avidity CTL ex vivo for effective adoptive therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4284 While adoptive T cell therapy is a promising treatment modality for cancer, the optimal approach to generate T cell grafts ex vivo is currently unknown. CD4+ T cells help generate effective immune responses by sustaining CD8+ T cell proliferation, preventing exhaustion, and establishing long-lived functional memory. Incorporation of CD4+ T cell help to expand CD8+ T cells may provide a novel strategy to generate CTL grafts for adoptive therapy. In mouse models, common γ-chain receptor cytokines and CD40/CD40L can mediate CD4+ T cell help. However, CD4+ T cell help in humans has yet to be fully defined. We therefore developed an in vitro model for human CD4+ T cell help, which utilizes a novel artificial APC, aAPC/mOKT3. K562-based aAPC/mOKT3 expresses a membranous form of anti-CD3 mAb, CD54, CD58, CD80, and CD83 and stimulates CD3+ T cells regardless of HLA haplotype or antigen specificity. Using aAPC/mOKT3, we stimulated CD8+ T cells in the presence or absence of CD4+ T cells and found that CD8+ T cells expanded better when coincubated with CD4+ T cells, suggesting the presence of CD4+ T cell help. Coculture experiments using transwell plates suggested that the observed CD4+ T cell help of CD8+ T cell expansion involved both soluble factors and cell-cell contact. To identify molecules mediating the observed CD4+ T cell help, supernatants of CD4+/CD8+ T cell mixed and separate cultures were measured for a panel of soluble factors. IL-2 and IL-21 were detected at lower levels in mixed cultures, consistent with more consumption or less production of these cytokines. Blockade of either IL-2 or IL-21 in CD4+/CD8+ T cell mixed cultures resulted in a reduction of CD8+ T cell expansion, indicating that, for both cytokines, more consumption rather than less production occurred and that IL-2 and IL-21 may serve as mediators of CD4+ T cell help. However, the addition of IL-21 to CD8+ T cells stimulated with aAPC/mOKT3 in the presence of IL-2 did not improve CD8+ T cell expansion, suggesting that IL-2 plus IL-21 cannot solely replace CD4+ T cell help. We found that the presence of CD4+ T cells upregulated the expression of IL-21R on CD8+ T cells. When we introduced IL-21R on CD8+ T cells and stimulated with aAPC/mOKT3 in the presence of IL-2 and IL-21, CD8+ T cell proliferation was restored. These results suggest that CD4+ T cells help CD8+ T cells proliferate ex vivo by secreting both IL-2/IL-21 and upregulating IL-21R. When peripheral CD3+ T cells from normal donors were stimulated with aAPC/mOKT3, the number of both CD4+ and CD8+ T cells increased. However, in contrast to other pan T cell expansion systems, aAPC/mOKT3 preferentially expanded CD8+ T cells. No obvious skewing in the Vβ usage of both CD4+ and CD8+ T cell populations was revealed by TCR Vβ repertoire analysis, supporting “unbiased” T cell expansion by aAPC/mOKT3. Moreover, HLA-restricted antigen-specific CD8+ CTL with high functional avidity could be generated from CD3+ T cells initially expanded for 4 weeks using aAPC/mOKT3. Using aAPC/mOKT3, tumor-infiltrating lymphocytes (TIL) were successfully expanded without adding soluble mAb or allogeneic feeder cells. As in peripheral T cell cultures, CD8+ T cells predominantly expanded in all cultures, including those that initially contained a minimal percentage of CD8+ T cells. Importantly, Foxp3+ Treg cells did not proliferate. Expanded T cells highly expressed CD27 and CD28, which are associated with T cell survival and persistence in vivo. They also secreted high levels of IFN-γ and IL-2, lower amounts of IL-4, and no IL-10. These results demonstrate that the aAPC/mOKT3-based system can expand functional CD8+ TIL in the presence of autologous CD4+ T cells. In conclusion, we have determined that CD4+ T cell-dependent CD8+ T cell expansion required both soluble factors secreted by and cell contact with CD4+ T cells. Among the soluble factors secreted by CD4+ T cells, IL-2 and IL-21 were necessary. Furthermore, upregulation of IL-21R on CD8+ T cells by CD4+ T cells was critical for an optimized response to IL-21. Thus, in humans, CD4+ T cells help CD8+ T cells proliferate by secreting IL-2/IL-21 and upregulating IL-21R. Our aAPC enabled expansion of CD8+ TIL in the presence of CD4+ T cell help without using soluble mAb or allogeneic feeder cells. Taken together, these results demonstrate the indispensable role of CD4+ T cell help on expanding CD8+ T cells and suggest a novel strategy to generate anti-tumor T cells ex vivo for adoptive therapy. Disclosures: No relevant conflicts of interest to declare.

Description: ASCT improves progression-free and likely overall survival in patients with relapsed follicular lymphoma. Randomized trials of ASCT as upfront therapy have generally also found an improvement in progression-free survival, but this benefit has been counterbalanced by an increase in second malignancies. We report the long-term outcome of two sequential prospective clinical trials of autologous bone marrow transplantation for advanced stage follicular lymphoma patients under 60 in first remission. In the 1st study 83 previously untreated patients received 6–8 cycles of CHOP induction and in the 2nd study 20 patients received 4 cycles of high-dose CHOP induction (cyclophosphamide 1.5 g/m2 d1–2 with g-csf support). The median age of the patients was 42 (range 19–57). 17% had high FLIPI scores, 58% intermediate and 25% low; all patients were under 60 and 21% had missing data required in the FLIPI. 96% had stage III or IV disease. 77 of 83 patients on the 1st study, and 19 of 20 on the 2nd study (N=96 total), achieved a protocol-defined minimal disease state after CHOP induction (〈 2 cm masses,

Description: Control of alloreactivity without loss of immunity to pathogens and tumor antigens remains the major challenge in allogeneic hematopoietic stem cell transplantation (AHSCT). One platform for achieving this objective has been based upon the methodologies of stimulating donor T cells with recipient alloantigens and destroying, removing, and/or inactivating alloreactive donor T-cells. We have previously reported a clinical trial employing ex vivo inactivation of alloreactive donor T cells via CTLA-4 immunoglobulin-mediated co-stimulatory blockade of CD28 signaling, which allowed large doses of HLA-mismatched T cells to be infused at the time of AHSCT without excess graft-versus-host disease (GvHD) and with no clinically significant late viral infections in surviving patients. We have now refined this approach, exposing donor peripheral blood mononuclear cells (PBMCs) to irradiated stimulator PBMCs in the presence of clinical grade humanized anti-B7.1 and anti-B7.2 monoclonal antibodies, blocking the delivery of positive co-stimulatory signals to (predominantly CD4+) alloantigen-specific donor T cells. This strategy reduces proliferative responses to irradiated stimulator PBMCs to less than 1% of those seen with untreated cultured responder cells in fully HLA mismatched healthy volunteer donor pairs. The proliferative capacity of responder cells to mitogenic anti-CD3 and -CD28 antibodies following alloantigen-specific co-stimulatory blockade is not impaired. In order to demonstrate the retention of antigen-specific responses in donor CD4+ cells following recipient alloantigen-specific co-stimulatory blockade, we used a highly sensitive 5-color flow cytometric intracellular cytokine secretion assay following stimulation of PBMCs with virus-infected cell lysates. Donors in whom viral lysate-specific CD4+ cells could be detected in untreated PBMCs retain approximately two-thirds of VZV, HSV and CMV-specific Th1 cytokine+ (IFN-γ/IL2) CD4+ cells after anti-B-7 mediated alloantigen-specific co-stimulatory blockade. Th1 cytokine responses to the superantigen staphylococcal enterotoxin B are preserved in all donors. Furthermore, proliferative responses to CMV viral lysate are retained after effective allospecific co-stimulatory blockade in the majority of donors in whom CMV-specific Th1 cytokine+ CD4+ T cells could be detected in untreated PBMCs cultured in parallel. These data demonstrate that HLA-mismatched alloantigen-specific co-stimulatory blockade effectively reduces proliferative responses to alloantigens whilst retaining pathogen-specific CD4+ cells. Moreover, the capacity to secrete cytokines of these pathogen-specific T cells supports the hypothesis that they will be functional when adoptively transferred. An ongoing multicentre proof-of concept dose escalation study is underway in which this strategy is utilized to generate HLA mismatched donor T cells which are administered after haploidentical AHSCT to augment immune recovery without GvHD. Furthermore, the application of highly sensitive assays to quantify pathogen-specific immunity in donor T cells after alloantigen-specific co-stimulatory blockade may allow donor and recipient-specific T cell dosing strategies to be applied to minimize treatment-associated toxicity.