Multiparametric flow cytometry to characterize vaccine-induced polyfunctional T cell responses and T cell/NK cell exhaustion and memory phenotypes in mouse immuno-oncology models

Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4+ and CD8+ T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models

Bone marrow transplantation generates T cell–dependent control of myeloma in mice

Transplantation with autologous hematopoietic progenitors remains an important consolidation treatment for patients with multiple myeloma (MM) and is thought to prolong the disease plateau phase by providing intensive cytoreduction. However, transplantation induces inflammation in the context of profound lymphodepletion that may cause hitherto unexpected immunological effects. We developed preclinical models of bone marrow transplantation (BMT) for MM using Vk*MYC myeloma-bearing recipient mice and donor mice that were myeloma naive or myeloma experienced to simulate autologous transplantation. Surprisingly, we demonstrated broad induction of T cell-dependent myeloma control, most efficiently from memory T cells within myeloma-experienced grafts, but also through priming of naive T cells after BMT. CD8+ T cells from mice with controlled myeloma had a distinct T cell receptor (TCR) repertoire and higher clonotype overlap relative to myeloma-free BMT recipients. Furthermore, T cell-dependent myeloma control could be adoptively transferred to secondary recipients and was myeloma cell clone specific. Interestingly, donor-derived IL-17A acted directly on myeloma cells expressing the IL-17 receptor to induce a transcriptional landscape that promoted tumor growth and immune escape. Conversely, donor IFN-γ secretion and signaling were critical to protective immunity and were profoundly augmented by CD137 agonists. These data provide new insights into the mechanisms of action of transplantation in myeloma and provide rational approaches to improving clinical outcomes

Immunotherapy of multiple myeloma

Multiple myeloma (MM), a bone marrow-resident hematological malignancy of plasma cells, has remained largely incurable despite dramatic improvements in patient outcomes in the era of myeloma-targeted and immunomodulatory agents. It has recently become clear that T cells from MM patients are able to recognize and eliminate myeloma, although this is subverted in the majority of patients who eventually succumb to progressive disease. T cell exhaustion and a suppressive bone marrow microenvironment have been implicated in disease progression, and once these are established, immunotherapy appears largely ineffective. Autologous stem cell transplantation (ASCT) is a standard of care in eligible patients and results in immune effects beyond cytoreduction, including lymphodepletion, T cell priming via immunogenic cell death, and inflammation; all occur within the context of a disrupted bone marrow microenvironment. Recent studies suggest that ASCT reestablishes immune equilibrium and thus represents a logical platform in which to intervene to prevent immune escape. New immunotherapies based on checkpoint inhibition targeting the immune receptor TIGIT and the deletion of suppressive myeloid populations appear attractive, particularly after ASCT. Finally, the immunologically favorable environment created after ASCT may also represent an opportunity for approaches utilizing bispecific antibodies or chimeric antigen receptor (CAR) T cells

Flt-3L expansion of recipient CD8α+ dendritic cells deletes alloreactive donor T cells and represents an alternative to post-transplant cyclophosphamide for the prevention of GVHD

Allogeneic bone marrow transplantation (BMT) provides curative therapy for leukemia via immunological graft-versus-leukemia (GVL) effects. In practice, this must be balanced against life threatening pathology induced by graft-versus-host disease (GVHD). Recipient dendritic cells (DC) are thought to be important in the induction of GVL and GVHD.We have utilized preclinical models of allogeneic BMT to dissect the role and modulation of recipient DC in controlling donor T cell mediated GVHD and GVL.We demonstrate that recipient CD8a+ DC promote activation-induced clonal deletion of allospecific donor T cells after BMT. We compared pre-transplant fms-like tyrosine kinase-3 ligand (Flt-3L) treatment to the current clinical strategy of post-transplant cyclophosphamide (PT-Cy) therapy. Our results demonstrate superior protection from GVHD with the immunomodulatory Flt-3L approach, and similar attenuation of GVL responses with both strategies. Strikingly, Flt-3L treatment permitted maintenance of the donor polyclonal T cell pool, where PT-Cy did not.These data highlight pre-transplant Flt-3L therapy as a potent new therapeutic strategy to delete alloreactive T cells and prevent GVHD, which appears particularly well suited to haploidentical BMT where the control of infection and the prevention of GVHD are paramount

TIGIT immune checkpoint blockade restores CD8 T cell immunity against multiple myeloma

Immune-based therapies hold promise for the treatment of multiple myeloma (MM), but so far immune checkpoint blockade targeting programmed cell death protein 1 (PD-1) has not proven effective as single agent in this disease. T cell immunoglobulin and ITIM domains (TIGIT) is another immune checkpoint receptor known to negatively regulate T cell functions. In this study, we investigated the therapeutic potential of TIGIT blockade to unleash immune responses against MM. We observed that, in both mice and humans, MM progression was associated with high levels of TIGIT expression on CD8 T cells. TIGIT CD8 T cells from MM patients exhibited a dysfunctional phenotype, characterized by decreased proliferation and inability to produce cytokines in response to anti-CD3/CD28/CD2 or myeloma antigen stimulation. Moreover, when challenged with Vk*MYC mouse MM cells, TIGIT-deficient mice showed decreased serum M-protein levels associated with reduced tumor burden and prolonged survival, indicating that TIGIT limits anti-myeloma immune responses. Importantly, blocking TIGIT using monoclonal antibodies (mAbs) increased the effector function of MM patient CD8 T cells and suppressed MM development. Altogether our data provide evidence for an immune-inhibitory role of TIGIT in MM and support the development of TIGIT-blocking strategies for the treatment of MM patients