In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ ...regulatory T cells (Tregs), have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s) of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs) directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These "unstable" Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR(-/-), and the protective effects of DTA-1 were reduced in reconstituted RAG1(-/-) mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist anti-GITR mAbs as an immunotherapeutic strategy for cancer.
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor formation, facilitate immune escape, and enable tumor progression. MDSCs are important ...contributors to the development of an immunosuppressive tumor microenvironment that blocks the action of cytotoxic antitumor T effector cells. Heterogeneity in these cells poses a significant barrier to studying the in vivo contributions of individual MDSC subtypes. Herein, we show that granulocyte-macrophage colony stimulating factor, a cytokine critical for the numeric and functional development of MDSC populations, promotes expansion of a monocyte-derived MDSC population characterized by expression of CD11b and the chemokine receptor CCR2. Using a toxin-mediated ablation strategy to target CCR2-expressing cells, we show that these monocytic MDSCs regulate entry of activated CD8 T cells into the tumor site, thereby limiting the efficacy of immunotherapy. Our results argue that therapeutic targeting of monocytic MDSCs would enhance outcomes in immunotherapy.
Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, ...are needed. Melanoma vaccine design has mainly focused on targeting CD8+ T cells. Activation of effector CD8+ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.
VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8+ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8+ T cell effector responses, and depends on signaling through activating Fcγ receptors.
This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8+ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.
Respiratory syncytial virus (RSV), a major cause of illness and death in infants worldwide, could be prevented by vaccination during pregnancy. The efficacy, immunogenicity, and safety of a bivalent ...RSV prefusion F protein-based (RSVpreF) vaccine in pregnant women and their infants are uncertain.
In a phase 2b trial, we randomly assigned pregnant women, at 24 through 36 weeks' gestation, to receive either 120 or 240 μg of RSVpreF vaccine (with or without aluminum hydroxide) or placebo. The trial included safety end points and immunogenicity end points that, in this interim analysis, included 50% titers of RSV A, B, and combined A/B neutralizing antibodies in maternal serum at delivery and in umbilical-cord blood, as well as maternal-to-infant transplacental transfer ratios.
This planned interim analysis included 406 women and 403 infants; 327 women (80.5%) received RSVpreF vaccine. Most postvaccination reactions were mild to moderate; the incidence of local reactions was higher among women who received RSVpreF vaccine containing aluminum hydroxide than among those who received RSVpreF vaccine without aluminum hydroxide. The incidences of adverse events in the women and infants were similar in the vaccine and placebo groups; the type and frequency of these events were consistent with the background incidences among pregnant women and infants. The geometric mean ratios of 50% neutralizing titers between the infants of vaccine recipients and those of placebo recipients ranged from 9.7 to 11.7 among those with RSV A neutralizing antibodies and from 13.6 to 16.8 among those with RSV B neutralizing antibodies. Transplacental neutralizing antibody transfer ratios ranged from 1.41 to 2.10 and were higher with nonaluminum formulations than with aluminum formulations. Across the range of assessed gestational ages, infants of women who were immunized had similar titers in umbilical-cord blood and similar transplacental transfer ratios.
RSVpreF vaccine elicited neutralizing antibody responses with efficient transplacental transfer and without evident safety concerns. (Funded by Pfizer; ClinicalTrials.gov number, NCT04032093.).
Regulatory T cells (T
) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8
T cells. To better understand the mechanisms involved, we used ex vivo ...three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from T
-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor-β (TGF-β) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident T
in mice. T
isolated from B16 tumors alone were sufficient to suppress CD8
T cell-mediated killing, which depended on surface-bound TGF-β on the T
Immunosuppression of CD8
T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between T
and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-β-dependent manner and highlight potential ways to inhibit intratumoral T
therapeutically.
Harnessing the adaptive immune response to treat malignancy is now a clinical reality. Several strategies are used to treat melanoma; however, very few result in a complete response. CD4(+) T cells ...are important and potent mediators of anti-tumor immunity and adoptive transfer of specific CD4(+) T cells can promote tumor regression in mice and patients. OX40, a costimulatory molecule expressed primarily on activated CD4(+) T cells, promotes and enhances anti-tumor immunity with limited success on large tumors in mice. We show that OX40 engagement, in the context of chemotherapy-induced lymphopenia, induces a novel CD4(+) T cell population characterized by the expression of the master regulator eomesodermin that leads to both terminal differentiation and central memory phenotype, with concomitant secretion of Th1 and Th2 cytokines. This subpopulation of CD4(+) T cells eradicates very advanced melanomas in mice, and an analogous population of human tumor-specific CD4(+) T cells can kill melanoma in an in vitro system. The potency of the therapy extends to support a bystander killing effect of antigen loss variants. Our results show that these uniquely programmed effector CD4(+) T cells have a distinctive phenotype with increased tumoricidal capability and support the use of immune modulation in reprogramming the phenotype of CD4(+) T cells.
Cyclophosphamide (CTX), a commonly used chemotherapeutic agent can enhance immune responses. The ability of CTX to promote the proliferation of effector T cells and abrogate the function of ...regulatory T cells (Tregs) has been described. In this study, we examined the effects of CTX treatment on dendritic cell (DC) subsets and the subsequent outcome on the effector and suppressive arms of adaptive immunity. In secondary lymphoid tissues, tissue-derived migratory DCs (migratory DCs), lymphoid tissue–resident DCs (resident DCs), and plasmacytoid DCs (pDCs) are well described. CTX has profound and selective cytotoxic effects on CD8+ resident DCs, but not skin-derived migratory DCs or pDCs in lymph nodes (LNs) and spleen, causing an imbalance among these DC subsets. CTX treatment increases the potency of DCs in antigen presentation and cytokine secretion, and partially inhibits the suppressor activity of Tregs. Adoptive transfer of CD8+ DCs can reconstitute this population in regional draining LNs and abrogate the immune-enhancing effects of CTX in vivo. These findings demonstrate that CTX may improve immune responses by preferentially depleting CD8+ lymphoid-resident DCs, which leads to diminished Treg suppression and enhanced effector T-cell function in vivo.
One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an ...alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.
Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in ...mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.
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