These data demonstrate the potential for allogeneic immunotherapy with donor lymphocytes both to reduce relapse risk and to induce durable antitumor responses in patients with Hodgkin's lymphoma after hematopoietic stem-cell transplantation that incorporates in vivo T-cell depletion.

Exhaustion of chronically stimulated CD8+ T cells is a significant obstacle to immune control of chronic infections or tumors. Although co-inhibitory checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) antibody can restore functions to exhausted T cell populations, recovery is often incomplete and dependent upon the pool size of a quiescent T-bethigh subset that express lower levels of PD-1. In a model where unhelped, HY-specific CD8+ T cells gradually lose function following transfer to male BMT recipients, we have explored the effect of shifting the balance away from co-inhibition and toward co-stimulation by combining anti-PD-L1 with agonistic antibodies to the tumor-necrosis factor receptor superfamily members, OX40 and CD27. Several weeks following T cell transfer, both agonistic antibodies but especially anti-CD27 demonstrated synergy with anti-PD-L1 by enhancing CD8+ T cell proliferation and effector cytokine generation. Anti-CD27 and anti-PD-L1 synergised by downregulating the expression of multiple quiescence-related genes concomitant with a reduced frequency of T-bethigh cells within the exhausted population. However, in the presence of persistent antigen, the CD8+ T cell response was not sustained and the overall size of the effector cytokine-producing pool eventually contracted to levels below that of controls. Thus, CD27-mediated co-stimulation can synergize with co-inhibitory checkpoint blockade to switch off molecular programs for quiescence in exhausted T cell populations but at the expense of losing precursor cells required to maintain a response.

Allogeneic blood or BM transplantation (BMT) is the most commonly applied form of adoptive cellular therapy for cancer. In this context, the ability of donor T cells to respond to recipient antigens is coopted to generate graft-versus-tumor (GVT) responses. The major reason for treatment failure is tumor recurrence, which is linked to the eventual loss of functional, host-specific CTLs. In this study, we have explored the role of recipient antigen expression by nonhematopoietic cells in the failure to sustain effective CTL immunity. Using clinically relevant models, we found that nonhematopoietic antigen severely disrupts the formation of donor CD8 + T cell memory at 2 distinct levels that operate in the early and late phases of the response. First, initial and direct encounters between donor CD8 + T cells and nonhematopoietic cells blocked the programming of memory precursors essential for establishing recall immunity. Second, surviving CD8 + T cells became functionally exhausted with heightened expression of the coinhibitory receptor programmed death-1 (PD-1). These 2 factors acted together to induce even more profound failure in long-term immunosurveillance. Crucially, the functions of exhausted CD8 + T cells could be partially restored by late in vivo blockade of the interaction between PD-1 and its ligand, PD-L1, without induction of graft-versus-host disease, suggestive of a potential clinical strategy to prevent or treat relapse following allogeneic BMT.

SummaryFollowing reduced intensity-conditioned allogeneic stem cell transplantation (RIC allo-SCT) for chronic lymphocytic leukaemia (CLL), there is an inverse relationship between relapse and extensive chronic graft-versus-host disease (GVHD). We evaluated outcomes in 50 consecutive patients with CLL using the approach of alemtuzumab-based RIC allo-SCT and preemptive donor lymphocyte infusions (DLI) for mixed chimerism or minimal residual disease (MRD), with the intention of reducing the risk of GVHD. Forty two patients had high-risk disease, including 30% with 17p deletion (17pÀ). Of patients who were not in complete remission (CR) entering transplant, 83% subsequently achieved MRD-negative CR. Both MRD detection and uncorrected mixed chimerism were associated with greater risks of treatment failure. Nine of sixteen patients receiving DLI for persistent or relapsed disease subsequently attained MRD-negative CR. With a median follow-up of 4Á3 years, 4-year current progression-free survival was 65% and overall survival was 75% (60% and 61% in respectively, patients with 17pÀ). DLI was associated with a 29% cumulative incidence of severe GVHD and mortality of 6Á4%. At last follow-up, 83% of patients in CR were off all immunosuppressive treatment. In conclusion, the directed delivery of allogeneic cellular therapy has the potential to induce durable remissions in high-risk CLL without incurring excessive GVHD.

Summary The clinical significance of mixed chimerism following allogeneic haematopoietic stem cell transplantation (HSCT) remains controversial. Its relevance and incidence are probably influenced by the conditioning regimen and incorporation of T‐cell depletion. The presence of recipient chimerism levels >40–50% following T‐cell replete reduced intensity transplantation correlates with a high risk of graft rejection, regardless of donor‐lymphocyte infusions, but it is unclear whether this finding translates to T‐cell depleted transplants. We conducted a retrospective single‐institution analysis of patients receiving alemtuzumab‐based HSCT. 27/152 (18%) evaluable cases had predominantly recipient T‐cell chimerism at 3 months or beyond. By contrast, coincident chimerism in the granulocyte lineage was predominantly of donor origin (median 100%) in all but one patient. Donor lymphocyte infusion effectively converted predominantly recipient T‐cell chimerism to ful donor chimerism in all evaluable cases including three cases with no detectable donor T cells. The only graft failure occurred in the patient with predominantly recipient myeloid chimerism in whom rejection occurred rapidly before donor lymphocytes could be administered. We conclude that predominant or complete recipient T‐cell chimerism following alemtuzumab‐based regimens does not have the same clinical implications as that following T‐cell replete transplants and can be effectively converted with donor lymphocytes without the need for lympho‐depleting agents or re‐conditioning.

3009 Background: Interactions between tumour cells and host cells within the microenvironment are important in promoting the development of cancer. Tumor niches provide crucial anti-apoptotic and anti-proliferative signals that drive tumor chemoresistance. The CXCR4-CXCL12 chemokine axis forms a critical component of this niche. CXCL12 produced by stromal cells has direct pro-survival effects upon tumor cells, promotes metastasis and recruits CXCR4-expressing regulatory T cell populations that block anti-tumour immunity. In this study, we have tested the hypothesis that targeting therapeutic T cells to CXCR4-dependent niches will improve eradication of tumours in mice. Methods: The murine CXCR4 gene was inserted into retroviral vector, pMP71. Murine T cells were transduced with CXCR4 or control vector and tested for homing in vitro to CXCL12 through chemotaxis assays. In vivo imaging of the putative endosteal bone marrow (BM) niche was performed by multiphoton imaging through cranial frontal bones in osteoblast (collagen 1-α-GFP) reporter mice. In vivo trafficking, competitive transfer and memory recall experiments were performed following transfer of transduced T cells to syngeneic, sub-lethally irradiated mice. Anti-tumour reactivity of CXCR4-transduced T cells was tested in models of allogeneic BM transplantation (BMT). Results: CXCR4-transduced T cells demonstrated enhanced migration towards CXCL12 in vitro. No differences in viability, phenotype or function were observed in CXCR4-transduced versus control T cells in the presence or absence of CXCL12. In competitive assays, CXCR4-transduced CD8 T cells demonstrated a 2-fold greater capacity than controls to home to the BM by 24h after transfer to sub-lethally-irradiated recipients. Multiphoton imaging through cranial frontal bones indicated that fluorescently labelled CXCR4-transduced T cells were closer than control cells to the endosteum (13 μm versus 17 μm, p<0.01). By 14 days, the numbers of CXCR4-transduced CD8 T cells in the BM were 15-fold greater than controls. To test immunity to model antigen, CXCR4 or control vector-transduced OT-1 TCR-transgenic CD8 cells were transferred to sub-lethally irradiated mice before challenge with OVA peptide-loaded dendritic cells. Pre-vaccination, CXCR4-transduced OT-1 cells demonstrated greater engraftment than controls in the BM and spleen. Seven days following vaccination, CXCR4 OT-1 cells demonstrated a greater capacity than control cells to generate IFN-γ to OVA-peptide. Four weeks following vaccination, CXCR4-transduced CD8 T cells showed increased frequencies of cells with a CD44highIL-7Rαhigh memory phenotype than controls, with a greater proportion of cells undergoing proliferation as evaluated by BrdU incorporation. To test T cell immunity against a tumor that exploits the CXCR4-CXCL12 axis to recruit regulatory T cells, B6 BM and CXCR4- or control transduced B6 T cells were transferred to irradiated BALB/c recipients given A20 tumor. Tumor growth was delayed to a greater extent following transfer of CXCR4-compared to control-transduced donor T cells. Conclusion: Over-expression of CXCR4 in CD8 T cells potentiates engraftment, initial effector function and generation of memory cells. Disclosures: Stauss: Cell Medica: Scientific Advisor Other.

1911 A major limitation of adoptive T cell therapies for cancer is the failure to maintain durable anti-tumor immunity. Graft-versus-tumor responses following bone marrow transplantation (BMT) may only be short-lived due to 1) defects in memory precursor generation and 2) exhaustion of surviving CTL that results from direct recognition of alloantigen upon non-hematopoietic cells {Flutter et al. JCI 2010}. In this study, we have explored the potential for enhancing co-stimulatory signals either alone, or in combination with co-inhibitory PD-1-PD-L1 blockade to improve the long term CTL response. Signalling through OX40, a TNF-receptor family member, has been shown to have an important role in long-term immunity, including an enhancement in the generation of CD8 T cell memory precursors. The mechanisms of action are complex and may include both direct effects on CD8 cells and indirect effects on CD4 helper cells or via inhibition of Treg. In initial experiments, we evaluated the effects of early enforced OX40 co-stimulation following delayed transfer of donor T cells to haplo MHC-mismatched chimeras, 10 weeks following nonmyeloablative BMT. OX40 expression peaked on transferred CD4 and CD8 T cells in the first 1–2 weeks following transfer and was sustained thereafter, especially in the CD4 subset. 48 hours after T cell transfer, recipient mice were treated with agonistic anti-OX40 antibody (OX86) or isotype control. OX86 treatment led to a 9-fold increase in the expansion of CTL in comparison to isotype control treated mice, enhanced production of Granzyme B and IFNγ and led to more rapid eradication of host hematopoietic targets or host tumor cells. Moreover, OX86 antibody acted directly on CD8 T cells and bypassed the requirement for help from donor CD4 cells. However, although enforced OX40 co-stimulation boosted the primary effector response, it did not increase numbers of memory precursor cells, as assessed by survival and recall responses following transfer to antigen free hosts, and was unable to prevent eventual exhaustion of surviving donor CTL as tested at 60 days following transfer. Similarly, OX86 was unable to prevent exhaustion of CD8 cells transgenic for the male antigen-specific Matahari (Mh) TCR following adoptive transfer to male BMT recipients reconstituted with female BM. We have shown previously that the functions of exhausted donor CD8 cells are partially restored by blockade of the co-inhibitory PD-1 pathway in both haplo mismatched and MHC-matched mHAg mismatch models. We hypothesized that provision of co-stimulatory signals when exhaustion had become established would increase the effectiveness of co-inhibitory blockade. Therefore, 6 weeks after Mh CD8 T cell transfer to male BMT recipients, we examined the effect of OX86, with or without additional blockade of the PD-1 pathway. Only a minority of Mh CD8 cells from animals receiving isotype control antibody were proliferating in vivo as measured by BrdU incorporation over a 7 day pulse (20 +/−3% BrdU+) and few cells were able to produce IFNγ following antigen stimulation in vitro (3.5+/−1.4 x104 IFNγ+ cells/spleen). OX86 alone offered no restoration of function (15 +/− 2% BrdU+; 3.3+/−0.4 x104 IFNγ+ cells; p=ns). Blockade of PD-L1 modestly increased turnover of cells (37 +/− 6 % BrdU+; p<0.01 vs isotype), but in the absence of CD4 cells, did not significantly increase production of IFNγ (4.4+/−0.9 x104 IFNγ+ cells; p=ns). However, in vivo administration of OX86 combined with anti-PD-L1 blockade dramatically increased turnover of Mh CD8s (77 +/− 8% BrdU+; p<0.001 vs anti-PD-L1 alone, OX86 alone or Isotype) and enhanced their effector function ∼ 9-fold (27.4 +/− 6.8 x104 IFNγ+ cells/spleen; p<0.01 vs all others). In conclusion, forced co-stimulation via OX40 alone is unable either to prevent CTL exhaustion or restore CD8 T cell function when exhaustion has become established. In contrast, the marked synergy observed when agonistic OX40 signals are combined with co-inhibitory blockade, is consistent with a model in which the PD-1 pathway acts at a critical checkpoint that regulates the response to co-stimulation. Thus, these data suggest a novel approach to restoring the functions of exhausted anti-tumor CTL by modulating co-stimulatory and co-inhibitory pathways simultaneously. Disclosures: No relevant conflicts of interest to declare.