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Abstract: Herpes simplex virus 1 infection of the eye can result in stromal keratitis, a chronic immunoinflammatory lesion that is a significant cause of human blindness. A key to controlling the severity of lesions is to identify cellular and molecular events responsible for tissue damage. This report evaluates the role of lymphotoxin-α, a proinflammatory cytokine that could be involved during stromal keratitis. We demonstrate that after infection, both lymphotoxin-α and lymphotoxin-β transcripts are detectable at high levels 48 h postinfection, suggesting roles for the secreted homotrimer lymphotoxin-α3 and the membrane-bound lymphotoxin-α1β2 heterotrimer in stromal keratitis. Using a corneal stromal fibroblast cell line, lymphotoxin-α3 and lymphotoxin-α1β2 were found to have proinflammatory roles by stimulating production of chemokines. Treatment of mice with a depleting anti-lymphotoxin-α mAb during the clinical phase of the disease significantly attenuated stromal keratitis lesions. In treated mice, expression of proinflammatory molecules and chemokines was reduced, as were numbers of cornea-infiltrating proinflammatory cells, particularly Th1 cells. The protective effect of anti-lymphotoxin-α mAb was highly reduced with a mutant version of the mAb that lacks Fc receptor binding activity, indicating that depletion of lymphotoxin-expressing cells was mainly responsible for efficacy, with LT-α3 contributing minimally to inflammation. These data demonstrate that lymphotoxin-expressing cells, such as Th1 cells, mediate stromal keratitis. [Copyright &y& Elsevier]

Abstract: Tumor necrosis factor-superfamily (TNF-SF) members, lymphotoxin (LT)-α and LTβ, are proinflammatory cytokines associated with pathology in rheumatoid arthritis. LTα3 homotrimers are secreted, whereas LTα1β2 heterotrimers are expressed on the surface of activated lymphocytes. As many TNF-SF members are actively cleaved from cell membranes, we determined whether LTαβ heterotrimers are also cleaved, and are biologically active in rheumatoid arthritis (RA) patients. LTαβ heterotrimers were detected in culture supernatants from activated human T-helper (Th) 0, Th1, and Th17 cells, together with LTα3 and TNFα. The heterotimers were actively cleaved from the cell surface by ADAM17 metalloproteinase (MMP) and MMP-8, and cleavage was inhibited by TAPI-1, a TNF-α converting enzyme (TACE) inhibitor. Soluble LTαβ was detected in serum from both normal donors and RA patients, and was elevated in synovial fluid from RA patients compared to osteoarthritis (OA) patients. Levels of LTαβ in RA patient synovial fluid correlated with increased TNFα, IL-8, IL-12, IL-1β, IFN-γ, and IL-6 cytokines. Moreover, recombinant LTα1β2-induced CXCL1, CXCL2, IL-6, IL-8, VCAM-1, and ICAM-1 from primary synovial fibroblasts isolated from RA patients. Therefore, soluble LTαβ in synovial fluid is associated with a proinflammatory cytokine milieu that contributes to synovitis in RA. [Copyright &y& Elsevier]

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic. [Display omitted] • CD226 is required for response to anti-PD-(L)1 or anti-TIGIT antibody treatment • Tumor-infiltrating lymphocytes differentially express CD226 and CD28 • PD-1 and TIGIT impair CD226 phosphorylation through distinct mechanisms • Convergence of PD-1 and TIGIT to inhibit CD226 provides rationale for dual blockade Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Banta et al. find that PD-1 and TIGIT disrupt activation of the costimulatory receptor CD226 through distinct mechanisms, providing mechanistic rationale for the dual blockade of PD-(L)1 and TIGIT in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Blockade of immune checkpoints PD-1 and TIGIT has demonstrated activity in mouse tumor models and human patients with cancer. Although these coinhibitory receptors can restrict signaling in CD8 + T cells by regulating their associated co-stimulatory receptors CD28 and CD226, the functional consequences of combining PD-1 and TIGIT blockade remain poorly characterized. In mouse tumor models, we show that combination blockade elicited CD226-driven clonal expansion of tumor antigen-specific CD8 + T cells. The expanded clones emerged from a population of stem-like cells in draining lymph nodes, entering the blood as a previously unidentified single-phenotype, multiclonal population. Upon reaching the tumor, these transiting cells expanded further and differentiated into effector or exhausted T cells, with combination blockade restricting entry into the exhaustion pathway by favoring co-stimulation. Thus, PD-1 and TIGIT inhibition helps shape the repertoire of tumor-reactive CD8 + T cells in draining lymph nodes and determines their immunological fate in the tumor to enhance therapeutic benefit. Analysis of clinical trial samples suggests a similar mechanism may also occur in patients with cancer., Competing Interests: Competing interests: The authors declare the following competing interests: all authors are employees of Genentech, a member of the Roche group, which develops and markets drugs for profit., (© 2024. The Author(s).)

An insufficient quantity of functional T cells is a likely factor limiting the clinical activity of T-cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T-cell dependent (TDB) antibodies. The activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in the upregulation of the IL-2/15Rβ (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to IL-15. XmAb24306 enhanced T-cell bispecific antibody-induced CD8+ and CD4+ T-cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T-cell numbers were normalized, suggesting that T-cell expansion is the main mechanism of the observed benefit. Pretreatment of immunocompetent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in a significant increase of T cells in the blood, spleen, and tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis that the number of tumor-infiltrating T cells is rate limiting for the activity of solid tumor-targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T-cell bispecific antibodies support clinical evaluation of this novel immunotherapy combination., (©2024 American Association for Cancer Research.)

Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities., Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials., Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range ∼1.0-4.1 days) compared to wild-type IL-15 (half-life ∼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1 + lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data., Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine., Competing Interests: RY, PH, EC, SS, AS, ML, ASe, PC, YL, LJ, IH, ES, AK, and VS were employees of Genentech Inc., and hold stock in the company. SS, NK, LB, IL, RR, KA, CB, JD, MB are employees of Xencor Inc., and hold stock in the company., (Copyright © 2024 Yadav, Schubbert, Holder, Chiang, Kiabi, Bogaert, Leung, Rashid, Avery, Bonzon, Desjarlais, Sanjabi, Sharma, Lepherd, Shelton, Chan, Liu, Joslyn, Hosseini, Stefanich, Kamath, Bernett and Shivva.)

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone 1 . However, there remains little consensus on the mechanism(s) of response with this combination 2 . Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8 + T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]., Methods: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8 +  tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data., Results: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (K D ) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound  89 Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89 Zr immuno-PET reagents., Conclusion: 89 Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Recent advances in understanding the roles of immune checkpoints in allowing tumors to circumvent the immune system have led to successful therapeutic strategies that have fundamentally changed oncology practice. Thus far, immunotherapies against only two checkpoint targets have been approved, CTLA-4 and PD-L1/PD-1. Antibody blockade of these targets enhances the function of antitumor T cells at least in part by relieving inhibition of the T cell costimulatory receptor CD28. These successes have stimulated considerable interest in identifying other pathways that may bte targeted alone or together with existing immunotherapies. One such immune checkpoint axis is comprised of members of the PVR/nectin family that includes the inhibitory receptor T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT). Interestingly, TIGIT acts to regulate the activity of a second costimulatory receptor CD226 that works in parallel to CD28. There are currently over two dozen TIGIT-directed blocking antibodies in various phases of clinical development, testament to the promise of modulating this pathway to enhance antitumor immune responses. In this review, we discuss the role of TIGIT as a checkpoint inhibitor, its interplay with the activating counter-receptor CD226, and its status as the next advance in cancer immunotherapy., Competing Interests: Competing interests: EYC and IM are employees of Genentech, a member of the Roche group, which develops and markets drugs for profit., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8 + T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8 + T cells. Crosslinking of CD96 on human or mouse CD8 + T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8 + T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8 + T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8 + T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8 + T cell activation and effector function., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL1 1 , the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies., (© 2019 Husain et al.)

Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity., (Published by Elsevier Inc.)

Voltage-gated Kv1.3 and Ca 2+ -dependent KCa3.1 are the most prevalent K + channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.

Immunotherapies that harness the activity of the immune system against tumors are proving to be an effective therapeutic approach in multiple malignancies. Indeed, through accumulation of genetic mutations, many tumors express antigens that can potentially elicit specific tumor immunity. However, tumors can also suppress these responses by activating negative regulatory pathways and checkpoints such as PD-1/PD-L1 and CTLA-4. Blocking these checkpoints on T cells has provided dramatic clinical benefit, but only a subset of patients exhibit clear and durable responses, suggesting that other mechanisms must be limiting the immune response. We discuss here the role of TIGIT, an inhibitory receptor expressed by lymphocytes, in limiting antitumor responses and we review its mechanisms of action during the cancer immunity cycle., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The orbits of giant extrasolar planets often have surprisingly small semimajor axes, large eccentricities, or severe misalignments between their orbit normals and their host stars' spin axes. In some formation scenarios invoking Kozai-Lidov oscillations, an external planetary companion drives a planet onto an orbit having these properties. The mutual inclinations for Kozai-Lidov oscillations can be large and have not been confirmed observationally. Here we present evidence that observed eccentric warm Jupiters with eccentric giant companions have mutual inclinations that oscillate between 35° and 65°. Our inference is based on the pairs' observed apsidal separations, which cluster near 90°. The near-orthogonality of periapse directions is effected by the outer companion's quadrupolar and octupolar potentials. These systems may be undergoing a stalled version of tidal migration that produces warm Jupiters over hot Jupiters, and they provide evidence for a population of multiplanet systems that are not flat and have been sculpted by Kozai-Lidov oscillations., (Copyright © 2014, American Association for the Advancement of Science.)

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1β2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1β2 trigger signaling via LTβ Receptor (LTβR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα1β2 possesses two binding sites for LTβR with distinct affinities and that dimerization of LTβR by LTα1β2 is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα1β2, LTβR, and the fab fragment of an antibody that blocks LTβR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα1β2 reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTβR.