Supplementary Figure 5. TCR-T cells lack recognition of normal tissue cell lines.

Abstract FOXM1 is highly expressed in various cancer types and considered a key driver of cancer progression. Accordingly, we evaluated the immunogenicity of FOXM1 and investigated the feasibility of targeting this transcription factor using T-cell receptor (TCR) engineering. We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously processed and presented, and resulted in T-cell activation and cytotoxic T-cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose–response and X-scan, respectively. Most importantly, adoptive transfer of TCR-engineered T cells led to a significant reduction in tumor growth, as well as significantly prolonged survival in a tumor-bearing immunocompromised murine model. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this antigen using TCR engineering.

Supplementary Figure 2. FOXM1 epitopes are eluted on H1975.

Microbial immunotherapies show promise against cancer, yet broad efficacy and mechanistic insight remain elusive. Here, we introduce SPIKE 1.0 (S1.0), a metabolically engineered bacterium that converts tryptophan into immunomodulatory hydroxyindoles via tryptophan monooxygenase to remodel the tumor microenvironment (TME). A single systemic dose of S1.0 elicited potent, durable antitumor responses across multiple murine models, including humanized mice, with minimal toxicity. S1.0 enhanced inflammatory signaling, activated innate and adaptive immunity, and promoted T cell persistence, memory, and resistance to exhaustion. It outperformed checkpoint inhibitors and synergized with chemotherapy. Multi-omics profiling revealed that S1.0 rewired amino acid metabolism in tumor-infiltrating immune cells and disrupted immunosuppressive networks. These results establish S1.0 as a scalable, cost-effective microbial immunotherapy with broad translational potential for solid tumors.

FOXM1 is highly expressed in various cancer types and considered a key driver of cancer progression. Accordingly, we evaluated the immunogenicity of FOXM1 and investigated the feasibility of targeting this transcription factor using T-cell receptor (TCR) engineering. We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously processed and presented, and resulted in T-cell activation and cytotoxic T-cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose-response and X-scan, respectively. Most importantly, adoptive transfer of TCR-engineered T cells led to a significant reduction in tumor growth, as well as significantly prolonged survival in a tumor-bearing immunocompromised murine model. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this antigen using TCR engineering.

Supplementary Figure 7. FOXM1-specific TCR-T do not lead to toxicity in vivo.

UV radiation (UVR) drives high mutational burdens, yet precursor melanocytes accumulate these mutations without triggering immune clearance. Here, we investigated whether melanocyte-intrinsic transcriptional program(s) underlie immune tolerance to mutations resulting from UVR exposure. In primary human melanocytes, expression of PD-L1 (CD274) was dependent on microphthalmia-associated transcription factor (MITF), a crucial regulator of melanocyte development and an intermediate in the UV-tanning pathway. MITF directly activated PD-L1 transcription by binding a conserved upstream enhancer containing functional E-box elements. MITF determined both baseline melanocytic PD-L1 expression in healthy skin and its induction following UVR, independent of interferon signaling. Melanocyte-restricted Pd-l1 deletion in mice triggered CD8+ T cell infiltration and depigmentation after long-term UVB exposure, recapitulating features of human vitiligo. PD-L1-deficient human induced pluripotent stem cell (iPSC)-derived melanocytes underwent increased apoptosis and were more susceptible than PD-L1-intact melanocytes to gp100-specific CD8+ T cell killing. Thus, a melanocyte-intrinsic MITF-PD-L1 tolerance program protects melanocytes from autoimmune destruction, potentially facilitating early immune evasion during melanoma development and conversely underlying the responsiveness of melanoma to PD-1/PD-L1 blockade.

Cell-based immune therapies ranging from CAR-T cells to tumor infiltrating lymphocytes (TILs) and endogenous T-cell products, have produced unprecedented clinical responses in hematologic malignancies and are currently under active investigation for solid tumors. Nevertheless, several key challenges continue to limit the durability and breadth of clinical benefit. IL-7 is a pleiotropic cytokine that increases both the number and function of lymphocytes. Although not yet clinically approved, IL-7 has been used in over 620 adult and pediatric patients for a variety of reasons including, for example, to hasten bone marrow recovery after allogenic stem cell transplantation, to reverse lymphopenia due to HIV and idiopathic etiologies, to treat patients with various malignancies, and to boost vaccine responses. IL-7 is generally well-tolerated and effective in producing a durable increase in the number and function of CD4 and CD8 T cells. Recently, IL-7 has been used clinically in multiple myeloma patients receiving CAR-T cell therapy, in patients with urothelial cancer who are receiving checkpoint inhibitors, in patients undergoing endogenous lymphocyte cell therapy, and in critically-ill lymphopenic patients with COVID-19. The authors, all of whom have used IL-7 clinically, discuss how IL-7 effectively addresses all the major problems currently limiting adoptive cell therapies. Peering into the future, we believe that IL-7 will be a major advance as an adjuvant treatment in many cell therapies and hope that this commentary will expedite IL-7’s testing in multiple clinical settings.

Supplementary Table 1. Predicted peptides with corresponding HLA alleles.

Supplementary Figure 6. FOXM1-restricted TCR-T cells are specific.

Oncogenic KRAS mutations drive a substantial proportion of lung cancers and are linked to poor prognosis, positioning KRAS as a compelling target for cellular immunotherapy. Thirty-five percent of lung adenocarcinomas harbor the KRAS G12C, G12V, G12D mutations. Here, we developed HLA-A*03:01- and HLA-A*11:01-restricted T cell receptors (TCR) targeting the most prevalent G12C and G12V KRAS hotspot mutations in lung adenocarcinoma. Predicted high affinity peptides were screened using our TCR discovery and validation pipeline, and functional assessment was performed to determine sensitivity, specificity, and cytotoxic potential of TCR-engineered T cells. We discovered and validated 5 novel TCRs targeting KRAS G12C and G12V 9-mers, each of which demonstrated an ability to recognize and lyse tumor cells endogenously presenting mutant KRAS on HLA-A*03:01 or HLA-A*11:01. Notably, several TCRs demonstrated distinct modes of cross-reactivity, including peptide degeneracy across KRAS G12 variants, HLA degeneracy across HLA-A*03:01 and HLA-A*11:01, or dual degeneracy across both KRAS and HLA, thereby broadening the treatable target populations. TCRs that recognize the KRAS hotspot shared sequence motifs were found in several lung cancer patients. Our study highlights the successful generation of multi-valent KRAS-specific TCRs and supports the feasibility of targeting shared KRAS neoantigens through TCR engineering in lung cancer.

Supplementary Figure 1. FOXM1 is minimally expressed in healthy organs.

T cells have important functions in development and disease processes through T cell receptor (TCR)-dependent activities. Many tools were developed to predict the binding between TCRs and antigens. However, one of the uncertainties is whether such tools can decipher how small changes in the TCRs or antigenic peptides contribute to binding. We develop a deep learning model, pMTnet-omni, which not only predicts the binding vs. non-binding of TCRs towards pMHCs, but also distinguishes the stronger vs. weaker binding of TCRs similar in sequence. We leverage this capability to interpret the biological rules that govern TCR-antigen pairing. This also enables pMTnet-omni to accurately predict variant TCRs with desired stronger or weaker binding to the antigen, in conjunction with a Lab-in-the-Loop (LiL) mechanism. We show that pMTnet-omni can also predict binding of TCRs towards similar pMHCs. Overall, we provide a flexible toolkit for research and translational applications involving antigens and TCRs.

B cells constitute ∼15% to 20% of tumor-infiltrating lymphocytes in melanoma. Their presence in the tumor microenvironment correlates with improved survival and enhanced response to immune checkpoint blockade therapy. Yet, the functional contribution of B cells to melanoma immunity remains unclear. In this study, we showed that both genetic and antibody-mediated B cell depletion significantly promoted melanoma progression in mice. Immune profiling revealed that, although B cell percentages were reduced, IL-10-producing B regulatory cells (Bregs) persisted after depletion. However, the persistence of Bregs alone cannot explain the impact of B cell depletion on enhancing melanoma growth, as codepletion of B cells and CD4+ T cells, despite similar Breg levels, did not promote melanoma progression. B cell depletion also resulted in the accumulation of PD-1+ B cells, CD4+ T cells, and monocytic myeloid-derived suppressor cells into the tumor microenvironment, alongside a reduction in IFN-γ+CD8+ T cells, CXCL13+CD8+ T cells, and M1-like macrophages. Notably, plasma cell deficiency did not affect tumor growth, indicating that B cell-mediated antitumor activity is independent of antibody production. The tumor-promoting effect of B cell loss was at least partially CD4+ T cell dependent, as codepletion of B cells and CD4+ T cells reversed this phenotype and B cell depletion did not enhance tumor growth in Nu/Nu mice lacking mature T cells. Taken together, our findings reveal an antitumor role of B cells in melanoma and demonstrate that their loss promotes tumor progression through reprogramming of the tumor immune microenvironment.

CD8+ T cell-mediated cytotoxicity classically occurs through engagement of an alpha-beta T cell receptor (TCRαβ) with a peptide-class I major histocompatibility complex (pMHC). However, it is also known that cytotoxic CD8+ T lymphocytes (CTLs) can kill tumor cells in a pMHC-independent manner. The relative physiologic contribution and biological significance of pMHC-independent CTL killing remain unclear, and a receptor shared between CTLs and natural killer (NK) cells is generally invoked as the mechanism by which this occurs. In this study, we used acute myeloid leukemia (AML) as a model to examine mechanisms of pMHC-independent cytotoxicity and found a paradoxical TCR-dependent, MHC-independent mechanism that requires CD64. Utilizing knockouts of potential AML ligands and CTL receptors, we demonstrate that pMHC-independent cytotoxicity is a potent mechanism of CTL-mediating killing of AML cells and is largely restricted to CD64-expressing cells through an IFNγ-regulated process. Notably, we found that pMHC-independent CTL killing is not due to activation of commonly implicated NK activating receptors but rather requires an activated TCRαβ/CD3 complex. Thus, we identify a CD64-dependent, pMHC-independent, TCR-dependent mode of CTL cytotoxicity that appears highly enriched for in AML.

Supplementary Figure 9. FOXM1 TCR-T cells are detectable in the periphery at time of sacrifice.

Cellular therapies have revolutionized the treatment of hematologic malignancies and are now emerging as potentially promising interventions for solid tumors. While considerable learnings can be leveraged from hematologic applications to inform the development of cell therapies in solid tumors, a number of biological and operational challenges-such as target-antigen heterogeneity; off-tumor target-mediated toxicity; immunosuppressive microenvironment; limited trafficking and persistence; tissue accessibility; and significantly larger patient populations-necessitate innovative clinical, manufacturing, regulatory, and operational strategies to deliver effective cell therapies for solid tumors. This white paper, informed by the 2025 Summit on Advancing Cell Therapy for Solid Tumors-co-sponsored by American Society for Transplantation and Cellular Therapy (ASTCT) and Society for Immunotherapy of Cancer (SITC)-proposes a framework for cellular therapy development in solid tumors, with an emphasis on logistical, operational, and regulatory considerations unique to this setting. The paper lays out an innovative, collaborative operational model to leverage collective experience and knowledge; emphasize standardization; invest in both prospective and retrospective banking of materials; engage in regulatory data-driven recalibration; engage payers before therapy begins; and establish regional manufacturing hubs and tailored accreditation pathways to support scalability and quality assurance. Operational innovations that can streamline access include hub-and-spoke clinical networks-where a central specialized facility is connected to smaller, more accessible locations-as well as early patient referrals. To safely implement new therapies, it is essential for providers to undergo enhanced training to manage cellular therapy-specific and delayed toxicities. Regulatory recalibrations-including streamlined evaluation of long-term follow-up requirements and proactive payer engagement for comprehensive reimbursement-are equally vital. Finally, cell therapy experts must lead cross-disciplinary education to ensure equitable and safe access as indications for cellular therapy expand across solid-tumor types, autoimmune diseases, and other disease types. Collaborative efforts across clinical, operational, regulatory, and policy initiatives are vital in order to unlock the full potential of cellular therapy for diverse patient populations.

Clinical trials of adoptive cellular therapy demonstrate that a key characteristic associated with durable responses is in vivo expansion and persistence of transferred T cells. Strategies to develop a less differentiated, stem/memory population in the infusion product and peri-infusional regimens to promote the maintenance of desired T cell states following adoptive transfer would be desirable. Endogenous T cell therapy studies have routinely achieved memory T cells enriched for expression of interleukin (IL)-7 receptor; to eliminate the conventional requirement for immunosuppressive lymphodepletion and its attendant life-threatening toxicities, we performed the first-in-human use of IL-7 in combination with adoptively transferred antigen-specific memory CD8 T cells in a patient with refractory metastatic uveal melanoma. Single-cell immune repertoire profiling of serial peripheral blood sampling revealed substantial in vivo proliferation and expansion of a stem cell memory population in the endogenous T cell therapy product that achieved a >79% predominance of total circulating T cells by 3 weeks post-infusion in this non-lymphodepleted recipient. Although the patient’s disease ultimately progressed, these findings demonstrate safety and proof of concept for an IL-7 treatment regimen for expansion of adoptively transferred T cells in vivo and induced memory differentiation in a heavily pretreated patient with refractory solid malignancy.

Supplementary Figure 4. FOXM1-specific TCR-T sustain Tem phenotype while upregulating checkpoint molecules upon antigen exposure.