Discussion

Co-signaling molecules are pivotal in modulating T cell activation, guiding their differentiation, orchestrating effector functions, and influencing T cell survival. These molecules are often strategically positioned alongside TCR molecules at the immunological synapse, becoming activated following the TCR’s interaction with specific peptide-major histocompatibility complex (MHC) complexes presented by antigen-presenting cells (APCs). In an appropriate interaction with TCR signaling, they either amplify or attenuate T cell activation and subsequent responses. This complicated interaction results in a unique expression of costimulatory and co-inhibitory molecules, marked by overlapping yet distinct spatiotemporal patterns [31]. Our understanding of individual co-signaling molecules has significantly deepened, especially regarding their role in various T cell response phases. However, comprehending how these multiple pathways synergize remains an area of limited insight. The fundamental mechanics of T cell co-signaling are critical for developing advanced cosignaling-based immunotherapies. PD-1 expression, in particular, has been validated as an essential and central target when it comes to immune modulator therapies for cancer and an important backbone for future combinatorial strategies [32]. This study was designed to explore the relationship between PD-1 expression and the co-expression of T cell co-signaling molecules, a relationship pivotal to refining co-signaling immunotherapy strategies. For this purpose, we analyzed the mRNA expression patterns of PD-1 and its co-expression with various co-inhibitory and co-stimulatory molecules, utilizing Pearson’s correlation coefficient as our analytical tool.

The foremost observation from our analysis elucidates that the co-expression patterns of PD-1 with both co-inhibitory and costimulatory molecules exhibit considerable heterogeneity across different cancer types. Notably, the coexpression of co-inhibitory molecules demonstrated a dominant presence in melanoma, while that of co-stimulatory molecules was more pronounced in urothelial cancer. In contrast, pancreatic adenocarcinoma presented a more restricted co-expression profile, characterized by a significant correlation of PD1 expression solely with CD28. Intriguingly, ovarian cancer displayed no notable correlations in this regard. This finding highlights the involved and diverse nature of immune responses across various cancer forms, thereby accentuating the critical need for tailored immunotherapy approaches specific to each cancer type. Furthermore, our study reveals a heightened state of immune activation or exhaustion, a characteristic prominently observed in both melanoma and urothelial carcinoma, which are current targets of immunotherapeutic interventions. This discussion will cover more profoundly the distinctions of our findings, mainly focusing on the roles and implications of immune-inhibitory and immuno-stimulatory molecules within the context of these malignancies.

Our investigation revealed a notable correlation in the expression levels of LAG3 and PD-1 in both melanoma and urothelial carcinoma. LAG-3 functions as a surface receptor on activated T cells, primarily binding to MHC-II, although other ligands have been identified as well. This significant correlation could be attributed to the fact that both LAG-3 and PD-1 are co-inhibitory molecules, often indicative of T-cell exhaustion. Prolonged exposure of T cells to antigens, such as in cancer, leads to the activation of various inhibitory receptors, including LAG-3 and PD-1 [33]. These results are consistent with findings suggesting that concurrent blockage of PD-1 and LAG3 can synergistically reverse T-cell exhaustion [34, 35]. In a clinical context, this understanding has been applied in the recent approval of a combination therapy comprising anti-LAG3 relatlimab and antiPD-1 nivolumab, specifically for the treatment of patients with metastatic melanoma [23].

Another important finding from our study is the moderate positive correlation between TIGIT and PD-1. TIGIT, a transmembrane protein receptor, operates as an immunological checkpoint on T and natural killer (NK) cells, facilitated by its cytoplasmic tail containing two immunoreceptor tyrosine-based inhibitory motifs (ITIM). The receptor’s primary ligands, CD155 and CD112, are predominantly expressed on APCs, with nectin-2 emerging as a more recent ligand [36, 37]. This correlation aligns with prior observations where TIGIT expression was linked with PD-1 in various contexts, including mouse tumor models and human non-small cell lung cancer (NSCLC) and colon cancer [38]. This association is further corroborated by findings from Chauvin et al., who reported co-expression of TIGIT and PD-1 in advanced melanoma tumors [39]. The therapeutic potential of targeting TIGIT is emphasized by a recent phase 1 trial, where Tiragolumab, an anti-TIGIT humanized IgG1 monoclonal antibody inhibiting TIGIT-CD155 interaction, showed promising efficacy in patients with immunotherapy-naive NSCLC and esophageal cancer [40].

The phase III KEYVIBE-010 study (NCT05665595), a randomized, double-blind trial, aimed to assess the effectiveness and safety of vibostolimab (Anti-TIGIT) + pembrolizumab (AntiPD1) (MK-7684A) compared to pembrolizumab monotherapy as adjuvant therapy in patients with resected high-risk stage IIB-IV melanoma.

This study’s findings reveal a moderate correlation between TIM-3 and PD-1 expression, specifically in melanoma. TIM-3, recognized as an immunoinhibitory molecule, was first identified on CD4+ Th1 (helper) and CD8+ Tc1 (cytotoxic) T-cells, and its presence was later confirmed on various other cell types [41-46]. The molecule’s primary ligand, galectin-9, initiates the apoptosis of effector T-cells [47, 48]. TIM-3 expression is a marker of T cell exhaustion, frequently observed in chronic infections [49]. This study’s findings are in line with those reported by Fourcade et al. [50], who noted a similar upregulation of TIM-3 in conjunction with PD-1 expression in advanced melanoma patients. This correlation may shed light on potential efficacy observed in preclinical and in vivo models when simultaneously targeting both PD-1 and TIM-3 pathways, as opposed to targeting each pathway individually, in the treatment of malignant tumors [50-53].

In this study, a remarkable correlation was identified between VISTA expression and PD-1 in urothelial carcinoma. VISTA, serving both as a ligand and a receptor on T cells, plays a pivotal role in inhibiting T cell proliferation and fostering the transformation of naive T cells into regulatory T cells [54]. The observed positive correlation can potentially be attributed to VISTA’s structural resemblance to the B7 superfamily, positioning it as a PD1 homolog [55, 56]. The co-expression of PD-1 and VISTA in urothelial carcinoma represents a novel area of research. While the precise nature of this relationship remains challenging to elucidate, it could be linked to the strong association between PDL1, the primary ligand of PD-1, and VISTA. A recent 2022 study proposed that a combination therapy targeting VISTA and PD-L1 might offer a new avenue for enhancing immunosuppression in cancer treatments [57].

CTLA-4 emerged as another immunoinhibitory molecule in our study, displaying a co-expression pattern with PD-1. Considered the godfather of immune checkpoints, CTLA-4 was the first clinically targeted clinical immune checkpoint receptor. It predominantly controls the initial phase of T cell activation, while PD-1 is more involved in regulating T cells within tissues and tumors [58]. The observed association between PD-1 and CTLA-4 expression might be partially attributed to the PD-1 receptor’s classification within the CD28/CTLA-4 family [6]. This study’s findings mirror those of the previous research, which observed parallel expression levels of CTLA-4 and PD-1 in tumor tissues, markedly exceeding those in normal tissues in patients with urothelial carcinoma [59]. Our results support the rationale for combining anti-CTLA4 (ipilimumab) with anti-PD1 (nivolumab) in urothelial carcinoma. This has been demonstrated clinically in the checkmate 032 phase I/II multicenter trial, where nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every three weeks for four doses, followed by nivolumab monotherapy 3 mg/kg every two weeks showed a better objective response rate than nivolumab 3 mg/kg monotherapy every two weeks at a median follow-up of 22 months (38% vs. 26.9%) [60].

In our study, we observed a significant correlation between the expressions of CD27 and CD137, both co-stimulatory molecules, along with PD-1 expression in melanoma and urothelial carcinoma patients. These receptors are part of the tumor necrosis factor (TNF) receptor superfamily, playing crucial roles in immune response modulation. CD27, exclusively located on lymphocytes, enhances immune function and aids in the differentiation of effector T cells [61-63]. CD137, on the other hand, is found in a broader range of immune cells, including T cells, B cells, NK cells, monocytes, and neutrophils. The activation and proliferation of these cells, mediated by CD137, contribute to augmenting the efficacy of anti-tumor therapies [64, 65]. Current early-phase clinical trials are exploring the potential of varlilumab, a CD27 agonist, and utomilumab, a CD137 agonist, either as monotherapy treatments or in combination with anti-PD-1 drugs [49, 66]. A notable example is the ongoing clinical research involving CDX-527, a bispecific antibody. This antibody has demonstrated synergistic effects in mice models, effectively inhibiting PD-1 signaling while robustly triggering CD27-mediated T-cell co-stimulation via PD-L1 crosslinking [67]. This supports our finding of the positive association between CD27 and PD-1. In terms of CD137, our results align with existing studies that have identified a concurrent expression of CD137 and PD-1 on CD8+ tumor-infiltrating lymphocyte (TIL) subpopulations in melanoma [68]. Further validating the interconnection between these co-stimulatory pathways and their role in cancer immunotherapy.

Another notable finding in urothelial carcinoma is that OX40 expression directly correlates with PD-1 expression. OX40, also known as CD134 and a member of the TNF receptor superfamily, is a crucial player in T-cell activation. Its upregulation has been linked to enhanced patient outcomes [69, 70]. This particular association between OX40 and PD-1 in our study presents a contrast to the findings of Zhu et al. (2021) [71]. While Zhu et al. did not specifically focus on the direct co-expression relationship between PD-1 and OX40, their analysis of CD8+ T cells in bladder cancer patients indicated notable differences in the expression levels of CD8+ PD-1+ and CD8+ OX40+ cells. This divergence points out the complexity of immune responses in urothelial carcinoma and highlights the potential for immunotherapeutic targeting based on specific co-expression patterns.

The exploration of urothelial carcinoma in this study uncovered a significant correlation between HVEM, a co-stimulatory molecule, and PD-1 co-expression. HVEM also referred to as TNFRSF14, is a multifaceted TNFR superfamily protein capable of binding to both co-stimulatory and co-inhibitory receptors [72]. This particular correlation between HVEM and PD-1 is a novel discovery, as such an association has not been previously reported in any cancer type, either directly or indirectly.

In our analysis of pancreatic adenocarcinoma, only the expression of CD28 demonstrated a moderate positive correlation with PD-1 expression. This correlation might be related in part to PD-1+ CD8 T cells, which proliferate in response to PD-1 therapy, and express CD28 [73-75].

This research is distinguished by its extensive examination of PD-1 co-expression with a wide array of co-inhibitory and co-stimulatory molecules across four cancer types that are known to vary in terms of the immunogenicity of the TME and the likelihood of response to PD1 blockade. The use of a substantial and real-world clinical and transcriptomic dataset from the Total Cancer Care Protocol and Avatar® project that is active across 18 cancer centers within the U.S. allowed for the analysis of a diverse and robust patient population. This analysis facilitated a deeper understanding of the TME in different cancers, significantly broadening the applicability of our findings. However, the study’s retrospective design and reliance on pre-existing data present certain limitations. This approach may not fully capture the complete spectrum of patient demographics or the diversity of cancer subtypes.

Moreover, the primary focus on transcriptomic data, while valuable, overlooks potential post-transcriptional modifications and proteinlevel interactions that are crucial in cancer development and progression. Consequently, these findings underline the need for future research, both preclinical and prospective clinical studies that can validate and build upon these results. Such studies should aim to determine the clinical impact of immunotherapies designed around specific co-expression patterns. Furthermore, while we used PD1 as a backbone in this study, a more comprehensive analysis looking for dominant immune checkpoints independent of PD1 in certain maliganancies can be conducted. An immersed exploration into post-transcriptional mechanisms and protein interactions within the TME could yield further insights. Additionally, expanding the research to encompass a wider variety of cancers and a more diverse patient demographic would enhance the relevance and impact of the findings, paving the way for more personalized and effective cancer treatments.

In conclusion, melanoma and urothelial carcinoma as more immunogenic tumors reflected a PD-1 plus an immunoinhibitory dominant phenotype. The less immunogenic ovarian and pancreatic carcinomas reflected a trend toward a PD-1 plus an immunostimulatory phenotype. The dominance of the coexpression of co-inhibitory molecules in melanoma and to a certain extent in urothelial carcinoma may reflect the prevalence of late exhausted T cells, as compared to less differentiated T cells in ovarian and pancreatic carcinomas. Further validation of our findings is warranted as these may inform potential combination strategies suggested that may be effective in the tested malignancies. Future directions should focus on preclinical and clinical testing in an effort to validate these findings. This is in addition to expanding this research to include a broader range of cancers and exploring the functional impact of these co-expression patterns within the TME.

Acknowledgments

Author Contributions: Conceptualization, Ahmad Tarhini; Data curation, Ahmad Tarhini, Dale Hedges, Aik Choon Tan, Paulo Rodriguez, Vineeth K. Sukrithan, Aakrosh Ratan, Martin McCarter, John Carpten, Howard Colman, Alexandra Ikeguchi,

Igor Puzanov, Susanne Arnold, Michelle Churchman, Patrick Hwu, Jose Conejo-Garcia, William Dalton, George Weiner and Islam Eljilany; Formal analysis, Ahmad Tarhini and Dale Hedges; Funding acquisition, Ahmad Tarhini; Investigation, Ahmad Tarhini and Islam Eljilany; Methodology, Ahmad Tarhini and Dale Hedges; Project administration, Ahmad Tarhini; Resources, Ahmad Tarhini; Supervision, Ahmad Tarhini; Writing – original draft, Ahmad Tarhini and Islam Eljilany; Writing – review & editing, Ahmad Tarhini, Dale Hedges, Aik Choon Tan, Paulo Rodriguez, Vineeth K. Sukrithan, Aakrosh Ratan, Martin McCarter, John Carpten, Howard Colman, Alexandra Ikeguchi, Igor Puzanov, Susanne Arnold, Michelle Churchman, Patrick Hwu, Jose Conejo-Garcia, William Dalton, George Weiner and Islam Eljilany.

Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (IRB; IRB# Pro00014441) of the Total Cancer Care Protocol (NCT03977402) and Avatar® project.

Informed Consent Statement: Written informed consent was obtained at the participating institutions from all subjects involved in the original study protocol within which this project was nested.

Data Availability Statement: All results relevant to this study are included in the article. Data are available upon reasonable requests to the corresponding author.

Conflicts of Interest: I.E., D.H., M.L.C., J.C., A.P.I.,W.S.D., G.J.W., and P.H. declare no conflicts of interest. V.S. declares receiving research funding from Eli Lilly and Company and Crinetics Pharmaceuticals. Consulting for GE Health. Advisory Board member of Exelixis. Receiving speaking feesfrom Lantheus Pharmaceuticals. M.M. declares contracted research grants with Merck, Taiho, and the National Comprehensive Cancer Network.

IP declares Program Leader (1.2 cal months) for NIH/NCI Cancer Center Support Grant (P30CA016056); consulting with Nouscom, Iovance, Nektar, and Regeneron; stock owner with Ideaya, Inc. H.C. declares advisory board/consultant: Best Doctors/Teladoc, Orbus Therapeutics, Bristol Meyers Squibb, Regeneron, Novocure, and PPD/Chimerix; research funding (site PI/institutional contract):

Orbus, GCAR, Array BioPharma, Karyopharm Therapeutics, Nuvation Bio, Bayer, Bristol Meyer Squib, and Sumitomo Dainippon Pharma. To date, S.M.A. is a member of ASCO COI. I.M.E.N. is on the scientific advisory board of Endectra, L.L., acts as deputy editor for the Journal of Medical Physics, and receives funding from NIH and DoD. J.R.C.G has stock options in Compass Therapeutics, Anixa Biosciences and Alloy Therapeutics; receives consulting fees from Alloy Therapeutics; has intellectual property with Compass Therapeutics and Anixa Biosciences; receives licensing fees from Anixa Biosciences; and is co-founder of Cellepus Therapeutics.A.A.T.declares contracted research grants with institution from Bristol Myers Squib, Genentech-Roche, Regeneron, Sanofi-Genzyme, Nektar, Clinigen, Merck, Acrotech,

Pfizer, Checkmate, OncoSec, Scholar Rock, InflaRx GmbH, Agenus; personal consultant/advisory board fees from Bristol Myers Squibb, Merck, Easai, Instil Bio, Clinigin, Regeneron,

Sanofi-Genzyme, Novartis, Partner Therapeutics, Genentech/ Roche, BioNTech, Concert AI, AstraZeneca outside the submitted work.

Funding: This work was supported by an ORIEN OUNDATION NOVA (New Oncologic Visionary Award) Grant and Community Foundation of Tampa Bay: 69-21295-01-01 (PI, A. Tarhini). Also, partly supported by Moffitt Cancer Center National Institute of Health (NIH) and  National Cancer Institute (NCI) Center Core Grant (P30) 5P30CA076292-22. AT

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