Delayed vaccine-induced CD8+ T cell expansion by topoisomerase I inhibition mediates enhanced CD70-dependent tumor eradication.

BACKGROUND: The survival of patients with cervical cancer who are treated with cisplatin in conjunction with the topoisomerase I inhibitor topotecan is enhanced when compared with patients treated with only one of these chemotherapeutics. Moreover, cisplatin-based and T cell-based immunotherapy have been shown to synergize, resulting in stronger antitumor responses. Here, we interrogated whether topotecan could further enhance the synergy of cisplatin with T cell-based cancer immunotherapy. METHODS: Mice bearing human papilloma virus 16 (HPV16) E6/E7-expressing TC-1 tumors were vaccinated with HPV16 E7 long peptides and additionally received chemotherapy consisting of cisplatin and topotecan. We performed an in-depth study of this combinatorial chemoimmunotherapy on the effector function and expansion/contraction kinetics of vaccine-induced CD8+ T cells in the peripheral blood and tumor microenvironment (TME). In addition, we interrogated the particular role of chemotherapy-induced upregulation of costimulatory ligands by tumor-infiltrated myeloid cells on T cell proliferation and survival. RESULTS: We show that E7 long peptide vaccination combined with cisplatin and topotecan, results in CD8+ T cell-dependent durable rejection of established tumors and 94% long-term survival. Although topotecan initially repressed the expansion of vaccine-induced CD8+ T cells, these cells eventually expanded vigorously, which was followed by delayed contraction. These effects associated with the induction of the proliferation marker Ki-67 and the antiapoptosis molecule Bcl-2 by intratumoral tumor-specific CD8+ T cells, which was regulated by topotecan-mediated upregulation of the costimulatory ligand CD70 on myeloid cells in the TME. CONCLUSIONS: Taken together, our data show that although treatment with cisplatin, topotecan and vaccination initially delays T cell expansion, this combinatorial therapy results eventually in a more robust T cell-mediated tumor eradication due to enhancement of costimulatory molecules in the TME.

OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum.

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.

A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection.

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Interleukin-6-mediated resistance to immunotherapy is linked to impaired myeloid cell function.

High serum levels of interleukin-6 (IL-6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)-associated tumor model expressing IL-6 to investigate the impact of tumor-expressed IL-6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor-produced IL-6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor-produced IL-6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T-cell-mediated killing. Furthermore, no overt differences in the frequencies of tumor-infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL-6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL-6-mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL-6-mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer.

Lack of myeloid cell infiltration as an acquired resistance strategy to immunotherapy.

BACKGROUND: Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments. METHODS: We exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients. RESULTS: Full tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-α and transforming growth factor-ß signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naïve hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8+ T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients. CONCLUSION: An immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities.

Novel TLR2-binding adjuvant induces enhanced T cell responses and tumor eradication.

BACKGROUND: Ligands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of TLR2-ligand Pam3CSK4 to synthetic long peptides (SLPs) was shown to strongly enhance the induction of antitumor immunity. To further improve cancer vaccination, we have previously shown that the novel TLR2-L Amplivant (AV), a modified Pam3CSK4, potentiates the maturation effects on murine DCs. In the current study, we further assessed the immunological properties of AV. METHODS: Naïve mice were vaccinated with a conjugate of either Pam3CSK4 or AV and an SLP to assess specific T cell priming efficiency in vivo. The potency of AV and Pam3CSK4, either as free compounds or conjugated to different SLPs, to mature murine DCs was compared by stimulating murine dendritic cells overnight followed by ELISA and flow cytometry analysis. Murine tumor experiments were carried out by vaccinating mice carrying established HPV16 E6 and E7-expressing tumors and subsequently analyzing myeloid and lymphoid cells infiltrating the tumor microenvironment. Furthermore, tumor outgrowth after vaccination was monitored to enable comparison of the efficiency to induce antitumor immunity by Pam3CSK-SLP and AV-SLP conjugates. To enhance therapeutic efficacy, AV-SLP conjugate vaccination was combined with ablative therapies to assess whether synergism between such therapies would occur. RESULTS: SLPs conjugated to AV induce stronger DC maturation, in vivo T cell priming and antitumor immunity compared to conjugates with Pam3CSK4. Interestingly, AV-SLP conjugates modulate the macrophage populations in the tumor microenvironment, correlating with a therapeutic effect in an aggressive murine tumor model. The potency of AV-SLP conjugates in cancer vaccination operates optimally in combination with chemotherapy or photodynamic therapy. CONCLUSION: These data allow further optimization of vaccination-based immunotherapy of cancer by use of the improved TLR2-ligand Amplivant.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

Inhibition of 14q32 microRNA miR-495 reduces lesion formation, intimal hyperplasia and plasma cholesterol levels in experimental restenosis.

BACKGROUND AND AIMS: We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32 microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis. METHODS: Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE-/- mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs). RESULTS: GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction. CONCLUSIONS: GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage influx, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis.

Tumor Eradication by Cisplatin Is Sustained by CD80/86-Mediated Costimulation of CD8+ T Cells.

Certain cytotoxic chemotherapeutic drugs are immunogenic, stimulating tumor immunity through mechanisms that are not completely understood. Here we show how the DNA-damaging drug cisplatin modulates tumor immunity. At the maximum tolerated dose (MTD), cisplatin cured 50% of mice with established murine TC-1 or C3 tumors, which are preclinical models of human papillomavirus (HPV)-associated cancer. Notably, the curative benefit of cisplatin relied entirely upon induction of tumor-specific CD8+ T cells. Mechanistic investigations showed that cisplatin stimulated tumor infiltration of inflammatory antigen-presenting cells (APC) expressing relatively higher levels of the T-cell costimulatory ligands CD70, CD80, and CD86. Cell death triggered by cisplatin was associated with the release of at least 19 proteins in the tumor environment that could act as damage-associated molecular patterns and upregulate costimulatory molecules, either alone or in concert, but the responsible proteins remain unknown. Essentially, the curative effect of cisplatin was abrogated in mice lacking expression of CD80 and CD86 on APCs. Furthermore, cisplatin treatment was improved by CTLA-4 blockade, which increases the availability of CD80/86 to bind to CD28. In contrast, there was no effect of CD27 stimulation, which replaces CD70 interaction. At the cisplatin MTD, cure rates could also be increased by vaccination with synthetic long peptides, whereas cures could also be achieved at similar rates at 80% of the MTD with reduced side effects. Our findings reveal an essential basis for the immunogenic properties of cisplatin, which are mediated by the induction of costimulatory signals for CD8+ T-cell-dependent tumor destruction. Cancer Res; 76(20); 6017-29. ©2016 AACR.

Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses.

Therapeutic vaccination with human papillomavirus type 16 synthetic long peptides (HPV16-SLPs) results in T cell-mediated regression of HPV16-induced premalignant lesions but fails to install clinically effective immunity in patients with HPV16-positive cervical cancer. We explored whether HPV16-SLP vaccination can be combined with standard carboplatin and paclitaxel chemotherapy to improve immunity and which time point would be optimal for vaccination. This was studied in the HPV16 E6/E7-positive TC-1 mouse tumor model and in patients with advanced cervical cancer. In mice and patients, the presence of a progressing tumor was associated with abnormal frequencies of circulating myeloid cells. Treatment of TC-1-bearing mice with chemotherapy and therapeutic vaccination resulted in superior survival and was directly related to a chemotherapy-mediated altered composition of the myeloid cell population in the blood and tumor. Chemotherapy had no effect on tumor-specific T cell responses. In advanced cervical cancer patients, carboplatin-paclitaxel also normalized the abnormal numbers of circulating myeloid cells, and this was associated with increased T cell reactivity to recall antigens. The effect was most pronounced starting 2 weeks after the second cycle of chemotherapy, providing an optimal immunological window for vaccination. This was validated with a single dose of HPV16-SLP vaccine given in this time window. The resulting proliferative HPV16-specific T cell responses were unusually strong and were retained after all cycles of chemotherapy. In conclusion, carboplatin-paclitaxel therapy fosters vigorous vaccine-induced T cell responses when vaccination is given after chemotherapy and has reset the tumor-induced abnormal myeloid cell composition to normal values.

New approaches in vaccine-based immunotherapy for human papillomavirus-induced cancer.

The identification of human papillomavirus as the etiological factor for cervical cancer provides an opportunity to treat these malignancies by vaccination. Although therapeutic vaccination against viral oncogenes regularly induces a specific T cell response, clinical effectivity remains low. Three factors are particularly important for clinical outcome: the balance between cytotoxic T cells and regulatory immune subsets, the balance between cytotoxic T cells and tumor cells and finally the killing efficiency of cytotoxic T cells within the tumor. To improve these three factors, therapeutic vaccination is combined with other treatments. Here, we review those studies that are based on understanding the inhibitory mechanisms that prevent unleashing the full power of therapeutic vaccine-induced T cells and utilize combinatorial interventions based on these insights.

Therapeutic Peptide Vaccine-Induced CD8 T Cells Strongly Modulate Intratumoral Macrophages Required for Tumor Regression.

Abundant macrophage infiltration of solid cancers commonly correlates with poor prognosis. Tumor-promoting functions of macrophages include angiogenesis, metastasis formation, and suppression of Th1-type immune responses. Here, we show that successful treatment of cervical carcinoma in mouse models with synthetic long peptide (SLP) vaccines induced influx of cytokine-producing CD8 T cells that strongly altered the numbers and phenotype of intratumoral macrophages. On the basis of the expression of CD11b, CD11c, F4/80, Ly6C, Ly6G, and MHC II, we identified four myeloid subpopulations that increased in numbers from 2.0-fold to 8.7-fold in regressing tumors. These changes of the intratumoral myeloid composition coincided with macrophage recruitment by chemokines, including CCL2 and CCL5, and were completely dependent on a vaccine-induced influx of tumor-specific CD8 T cells. CD4 T cells were dispensable. Incubation of tumor cells with T cell-derived IFNγ and TNFα recapitulated the chemokine profile observed in vivo, confirming the capacity of antitumor CD8 T cells to mediate macrophage infiltration of tumors. Strikingly, complete regressions of large established tumors depended on the tumor-infiltrating macrophages that were induced by this immunotherapy, because a small-molecule drug inhibitor targeting CSF-1R diminished the number of intratumoral macrophages and abrogated the complete remissions. Survival rates after therapeutic SLP vaccination deteriorated in the presence of CSF-1R blockers. Together, these results show that therapeutic peptide vaccination could induce cytokine-producing T cells with strong macrophage-skewing capacity necessary for tumor shrinkage, and suggest that the development of macrophage-polarizing, rather than macrophage-depleting, agents is warranted.

Vaccine-induced tumor necrosis factor-producing T cells synergize with cisplatin to promote tumor cell death.

PURPOSE: Cancer immunotherapy, such as vaccination, is an increasingly successful treatment modality, but its interaction with chemotherapy remains largely undefined. Therefore, we explored the mechanism of synergy between vaccination with synthetic long peptides (SLP) of human papillomavirus type 16 (HPV16) and cisplatin in a preclinical tumor model for HPV16. EXPERIMENTAL DESIGN: SLP vaccination in this preclinical tumor model allowed the elucidation of novel mechanisms of synergy between chemo- and immunotherapy. By analyzing the tumor immune infiltrate, we focused on the local intratumoral effects of chemotherapy, vaccination, or the combination. RESULTS: Of several chemotherapeutic agents, cisplatin synergized best with SLP vaccination in tumor eradication, without requirement for the maximum-tolerated dose (MTD). Upon SLP vaccination, tumors were highly infiltrated with HPV-specific, tumor necrosis factor-α (TNFα)- and interferon-γ (IFNγ)-producing T cells. Upon combined treatment, tumor cell proliferation was significantly decreased compared with single treated and untreated tumors. Furthermore, we showed that TNFα strongly enhanced cisplatin-induced apoptotic tumor cell death in a JNK-dependent manner. This is consistent with upregulation of proapoptotic molecules and with enhanced cell death in vivo upon combined SLP vaccination and cisplatin treatment. In vivo neutralization of TNFα significantly reduced the antitumor responses induced by the combined treatment. CONCLUSION: Taken together, our data show that peptide vaccination with cisplatin treatment leads to decreased tumor cell proliferation and TNFα-induced enhanced cisplatin-mediated killing of tumor cells, together resulting in superior tumor eradication.

Inhibition of CSF-1R supports T-cell mediated melanoma therapy.

Tumor associated macrophages (TAM) can promote angiogenesis, invasiveness and immunosuppression. The cytokine CSF-1 (or M-CSF) is an important factor of TAM recruitment and differentiation and several pharmacological agents targeting the CSF-1 receptor (CSF-1R) have been developed to regulate TAM in solid cancers. We show that the kinase inhibitor PLX3397 strongly dampened the systemic and local accumulation of macrophages driven by B16F10 melanomas, without affecting Gr-1(+) myeloid derived suppressor cells. Removal of intratumoral macrophages was remarkably efficient and a modest, but statistically significant, delay in melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly enhanced tumor control by immunotherapy using tumor-specific CD8 T cells. Elevated IFNγ production by T cells was observed in mice treated with the combination of PLX3397 and immunotherapy. These results support the combined use of CSF-1R inhibition with CD8 T cell immunotherapy, especially for macrophage-stimulating tumors.

Controlled local delivery of CTLA-4 blocking antibody induces CD8+ T-cell-dependent tumor eradication and decreases risk of toxic side effects.

PURPOSE: Blockade of CTLA-4 by antibodies has potentiated antitumor T-cell responses in both preclinical models and clinical trials. However, treatment with CTLA-4 blocking antibodies is associated with autoimmune and inflammatory side effects. In this study, we propose a novel administration method for CTLA-4 blocking antibodies as monotherapy. EXPERIMENTAL DESIGN: We use different preclinical mouse models of cancer to investigate the local administration of CTLA-4 blocking antibody and its effect on cancer progression and the antitumor T-cell response. RESULTS: By injecting the antibodies in a subcutaneous slow-release delivery formulation in the tumor area, we show that an eight-fold lower dose of antibody is as effective in inducing tumor eradication as systemic delivery. A lower dose and slow release of the antibody results in thousand-fold decreased levels of antibody in the serum, reducing adverse events and the risk of autoimmunity. The main target and effector cells of the CTLA-4 blockade treatment in the studied tumor models are tumor-specific endogenous CD8(+) T cells that are capable of eradicating also distant tumors, whereas CD4(+) T cells do not play a prominent role in the antibody-mediated tumor eradication. CONCLUSIONS: Injecting CTLA-4 blocking antibody in a slow-release formulation close to the tumor is an effective way of activating the antitumor T-cell response. This administration method is associated with very low serum levels of antibody, which decreases the risk of treatment-induced side effects. These results call for exploration of a similar delivery principle in clinical settings.

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections.

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccines.