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Although immune checkpoint blockade (ICB) has become the mainstay of treatment for advanced solid organ malignancies, success in revitalizing the host anticancer immune response remains limited. G-protein coupled receptors (GPCRs) are a broad family of cell-surface proteins that have been regarded as main players in regulating the immune system, namely by mediating the activity of T lymphocytes. Among the most novel immunoregulatory GPCRs include GPR171, lysophosphatidic acid receptors (LPARs), GPR68, cannabinoid receptor 2 (CB2), and prostaglandin E receptors, many of which have shown promise in mediating antitumor response via activation of cytotoxic T cells, inhibiting immunosuppressive lymphocytes, and facilitating immune cell infiltration within the tumor microenvironment across multiple types of cancers. This paper reviews our current understanding of some of the most novel GPCRs—their expression patterns, evolving roles within the immune system and cancer, potential therapeutic applications, and perspective for future investigation. [ABSTRACT FROM AUTHOR]

A correction to this paper has been published: https://doi.org/10.1007/s00262-021-02903-w [ABSTRACT FROM AUTHOR]

A correction to this paper has been published: https://doi.org/10.1007/s00262-021-02970-z [ABSTRACT FROM AUTHOR]

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses. [ABSTRACT FROM AUTHOR]

Although immunodeficiency is usually considered a prerequisite of oncogenesis, a detailed immune profile in cancer has not yet been described. Without such profiling, it is not surprising that there is a vast discrepancy in the responses of cancer patients to immunotherapy. Our results show that the integrity of the immune system deteriorates with cancer progression by displaying a trend toward decreasing levels of functional T cells, including CD4, naïve, and central memory T cells, and an expansion of hyporesponsive populations such as CD28− and CMV-specific T cells. One hundred and one patients constitute the study group for the observational study reported in this paper. Forty-eight patients with newly diagnosed stages III and IV and 53 patients with extensively treated stage IV disease. The costimulatory molecules CD27 and CD28 were downregulated in all patients. Among the proinflammatory cytokines (IL-6, TNF-α, IFN-γ), only IL-6 differed significantly among the groups, increasing as the cancer stage progressed. Plasma IL-7 did not differ among the participants. The relative deficits of naïve T cells in cancer patients may be associated with the downregulation of IL-7Rα expression rather than changes in the circulating levels of IL-7. The downregulation of IL-7Rα expression was shown to be associated with increased levels of intracellular CMV. The present study suggests that the immune impairment in patients with cancer is associated with multiple factors, such as the stage of cancer, consequence of CMV infection and impact of treatment. [ABSTRACT FROM AUTHOR]

Innate and adaptive immune responses have many interactions that are regulated by the balance of signals initiated by a variety of activatory and inhibitory receptors. Among these, the NKG2D molecule was identified as expressed by T lymphocytes, including most CD8+ cells and a minority of CD4+ cells, designated TNK cells in this paper. Tumor cells may overexpress the stress-inducible NKG2D ligands (NKG2DLs: MICA/B, ULBPs) and the NKG2D signaling has been shown to be involved in lymphocyte-mediated anti-tumor activity. Aberrant expression of NKG2DLs by cancer cells, such as the release of soluble form of NKG2DLs, can lead to the impairment of these immune responses. Here, we discuss the significance of NKG2D in TNK-mediated anti-tumor activity. Our studies demonstrate that NKG2D+ T cells (TNK) are commonly recruited at the tumor site in melanoma patients where they may exert anti-tumor activity by engaging both TCR and NKG2D. Moreover, NKG2D and TCR triggering was also observed by peripheral blood derived T lymphocyte- or T cell clone-mediated tumor recognition, both in melanoma and colorectal cancer (CRC) patients. Notably, heterogeneous expression of NKG2DLs was found in melanoma and CRC cells, with a decrease of these molecules along with tumor progression. Therefore, through the mechanisms that govern NKG2D engagement in anti-tumor activity and the expression of NKG2DLs by tumor cells that still need to be dissected, we showed that NKG2D expressing TNK cells are a relevant T cell subtype for immunosurveillance of tumors and we propose that new immunotherapeutic interventions for cancer patients should be aimed also at enhancing NKG2DLs expression by tumor cells. [ABSTRACT FROM AUTHOR]

Experimental evidences supporting the epidermal growth factor receptor (EGFR) as an important molecule for tumor metastasis had been accumulated. Currently, anti-EGFR monoclonal antibodies (mAbs) constitute a promising approach for the treatment of patients with metastatic tumors. However, the mechanisms associated with the potent anti-metastatic effect of these mAbs have not been completely elucidated due to the lack of appropriate syngeneic preclinical models. In this paper, we have investigated the effects of 7A7, an antibody specific to murine EGFR, on the metastatic properties of D122 murine lung carcinoma. 7A7 mAb significantly impaired metastatic spread of D122 cells in C57BL/6 mice by direct anti-proliferative and pro-apoptotic effects on tumor metastasis. 7A7 mAb capacity to inhibit EGFR activation on D122 cells could contribute to its anti-metastatic effect. In addition, 7A7 mAb was able to induce in vitro antibody-dependent cell-mediated cytotoxicity on D122 cells. Interestingly, 7A7 mAb treatment increased the number of natural killer cells, T lymphocytes and dendritic cells infiltrating the metastatic sites. More strikingly, depletion of CD8+ and CD4+ T cells in vivo completely abrogated the 7A7 mAb anti-metastatic activity whereas function of natural killer cells was irrelevant. This study supports an in vivo role for T cell response in the mechanism of action of anti-EGFR mAbs, suggesting the induction of an adjuvant effect. [ABSTRACT FROM AUTHOR]

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNγ production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials. [ABSTRACT FROM AUTHOR]

Esophageal cancer (EC) is a deadly malignancy. Small extracellular vesicles (sEVs) with programmed death ligand 1 (sEV-PDL1) induce immune escape to promote tumor progression. Furthermore, the imbalance between circulating follicular helper T (Tfh) and circulating follicular regulatory T (Tfr) cells is related to the progression of many malignant tumors. However, the role of the EC-derived sEV-PDL1 in circulating Tfh/Tfr is unknown. Circulating Tfh and Tfr cells were detected by flow cytometry. sEVs were isolated through differential centrifugation and cultured for cell expansion assays. Naïve CD4+ T cells were isolated, stimulated, and cultured with sEVs to evaluate the frequencies, phenotypes, and functions of Tfh and Tfr cells. The proportion of circulating Tfh in patients with EC was lower than that in healthy donors (HDs), whereas that of circulating Tfr was higher. The EC group showed significantly lower circulating Tfh/Tfr and a higher level of sEV-PDL1 than HDs. Notably, sEV-PDL1 was negatively correlated with circulating Tfh/Tfr in the EC group. In vitro assays, sEV-PDL1 inhibited Tfh expansion, enhanced the cytotoxic T lymphocyte-associated antigen 4+ (CTLA4+) Tfh cell percentage, decreased the levels of interleukin (IL)-21 and interferon-γ, and increased IL-10. sEV-PDL1 promoted the expansion and immunosuppressive functions of circulating Tfr; the increased percentages of CTLA4+ Tfr and inducible T cell co-stimulator+ Tfr were accompanied with high IL-10. However, applying an anti-PDL1 antibody significantly reversed this. Our results suggest a novel mechanism of sEV-PDL1-mediated immunosuppression in EC. Inhibiting sEV-PDL1 to restore circulating Tfh/Tfr balance provides a novel therapeutic approach for EC. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR T cell therapy in epithelial ovarian cancer and other cancers. [ABSTRACT FROM AUTHOR]

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from the immune killing of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19– lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19– lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR– T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells. [ABSTRACT FROM AUTHOR]

Background: Brain metastases are the most common intracranial tumors with an increasing incidence. They are an important cause of morbidity and mortality in patients with solid organ cancer and a focus of recent clinical research and experimental interest. Immune checkpoint inhibitors are being increasingly used to treat solid organ cancers. Methods: To determine whether immune checkpoint inhibitors were biologically effective in the brain, we compared melanoma brain metastasis samples where treatment with ipilimumab had occurred preoperatively to those who had not received any immune modulating therapy and looked for histopathological (invasion, vascularity, metastasis inducing proteins, matrix metalloproteinases, immune cell infiltration, tissue architecture) and advanced MRI differences (diffusion weighted imaging). Results: Co-localized tissue samples from the same regions as MRI regions of interest showed significantly lower vascularity (density of CD34 + vessels) in the core and higher T-cell infiltration (CD3 + cells) in the leading edge for ipilimumab-treated brain metastasis samples than for untreated cases and this correlated with a higher tumor ADC signal at post-treatment/preoperative MRI brain. Conclusions: Treatment of a melanoma brain metastasis with ipilimumab appears to cause measurable biological changes in the tumor that can be correlated with post-treatment diffusion weighted MRI imaging, suggesting both a mechanism of action and a possible surrogate marker of efficacy. [ABSTRACT FROM AUTHOR]

A correction to this paper has been published: https://doi.org/10.1007/s00262-021-02977-6 [ABSTRACT FROM AUTHOR]

Interleukin-36α (IL-36α) is essential for various inflammatory conditions, such as psoriasis and rheumatoid arthritis, whereas its role in tumor immunity is unclear. In this study, it was demonstrated that IL-36α could activate the NF-κB and MAPK signaling pathways in macrophages, leading to the expression of IL-1β, IL-6, TNF-α, CXCL1, CXCL2, CXCL3, CXCL5 and iNOS. Importantly, IL-36α has significant antitumor effects, altering the tumor microenvironment and promoting the infiltration of MHC IIhigh macrophages and CD8+ T cells while decreasing the levels of monocyte myeloid-derived suppressor cells, CD4+ T cells and regulatory T cells. This ultimately results in the inhibition of tumor growth and migration. Furthermore, IL-36α synergized with the PD-L1 antibody increased the immune cells infiltration and enhanced the anti-tumor effect of the PD-L1 antibody on melanoma. Collectively, this study reveals a new role for IL-36α in promoting anti-tumor immune responses in macrophages and suggests its potential for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Immunotherapy has revolutionized cancer therapy. Two recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or chimeric antigen receptor T (CAR-T) cells. Blinatumomab, an FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating effector-target cell contact and T-cell activation that results in effective elimination of target B cells. Although CD19 is expressed by essentially all B-cell malignancies at clinical presentation, relapses with loss or reduction in CD19 surface expression are increasingly recognized as a cause of treatment failure. Therefore, there is a clear need to develop therapeutics for alternate targets. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments. Target binding of the anti-CD22 and anti-CD3 moieties was confirmed by flow cytometry. CD22-BiTE promoted in vitro cell-mediated cytotoxicity in a dose and effector: target (E:T)-dependent fashion. Additionally, in an established acute lymphoblastic leukemia (ALL) xenograft mouse model, CD22-BiTE demonstrated tumor growth inhibition, comparable to blinatumomab. Further, the combination of blinatumomab and CD22-BiTE yielded increased efficacy in vivo when compared to the single agents. In conclusion, we report here the development of a new BiTE with cytotoxic activity against CD22+ cells which could represent an alternate or complementary therapeutic option for B-cell malignancies. [ABSTRACT FROM AUTHOR]

Brachyury is a transcription factor belonging to the T-box gene family and is involved in the posterior formation of the mesoderm and differentiation of chordates. As the overexpression of Brachyury is a poor prognostic factor in a variety of cancers, the establishment of Brachyury-targeted therapy would be beneficial for the treatment of aggressive tumors. Because transcription factors are difficult to treat with a therapeutic antibody, peptide vaccines are a feasible approach for targeting Brachyury. In this study, we identified Brachyury-derived epitopes that elicit antigen-specific and tumor-reactive CD4+ T cells that directly kill tumors. T cells recognizing Brachyury epitopes were present in patients with head and neck squamous cell carcinoma. Next, we focused on gemcitabine (GEM) as an immunoadjuvant to augment the efficacy of antitumor responses by T cells. Interestingly, GEM upregulated HLA class I and HLA-DR expression in tumor, followed by the upregulation of anti-tumor T cell responses. As tumoral PD-L1 expression was also augmented by GEM, PD-1/PD-L1 blockade and GEM synergistically enhanced the tumor-reactivity of Brachyury-reactive T cells. The synergy between the PD-1/PD-L1 blockade and GEM was also confirmed in a mouse model of head and neck squamous cell carcinoma. These results suggest that the combined treatment of Brachyury peptide with GEM and immune checkpoint blockade could be a promising immunotherapy against head and neck cancer. [ABSTRACT FROM AUTHOR]

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days. [ABSTRACT FROM AUTHOR]

Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1+ Tim-3− and PD-1+ Tim-3+ T cells in sick mice. Furthermore, PD-1+ Tim-3+ T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1+ Tim-3− T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1+ Tim-3− and PD-1+ Tim-3+ T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms. [ABSTRACT FROM AUTHOR]

Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy. [ABSTRACT FROM AUTHOR]

Human Vγ9Vδ2 T cells have attracted considerable attention as novel alternative antigen-presenting cells (APCs) with the potential to replace dendritic cells in antitumor immunotherapy owing to their high proliferative capacity and low cost. However, the utility of γδ T cells as APCs to induce CD8+ T cell-mediated antitumor immune response, as well as the mechanism by which they perform APC functions, remains unexplored. In this study, we found that activated Vγ9Vδ2 T cells were capable of inducing robust CD8+ T cell responses in osteosarcoma cells. Activated γδ T cells also effectively suppressed osteosarcoma growth by priming CD8+ T cells in xenograft animal models. Mechanistically, we further revealed that activated γδ T cells exhibited increased HSP90 production, which fed back to upregulate MyD88, followed by JNK activation and a subsequent improvement in CCL5 secretion, leading to enhanced CD8+ T cell cross-priming. Thus, our study suggests that Vγ9Vδ2 T cells represent a promising alternative APC for the development of γδ T cell-based tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy relies on improving T cell effector functions against malignancies, but despite the identification of several key transcription factors (TFs), the biological functions of these TFs are not entirely understood. We developed and utilized a novel, clinically relevant murine model to dissect the functional properties of crucial T cell transcription factors during anti-tumor responses. Our data showed that the loss of TCF-1 in CD8 T cells also leads to loss of key stimulatory molecules such as CD28. Our data showed that TCF-1 suppresses surface NKG2D expression on naïve and activated CD8 T cells via key transcriptional factors Eomes and T-bet. Using both in vitro and in vivo models, we uncovered how TCF-1 regulates critical molecules responsible for peripheral CD8 T cell effector functions. Finally, our unique genetic and molecular approaches suggested that TCF-1 also differentially regulates essential kinases. These kinases, including LCK, LAT, ITK, PLC-γ1, P65, ERKI/II, and JAK/STATs, are required for peripheral CD8 T cell persistent function during alloimmunity. Overall, our molecular and bioinformatics data demonstrate the mechanism by which TCF-1 modulated several critical aspects of T cell function during CD8 T cell response to cancer. Summary Figure: TCF-1 is required for persistent function of CD8 T cells but dispensable for anti-tumor response. Here, we have utilized a novel mouse model that lacks TCF-1 specifically on CD8 T cells for an allogeneic transplant model. We uncovered a molecular mechanism of how TCF-1 regulates key signaling pathways at both transcriptomic and protein levels. These key molecules included LCK, LAT, ITK, PLC-γ1, p65, ERK I/II, and JAK/STAT signaling. Next, we showed that the lack of TCF-1 impacted phenotype, proinflammatory cytokine production, chemokine expression, and T cell activation. We provided clinical evidence for how these changes impact GVHD target organs (skin, small intestine, and liver). Finally, we provided evidence that TCF-1 regulates NKG2D expression on mouse naïve and activated CD8 T cells. We have shown that CD8 T cells from TCF-1 cKO mice mediate cytolytic functions via NKG2D. [ABSTRACT FROM AUTHOR]

Recent studies have underscored the importance of gamma-delta (γδ) T cells in mediating potent MHC-unrestricted cytotoxicity in numerous malignancies. Here, we analyzed Vδ1 and Vδ2 γδ T cell subsets in newly diagnosed chronic myeloid leukemia (CML) patients (n = 40) who had initiated tyrosine kinase inhibitor (TKI) therapy including imatinib (n = 22), nilotinib (n = 14) and dasatinib (n = 4). Patient peripheral blood samples were analyzed at diagnosis and monitored prospectively at 3, 6, 12 and 18 months post-TKI. γδ T cells isolated from healthy donors and CML patients were used against K562, LAMA-84 and KYO-1 cell lines and against primary CML cells in cytotoxicity assays. We found large expansions of Vδ1 and Vδ2 T cells in patients at diagnosis compared to age-matched healthy donors (n = 40) (p < 0.0001). The γδ T cell reconstitution in patients on imatinib and also on nilotinib showed significant reductions of Vδ1 T cell and Vδ2 T cell absolute counts at 3 months compared to diagnosis. Importantly, Vδ1 and Vδ2 T absolute cell counts remained at normal levels from 3 months throughout the follow-up. Next, we observed susceptibility to specific lysis of primary CML tumor cells by Vδ1 T cells from healthy donors. Furthermore, we determined inherent cytotoxic reactivity by autologous patients' Vδ1 T lymphocytes against primary CML tumor cells. Finally, the TCR clonality profiles showed in CML patients mostly polyclonal repertoires regardless of the TKI. Our results provide further evidence into γδ T cell antileukemia immunity in CML that might be beneficial for long-term disease control and treatment outcome. [ABSTRACT FROM AUTHOR]

Engineered immune cell therapy to treat malignancies refractory to conventional therapies is modernizing oncology. Although αβ T cells are time-tested chassis for CAR, potential graft versus host disease (GvHD) apart from cytokine toxicity and antigen escape pose limitations to this approach. αβ T cell malignancy challenges isolation and expansion of therapeutic T cells. Moreover, αβ T cells may pose toxicity risk to inflammation sensitive vital tissues bearing the tumor. The HLA independent, multivalent, versatile and systemic anti-tumor immunity increases the desirability of γδ T cells as an alternate chassis for CAR. Indeed, CD19 γδ CAR T cell therapy to treat advanced lymphoma reached a milestone with the fast track status by FDA. However, reduced tumor-toxicity, homing, in vivo persistence and heterogeneity limits the translation of this therapy. The field is gaining momentum in recent years with optimization of gene delivery approaches and mechanistic insights into co-signaling requirements in γδ T cells. There is a renewed interest in customizing design of CAR guided by the biology of the host immune cells. Progress has been made in the current good manufacturing practice compatible expansion and engineering protocols for the δ1 and δ2 T cells. γδ CAR T cells may find its niche in the clinical situations wherein conventional CAR therapy is less suitable due to propensity for cytokine toxicity or off-tumor effect. As the therapy is moving towards clinical trials, this review chronicles the hitherto progress in the therapeutic engineering of γδ T cells for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Whereas neoadjuvant cisplatin-based chemotherapy (NAC) followed by a radical cystectomy remains the standard treatment for patients with muscle-invasive bladder cancer (MIBC), increasing evidence suggests that checkpoint inhibitors, either alone or in combination with chemotherapy, are effective in the (neo)adjuvant setting. The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in MIBC. Methods: Tumor tissue of 81 patients was used, including 60 patients treated with NAC and 21 patients undergoing upfront cystectomy. Multiplex immunohistochemistry was performed to assess CD3+, CD3+CD8+, CD3+CD8−FoxP3−, CD3+FoxP3+, and CD20+ cells. Patients were classified into a favorable or unfavorable outcome group based on the development of a recurrence within a year. Results: The density of intratumoral CD3+ T cells decreased following NAC in patients with a recurrence at one year, while it remained stable in patients without a recurrence (median fold change 0.6 [95CI 0.3; 1.0] versus 1.0 [95CI 0.6; 2.2]). This decrease was mainly attributable to a decrease in CD3+CD8−FoxP3− and CD3+FoxP3+ T cells and was not observed in patients with an early recurrence after upfront cystectomy. Additionally, in cystectomy tissue of patients treated with NAC, median CD3+ and CD3+CD8+ T cell densities were significantly lower in patients with versus patients without a recurrence (CD3: 261. cells/mm2 [95CI 22.4; 467.2]; CD8: 189.6 cells/mm2 [95CI 2.0;462.0]). Conclusion: T cell density decreases following NAC in MIBC patients with poor clinical outcome. Further research is needed to investigate whether this decrease in T cell density affects the efficacy of subsequent checkpoint inhibitors. Précis: The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in patients with MIBC. We reveal a decline in intratumoral CD3+ T cell density following NAC in patients with an early recurrence. [ABSTRACT FROM AUTHOR]

Wilms' tumor 1 (WT1) is a promising tumor-associated antigen for cancer immunotherapy. We developed an oral protein vaccine platform composed of WT1-anchored, genetically engineered Bifidobacterium longum (B. longum) and conducted an in vivo study in mice to examine its anticancer activity. Mice were orally treated with phosphate-buffered saline, wild-type B. longum105-A, B. longum 2012 displaying only galacto-N-biose/lacto-N-biose I-binding protein (GLBP), and WT1 protein- and GLBP-expressing B. longum 420. Tumor size reduced significantly in the B. longum 420 group than in the B. longum 105-A and 2012 groups (P < 0.00 l each), indicating B. longum 420's antitumor activity via WT1-specific immune responses. CD8+ T cells played a major role in the antitumor activity of B. longum 420. The proportion of CD103+CD11b+CD11c+ dendritic cells (DCs) increased in the Peyer's patches (PPs) from mice in the B. longum 420 group, indicating the definite activation of DCs. In the PPs, the number and proportion of CD8+ T cells capable of producing interferon-gamma were significantly greater in the B. longum 420 group than in the B. longum 2012 group (P < 0.05 or < 0.01). The production of WT1-specific IgG antibody was significantly higher in the B. longum 420 group than in the 2012 group (P < 0.05). The B. longum 420 group showed the most intense intratumoral infiltration of CD4+ and CD8+ T cells primed by activated DCs in the PPs of mice in the B. longum 420 group. Our findings provide insights into a novel, intestinal bacterium-based, cancer immunotherapy through intestinal immunity. [ABSTRACT FROM AUTHOR]

We extended our previous observations with other tumor models to study seven ovarian tumor cell lines—OVCAR3, OVCAR4, OVCAR8, SKOV3, Kuramochi, OAW28, and CaOV3. We found that NK cells targeted and killed poorly differentiated OVCAR8 and CAOV3; these two tumor lines express lower MHC-class I and higher CD44 surface receptors. OVCAR3 and OVCAR4 were more resistant to NK cell-mediated cytotoxicity, and SKOV3, Kuramochi and OAW28 had intermediate sensitivity to NK cell-mediated cytotoxicity, likely representing well-differentiated and moderately differentiated ovarian tumor cell lines, respectively. Similar trends were observed for secretion of IFN-γ by the NK cells when co-cultured with different ovarian tumor cell lines. Treatment with both IFN-γ and TNF-α upregulated MHC-class I in all ovarian tumor cell lines and resulted in tumor resistance to NK cell-mediated cytotoxicity and decreased secretion of IFN-γ in co-cultures of NK cells with tumors cells with the exception of OVCAR8 and CAOV3 which did not upregulate MHC-class I and remained sensitive to NK cell-mediated cytotoxicity and increased secretion of IFN-γ when co-cultured with NK cells. Similarly, treatment with NK cell supernatants induced resistance to NK cell-mediated cytotoxicity in OVCAR4 but not in OVCAR8, and the resistance to killing was correlated with the increased surface expression of MHC-class I in OVCAR4 but not in OVCAR8. In addition, OVCAR4 was found to be carboplatin sensitive before and after treatment with IFN-γ and NK cell supernatants, whereas OVCAR8 remained carboplatin resistant with and without treatment with IFN-γ and NK cell supernatants. Overall, sensitivity to NK cell-mediated killing correlated with the levels of tumor differentiation and aggressiveness, and more importantly, poorly differentiated ovarian tumors were unable to upregulate MHC-class I under the activating conditions for MHC-class I, a feature that was not seen in other tumor models and may likely be specific to ovarian tumors. Such tumors may also pose a significant challenge in elimination by the T cells; however, NK cells are capable of targeting such tumors and can be exploited to eliminate these tumors in immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

Histiocytic sarcoma (HS) is a rare and aggressive tumor in humans with no universally agreed standard of care therapy. Spontaneous canine HS exhibits increased prevalence in specific breeds, shares key genetic and biologic similarities with the human disease, and occurs in an immunocompetent setting. Previous data allude to the immunogenicity of this disease in both species, highlighting the potential for their successful treatment with immunotherapy. Quantification of CD3 tumor-infiltrating lymphocytes (TIL) in five cases of human HS revealed variable intra-tumoral T cell infiltration. Due to the paucity of human cases and lack of current model systems in which to appraise associations between anti-tumor immunity and treatment-outcome in HS, we analyzed clinical data and quantified TIL in 18 dogs that were previously diagnosed with localized HS and treated with curative-intent tumor resection with or without adjuvant chemotherapy. As in humans, assessment of TIL in biopsy tissues taken at diagnosis reveal a spectrum of immunologically "cold" to "hot" tumors. Importantly, we show that increased CD3 and granzyme B TIL are positively associated with favorable outcomes in dogs following surgical resection. NanoString transcriptional analyses revealed increased T cell and antigen presentation transcripts associated with prolonged survival in canine pulmonary HS and a decreased tumor immunogenicity profile associated with shorter survivals in splenic HS. Based on these findings, we propose that spontaneous canine HS is an accessible and powerful novel model to study tumor immunology and will provide a unique platform to preclinically appraise the efficacy and tolerability of anti-cancer immunotherapies for HS. [ABSTRACT FROM AUTHOR]

Information about several papers discussed at the symposium on cellular therapy of cancer, held in Manchester, England from December 2-3, 2006, is presented. The participants discussed several issues related to gene-modified T cell-based immunotherapies. Those who took part in the discussion included medical experts Balbino Alarcon, Janos Szollosi and Zelig Eshhar.