Furthermore, in an independent, highly lethal model of minimal residual lymphoma, increased MST was only observed following vaccine and IL-2 (Figure 5G). (IL-2S4B6). Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of survival, which directly correlated with 500% increase in effector CD8+ T-cell numbers. Notably, this dual regimen elicited large increases in both donor CD8+ T and NK cells, but not CD4+ T lymphocytes; the former 2 populations are essential for both vaccine efficacy and protection against opportunistic infections after HSCT. Indeed, IL-2S4B6-treated HSCT recipients infected with exhibited decreased bacterial levels. These preclinical studies validate a new strategy particularly well suited to the post-HSCT environment, which may augment adaptive and innate immune function in patients with malignant disease receiving autologous HSCT. Introduction Tumor relapse LRRK2-IN-1 remains the major cause of morbidity and mortality in patients with hematologic malignancies receiving autologous hematopoietic stem cell transplantation (HSCT) for hematolymphoid rescue. According to the Center for International Blood and Marrow Transplant Research, 80% of mortality after autologous HSCT (2010-2011) resulted from relapse of primary disease or infection in patients with myeloma, lymphoma, and leukemia.1 Multifaceted immunotherapeutic approaches combined with HSCT for patients with hematopoietic malignancy continue to hold large, but as yet unfulfilled, promise.2 Such enthusiasm for immune-based strategies rests in part from the notion that vaccination regimens can be used early after HSCT during reboot of the immune system to promote efficient antitumor and antipathogen immunity by taking advantage of minimal residual disease and the lymphopenia present.3-9 Nevertheless, generating successful protocols early after HSCT must account for the relative dearth of T cells, as well as the need for a vaccine with appropriate tumor or pathogen antigens to promote successful immunity. Heat shock protein gp96 is the resident endoplasmic reticulum protein chaperone and is intimately involved in MHC-I restricted antigen presentation.10-16 Following necrosis, gp96-peptide complexes are released and can be taken up by antigen presenting cells (APCs), leading to peptide delivery and their efficient activation.17,18 These APCs can therefore cross-present gp96-chaperoned peptides to CD8+ T lymphocytes,19,20 inducing their activation, expansion, and development of effector function. The vaccine used in the present studies consisted of tumor cells engineered to secrete a modified gp96 molecule lacking the endoplasmic reticulum KDEL (Lys-Asp-Glu-Leu) retention signal fused to the FC portion of murine IgG1 (gp96-Ig).21,22 This potent cell vaccine resulted in stimulation of multiple antigen-specific CD8+ T-cell populations in mice (tumor reactive)23-26 and primates (viral reactive),27,28 which prolonged survival in relevant preclinical models of cancer and acute infection, respectively. Moreover, recent studies found the majority of lung cancer patients vaccinated with a gp96-Ig-secreting tumor LRRK2-IN-1 cell vaccine generated a CD8+ interferon (IFN)-+ response (allo-reactive), and these individuals exhibited prolonged survival compared with nonresponders.29 Notably, gp96-Ig vaccination also stimulated natural killer (NK) cells in antitumor models, and this population was hypothesized to contribute to CD8+ T-cell expansion.30 Interleukin (IL)-2 therapy has demonstrated significant antitumor activity in experimental models and has diverse affects following HSCT, in part dependent on dose and time of infusion.31,32 However, because IL-2-induced expansion of T-regulatory cells (Treg) could inhibit antitumor immunity, an important advance for use of this cytokine would be to direct its activity primarily to antitumor effector vs Treg cells.33-35 Notably, recent findings have reported that IL-2 conjugated to a LRRK2-IN-1 particular anti-IL-2 monoclonal antibody (mAb) can augment antitumor responses.36,37 One cytokine-antibody complex using mAb clone S4B6 (IL-2S4B6), which activates the intermediate affinity IL-2 receptor ( and ), was found to stimulate the proliferation of predominately memory phenotype CD8+ T lymphocytes and NK cells2 populations essential for optimal gp96-Ig-induced antitumor responses.30 The preclinical studies presented here investigated the efficacy of vaccination with tumor cells secreting gp96-Ig together with an IL-2S4B6 complex in experimental mouse models of minimal residual lymphoma following syngeneic HSCT. The results obtained support the notion that the effect of gp96-Ig vaccination via cross-presentation early after autologous HSCT was to elicit tumor-reactive CD8+ T CRE-BPA cells, and together with directed IL-2 treatment, markedly augmented effector CD8+ T-cell levels. Global expansion of donor CD8+ T lymphocytes and NK cells, but not CD4+ T lymphocytes, following administration of this IL-2S4B6 complex contributed to prolonged survival of lymphoma-bearing HSCT recipients, as well as augmented antipathogen responsiveness early after HSCT. Materials.