Citations

Abstract B7 homolog-1 ( B7-H1) expressed by antigen-presenting cells or tumor cells is a negative regulator of T cell responses, however its function on T cells is not clear. Here we show that B7-H1 was upregulated in CD8 T cells during immunization and its expression was maintained on memory CD8 T cells. Although B7-H1 deficient naïve CD8 T cells responded normally to antigen stimulation and underwent normal antigen-independent homeostatic proliferation, the maximal expansion of CD8 T cells was blocked due to their high susceptibility to apoptosis. B7-H1-deficient effector CD8 T cells showed weaker effector function and cytotoxicity against tumor cells compared to that of normal CD8 T cells. Once activated, B7-H1-deficient CD8 T cells generated less memory T cells. This effect was independent of the primary expansion of the antigen-specific CD8 T cell population. Our results revealed previously unknown function of B7-H1 on CD8 T cells in mediating survival and differentiation of effector CD8 T cells into memory T cells.

Abstract Studies from cancer and chronic infections show that B7-H1 (PD-L1) engagement with its receptor PD-1 enhances apoptosis of effector T cells. It is not clear how B7-H1 regulates T cell apoptosis and the subsequent impact of B7-H1 on T cell memory generation. In immunized B7-H1 deficient mice we observed increased expansion of effector CD8 T cells and delayed T cell contraction followed by protective CD8 T cell memory capable of completely rejecting tumor metastases in lung tissue. Intracellular staining revealed that antigen-primed CD8 T cells in B7-H1 deficient mice express lower levels of the pro-apoptotic molecule Bim. Injection of B7-H1 or PD-1 blocking antibody into immunized mice reduced Bim levels in effector CD8 T cells. In vitro studies show that engagement of activated CD8 T cells by plate-bound B7-H1 fusion protein led to increased cell death and higher levels of Bim expression. Our results suggest that B7-H1 may negatively regulate CD8 T cell memory by enhancing the depletion of effector CD8 T cells through up-regulation of Bim. Our findings would provide a new strategy to target B7-H1 signaling in effector CD8 T cells to achieve protective immunity in the treatment of cancer and infectious diseases.

Abstract Spontaneous antitumor immunity remains challenging to detect and monitor largely due to the absence of established biomarkers. We selected the integrin CD11a as a biomarker to define antitumor T cell responses. CD11a is up-regulated on effector and memory T cells and is involved in the killing of tumor cells by cytotoxic T lymphocytes. In primary (subcutaneously inoculated) and metastatic (intravenously injected) tumors, CD11a high CD8 T cells were detected in the primary (skin) or metastatic (lung) tumor sites, but not in secondary lymphoid organs. Once tumors were established, CD11a high CD8 T cells were identified in tumor draining lymph nodes and peripheral blood. Phenotype analysis revealed that CD11a high CD8 T cells were proliferating (Ki67+) activated T cells (CD62L low, CD69 high, PD-1 high). CD11a high CD8 T cells expressed the transcription factor T-bet but not Foxp3. Live, but not dead, tumor cells, induced expansion of CD11a high CD8 T cells in the lung, suggesting that the access of tumor cells to target tissues is required to induce CD11a high CD8 T cell responses. Our results suggest that CD11a may provide a new T cell biomarker to measure ongoing natural tumor-reactive T cell responses and to predicate the efficacy of tumor immunotherapy.

Abstract Seven different anti-PD-1 and PD-L1 monoclonal antibodies are now widely used in the US to treat a variety of cancer types, but there have been no clinical trials comparing them directly. Furthermore, because many of these antibodies do not cross react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by FDA-approved antibodies. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor antigen (OVA), followed by subsequent B16-OVA tumor challenge. We found that anti-PD-1/L1-treated mice exhibited significantly better tumor rejection than controls, although both the control and antibody-treated mice generated comparable numbers of OVA-specific T cells at the time of priming. To determine what could mediate this strong antitumor immune response, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the anti-PD-1/L1-treated mice, the same subset of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of FDA-approved anti-PD-1/L1 monoclonal antibodies and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for future studies to optimize and better utilize anti-PD-1/L1 therapeutics.

AbstractImmune checkpoint inhibitors (ICIs), as a novel class of anticancer therapy, can be more efficacious and less toxic than chemotherapy, but their clinical success is confined to certain tumor types. Elucidating their targets, mechanisms and scope of action, and potential synergism with chemotherapy and/or targeted therapies are critical to widen their clinical indications. Treatment response to an ICI targeting programmed death‐1 (anti‐PD‐1) is sought to be understood here by conducting a preplanned correlative analysis of a phase II clinical trial in patients with small bowel adenocarcinoma (SBA). The cytolytic capacity of circulating immune cells in cancer patients using a novel ex vivo cytotoxicity assay is evaluated, and the utility of circulating biomarkers is investigated to predict and monitor the treatment effect of anti‐PD‐1. Baseline expression of Bim and NKG7 and upregulation of CX3CR1 in circulating T cells are associated with the clinical benefit of anti‐PD‐1 in patients with SBA. Overall, these findings suggest that the frequency and cytolytic capacity of circulating, effector immune cells may differentiate clinical response to ICIs, providing a strong rationale to support immune monitoring using patient peripheral blood.

Abstract Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Abstract Introduction Central nervous system relapse (secondary central nervous system lymphoma -SCNS) is an uncommon but devastating complication of aggressive B-cell lymphoma. Patients (Pts) with CNS-IPI 4-6 are at greatest risk (10.2% at 2 years). Intravenous high-dose methotrexate (HD-MTX) is widely used to mitigate SCNS risk but data supporting this practice are limited. Methods We performed a multicentre, retrospective study at 21 sites in Australia, Asia, North America and Europe. Chart or registry review was performed for consecutively diagnosed pts with diffuse large B-cell lymphoma (DLBCL) and CNS-IPI 4-6, high grade B-cell lymphoma (HGBL) with rearrangements of MYC+BCL2 and/or BCL6 and primary breast/testicular DLBCL irrespective of CNS-IPI. Pts were diagnosed between 2000-2020, 18-80 years at diagnosis, and treated with curative intent anti-CD20 based chemo-immunotherapy. Pts with CNS involvement at diagnosis were excluded. HD-MTX was defined as at least one cycle of intravenous MTX at any dose. Time to SCNS was calculated from date of diagnosis (all-pts), and from the end of frontline systemic lymphoma therapy, defined as 6x21 days from diagnosis (complete response (CR-pts)), until SCNS, systemic relapse, death, or censoring, whichever came first. Cumulative risk of SCNS was computed using the Aalen-Johansen estimator treating death and systemic relapses as competing events. Adjusted cumulative risks were obtained by using an inverse probability of treatment weighting approach. The average treatment effect was computed as the difference in adjusted 5-year risk of SCNS. Results - 2300 and 1455 pts were included in the all-pts and CR-pts analyses, respectively. Baseline demographics and details of therapy are summarised in Table 1. Except for a predominance of males, pts ≤60 years and pts with ECOG 0-1 in the HD-MTX vs no HD-MTX groups, the demographics and treatments were well balanced. At a median follow up of 5.9 years (range 0.0-19.1) and 5.5 years from diagnosis (range 0.0-18.7), 201/2300 and 84/1455 pts experienced CNS events in the all-pts and CR-pts analyses respectively. For all-pts(n=2300), CNS-IPI was 4-6 in 2052(89.2%), with R-CHOP-like therapy given to 93.8%. 410 pts (17.8%) received HD-MTX (265 HD-MTX alone, 145 in combination with intrathecal methotrexate (IT-MTX);435 received IT-MTX alone;1455 received neither. There were 32/410 and 169/1890 SCNS events, with median time from diagnosis to SCNS of 8.8 and 6.7 months in the HD-MTX and no HD-MTX groups respectively. 5-year OS was 70% (95% CI, 65-76%) and 55% (95% CI 53-57%) in HD-MTX and no HD-MTX groups respectively. There was no difference in the adjusted 5-year risk of SCNS between the HD-MTX and no HD-MTX groups (8.4% vs 9.1%, adjusted hazard ratio [HR] 0.71, p=0.100) (Figure 1). For CR-pts(n=1455), CNS-IPI was 4-6 in 1267(87.0%), with R-CHOP-like therapy given to 93.3%. 284 pts (19.5%) received HD-MTX (170 HD-MTX alone, 114 with IT-MTX);298 received IT-MTX alone;873 received neither. There were 16/284 and 68/1171 SCNS events, with median time from diagnosis to SCNS of 11.0 and 10.3 months in the HD-MTX and no HD-MTX groups respectively. 5-year OS was 74%(95% CI 67-81%) and 75%(95% CI 72-78%) in the HD-MTX groups and no HD-MTX groups respectively (adjusted HR 1.08, p=0.622). There was no difference in the 5-year risk of CNS relapse between the HD-MTX and no HD-MTX groups 5.0% vs 6.0% (adjusted HR 1.03, p=0.903) (Figure 2). Exploratory analysis of the impact of HD-MTX among the highest risk groups CNS IPI 5 (n=368), CNS-IPI 6 (n=59) and CNS-IPI 6 plus all pts with testicular, renal or adrenal involvement (n=349) did not reveal differences in SCNS rates in HD-MTX treated pts. Additional subgroup analyses will be presented at the meeting. Conclusions To our knowledge, this is the largest study of the efficacy of HD-MTX in reducing SCNS events focusing exclusively on high-risk pts. The overall incidence of CNS relapse observed was consistent with previous reports in similar patient cohorts at 9%. The use of HD-MTX did not lower SCNS rates overall or when analysis was confined to CR pts at completion of curative intent therapy to compensate for potential immortal bias associated with HD-MTX therapy. Despite the limitations of the non-randomized and retrospective design, it appears unlikely that HD-MTX is associated with a clinically meaningful reduction in SCNS rates in pts with high risk for SCNS. Figure 1 Figure 1. Disclosures Lewis: AstraZeneca: Consultancy, Honoraria; Novartis: Patents & Royalties: Conference attendance; Janssen: Honoraria, Patents & Royalties: Conference attendance; Roche: Consultancy, Honoraria. Villa: Janssen: Honoraria; Gilead: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria; Seattle Genetics: Honoraria; Celgene: Honoraria; Lundbeck: Honoraria; Roche: Honoraria; NanoString Technologies: Honoraria. Bobillo: F. Hoffmann-La Roche Ltd: Consultancy, Speakers Bureau; Gilead: Speakers Bureau. Ekstroem Smedby: Takeda: Consultancy; Janssen Cilag: Research Funding. Savage: Merck: Consultancy, Honoraria, Other: Institutional clinical trial funding; BMS: Consultancy, Honoraria, Other: Institutional clinical trial funding; AbbVie: Consultancy, Honoraria; Roche: Research Funding; Takeda: Other: Institutional clinical trial funding; Servier: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Astra-Zeneca: Consultancy, Honoraria; Beigene: Other: Institutional clinical trial funding; Genentech: Research Funding. Eyre: Beigene: Honoraria, Research Funding; Incyte: Consultancy; Secura Bio: Consultancy, Honoraria; Janssen: Honoraria; Gilead/KITE: Honoraria, Other: Travel support for conferences, Research Funding, Speakers Bureau; Loxo Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Other: Travel to conferences; AstraZeneca: Honoraria, Research Funding; Roche: Consultancy, Honoraria. Cwynarski: Incyte: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Atara: Consultancy; Celgene: Consultancy; Takeda: Consultancy, Other: travel to scientific conferences, Speakers Bureau; Kite, a Gilead Company: Consultancy, Other: travel to scientific conferences, Speakers Bureau; Janssen: Consultancy, Other: travel to scientific conferences; Roche: Consultancy, Other: travel to scientific conferences, Speakers Bureau; BMS/Celgene: Other: travel to scientific conferences. Stewart: Teva: Honoraria; Sandoz: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Abbvie: Honoraria; Janssen: Honoraria; Roche: Honoraria. Bishton: Gilead: Honoraria, Other: Travel grants; AbbVie: Honoraria, Other: Travel grants; Celgene/BMS: Honoraria, Other: travel grants; Celltrion: Honoraria, Other: Travel grants; Takeda.: Honoraria, Other: Travel grants . Fox: F. Hoffmann-La Roche Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees. Joffe: Epizyme: Consultancy; AstraZeneca: Consultancy. Eloranta: Janssen Pharmaceutical NV: Other: NV. Sehn: Genmab: Consultancy; Novartis: Consultancy; Debiopharm: Consultancy. Manos: Bristol-Myers Squibb: Other: Travel and meetings. Hawkes: Specialised Therapeutics: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Merck Sharpe Dohme: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Antigene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Speakers Bureau; Bristol Myers Squib/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck KgA: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accommodation expenses, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Minson: Novartis: Research Funding; Hoffman La Roche: Research Funding. Dickinson: Celgene: Research Funding; Amgen: Honoraria; Takeda: Research Funding; Gilead Sciences: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, accommodation, expenses, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Øvlisen: Abbvie: Other: Travel expenses. Gregory: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, Speakers Bureau; Janssen: Consultancy; Novartis: Consultancy. Ku: Roche: Consultancy; Antegene: Consultancy; Genor Biopharma: Consultancy. Talaulikar: Roche: Honoraria, Research Funding; Jansenn: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; EUSA Pharma: Honoraria, Research Funding. Maurer: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genentech: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Nanostring: Research Funding. El-Galaly: Abbvie: Other: Speakers fee; ROCHE Ltd: Ended employment in the past 24 months. Cheah: Roche: Consultancy, Honoraria, Other: advisory and travel expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: advisory; MSD: Consultancy, Honoraria, Other: advisory, Research Funding; Gilead: Consultancy, Honoraria, Other: advisory; Ascentage pharma: Consultancy, Honoraria, Other: advisory; Beigene: Consultancy, Honoraria, Other: advisory; AbbVie: Research Funding; Celgene: Research Funding; AstraZeneca: Consultancy, Honoraria, Other: advisory; Loxo/Lilly: Consultancy, Honoraria, Other: advisory; TG Therapeutics: Consultancy, Honoraria, Other: advisory.

Abstract Many tumors exhibit increased incorporation of sialic acids into cell-surface glycans, which impact the tumor microenvironment. Sialic acid immunoglobulin-like lectins (Siglec) are receptors that recognize sialic acids and modulate immune responses, including responses to tumors. However, the roles of individual sialyltransferases in tumorigenesis and tumor growth are not well understood. Here, we examined the sialyltransferase ST8Sia6, which generated α2,8-linked disialic acids that bind to murine Siglec-E and human Siglec-7 and -9. Increased ST8Sia6 expression was found on many human tumors and associated with decreased survival in several cancers, including colon cancer. Because of this, we engineered MC38 and B16-F10 tumor lines to express ST8Sia6. ST8Sia6-expressing MC38 and B16-F10 tumors exhibited faster growth and led to decreased survival, which required host Siglec-E. ST8Sia6 expression on tumors also altered macrophage polarization toward M2, including upregulation of the immune modulator arginase, which also required Siglec-E. ST8Sia6 also accelerated tumorigenesis in a genetically engineered, spontaneous murine model of colon cancer, decreasing survival from approximately 6 months to 67 days. Thus, ST8Sia6 expression on tumors inhibits antitumor immune responses to accelerate tumor growth.

Although the immune checkpoint role of programmed death ligand 1 (PD-L1) has been established and targeted in cancer immunotherapy, the tumor-intrinsic role of PD-L1 is less appreciated in tumor biology and therapeutics development, partly because of the incomplete mechanistic understanding. Here we demonstrate a potentially novel mechanism by which PD-L1 promotes the epithelial-mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) cells by suppressing the destruction of the EMT transcription factor Snail. PD-L1 directly binds to and inhibits the tyrosine phosphatase PTP1B, thus preserving p38-MAPK activity that phosphorylates and inhibits glycogen synthase kinase 3β (GSK3β). Via this mechanism, PD-L1 prevents the GSK3β-mediated phosphorylation, ubiquitination, and degradation of Snail and consequently promotes the EMT and metastatic potential of TNBC. Significantly, PD-L1 antibodies that confine the tumor-intrinsic PD-L1/Snail pathway restricted TNBC progression in immunodeficient mice. More importantly, targeting both tumor-intrinsic and tumor-extrinsic functions of PD-L1 showed strong synergistic tumor suppression effect in an immunocompetent TNBC mouse model. Our findings support that PD-L1 intrinsically facilitates TNBC progression by promoting the EMT, and this potentially novel PD-L1 signaling pathway could be targeted for better clinical management of PD-L1–overexpressing TNBCs.

PurposeTo develop a robust, accurate, and accelerated T1ρ quantification solution for submillimeter in vivo whole‐brain imaging.MethodsA multislice T1ρ mapping solution (MS‐T1ρ) was developed based on a two‐acquisition scheme using turbo spin echo with RF cycling to allow for whole‐brain coverage with 0.8‐mm in‐plane resolution. A compressed sensing–based fast imaging method, SCOPE, was used to accelerate the MS‐T1ρ acquisition time to a total scan time of 3 minutes 31 seconds. A phantom experiment was conducted to assess the accuracy of MS‐T1ρ by comparing the T1ρ value obtained using MS‐T1ρ with the reference value obtained using the standard single‐slice T1ρ mapping method. In vivo scans of 13 volunteers were acquired prospectively to validate the robustness of MS‐T1ρ.ResultsIn the phantom study, the T1ρ values obtained with MS‐T1ρ were in good agreement with the reference T1ρ values (R2 = 0.9991) and showed high consistency throughout all slices (coefficient of variation = 2.2 ± 2.43%). In the in vivo experiments, T1ρ maps were successfully acquired for all volunteers with no visually noticeable artifacts. There was no significant difference in T1ρ values between MS‐T1ρ acquisitions and fully sampled acquisitions for all brain tissues (p‐value > .05). In the intraclass correlation coefficient and Bland‐Altman analyses, the accelerated T1ρ measurements show moderate to good agreement to the fully sampled reference values.ConclusionThe proposed MS‐T1ρ solution allows for high‐resolution whole‐brain T1ρ mapping within 4 minutes and may provide a potential tool for investigating neural diseases.