Valentin Voillet’s research while affiliated with Fred Hutch Cancer Center and other places

Adoptive therapies using T cells genetically modified with T cell receptors (TCR)s have shown limited efficacy in the solid tumor setting. Although functional CD4 ⁺ and CD8 ⁺ T cells transduced with a TCR specific for HLA-A2-restricted melanoma-associated antigen A1 (MAGE-A1, T TCR−MA1−CD8αβ ) could be detected post-transfer and were safe in one patient who subsequently progressed, T TCR−MA1−CD8αβ were insufficient to sustain antitumor activity in “stress” mouse tumor models. Leveraging the obligate co-expression of CD8αβ required for engagement of CD4 ⁺ T cells expressing the TCR, we screened positive co-stimulatory signals tethered to the intracellular tail of CD8β and identified that CD28 reduced exhaustion, enhanced tumor infiltration and improved murine tumor control. Further modifications of the CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models. Thus, integrating co-stimulatory signals downstream of the TCR signaling complex can enhance TCR-engineered T cell function, independent of tumor-associated co-stimulatory ligand expression.

Acute myeloid leukemia (AML) that is relapsed and/or refractory post-allogeneic hematopoietic cell transplantation (HCT) is usually fatal. In a prior study, we demonstrated that AML relapse in high-risk patients was prevented by post-HCT immunotherapy with Epstein-Barr virus (EBV)-specific donor CD8 ⁺ T cells engineered to express a high-affinity Wilms Tumor Antigen 1 (WT1)-specific T-cell receptor (TTCR- C4). However, in the present study, infusion of EBV- or Cytomegalovirus (CMV)-specific T TCR-C4 did not clearly improve outcomes in fifteen patients with active disease post-HCT. TCRC4-transduced EBV-specific T cells persisted longer post-transfer than CMV-specific T cells. Persisting T TCR-C4 skewed towards dysfunctional natural killer-like terminal differentiation, distinct from the dominant exhaustion programs reported for T-cell therapies targeting solid tumors. In one patient with active AML post-HCT, a sustained T TCR-C4 effector-memory profile correlated with long-term T TCR-C4 persistence and disease control. These findings reveal complex mechanisms underlying AML-induced T-cell dysfunction, informing future therapeutic strategies for addressing post-HCT relapse.

Understanding acute myeloid leukemia (AML)-specific mechanisms of T cell dysfunction is key to improving T cell therapies. In solid tumors, reduced T cell persistence commonly links to the expression of exhaustion markers (PD1, CTLA4, TIM3, LAG3, TIGIT) targetable with checkpoint blockade. In AML, the presence of T cell exhaustion has been contested (Penter et al., Blood 2023), highlighting alternative T cell dysfunction mechanisms based on terminal differentiation of CD8+ T cells expressing NK-like (NKL) markers (Abbas et al., Nat Commun. 2021; Mazziotta et al., Blood 2024). Whether AML induces this skewing of antigen-specific T cells remains an open question. Wilms' Tumor 1 (WT1) protein is overexpressed and correlates with poor prognosis in high-risk AML. In a phase I/II clinical trial, we employed T cells engineered to express a high-affinity WT1-specific T cell receptor (TCRC4) to treat 15 HLA-A2+ patients with relapsed/refractory AML after allogeneic transplant. To mitigate graft-versus-host disease (GVHD) from the endogenous TCR, we used EBV or CMV-specific CD8+ T cells obtained from HLA-matched donors. This framework enabled us to longitudinally monitor post-transfer AML-specific T cells via high-dimensional multimodal approaches. We studied the impact of virus-specificity on persistence, finding higher persistence (p < 0.05) for EBV vs. CMV-specific TTCR-C4. To directly compare CMV and EBV-specific T cells, we reanalyzed mass cytometry data (Schmidt et al, Cell Rep 2023) from healthy individuals and others with non-AML cancers (n = 143). Differential abundance analysis showed increased (FDR < 0.05) terminally differentiated (Temra) CMV vs. EBV-specific CD8+ T cells suggesting that CMV-specific TTCR-C4 cells were affected by the substrate cell's terminal differentiation, compromising post-infusion persistence. Disease status at TTCR-C4 infusion also affected persistence, with lower persistence in patients with minimal residual disease (MRD)+ or overt AML vs. MRD- patients. To study TTCR-C4 post-transfer, we built a 24-color spectral flow-cytometry panel for peripheral blood samples at ~1, ~7, ~28 days, and 4 months post-transfer. Unsupervised clustering and differential abundance analysis showed TTCR-C4's progressive differentiation from proliferative (Ki67+) effector to non-proliferative (Ki67-) Temra cells with cytotoxic/NKL markers (CD45RA, CD57, KLRG1, GNLY) (p <0.05). This shift correlated with a drop in IFNg and TNF⍺ producing TTCR-C4, associating the NKL shift with T cell dysfunction. Single-cell RNA sequencing (scRNAseq) corroborated these results at the transcriptional level. Principal component analysis, trajectory, and velocity inference methods positioned TTCR-C4 between effector memory/activated and NKL/Temra cells in the CD8+ T cell differentiation spectrum. However, when comparing timepoints with detectable (+) (n = 9) and non-detectable (-) (n = 6) AML, we found that TTCR-C4 overexpressed NKL markers in AML+, suggesting that AML accelerates NKL/terminal differentiation in antigen-specific cells. To verify, we rechallenged CD8+ WT1-specific T cells in an in-vitro model with WT1+ HLA-A2+-transduced K562 cells every 3-4 days. By day 13, T cells lost control of K562 cells and skewed to an NKL transcriptional profile (bulk-RNAseq). To further prove that T cells do not undergo T cell exhaustion in AML vs. solid tumors, we reanalyzed AML, melanoma, pancreatic and lung cancer scRNAseq independent datasets documenting exhaustion as a typical cell state in solid tumors but not in AML. Data from one AML patient suggested that azacitidine treatment can prevent TTCR-C4 terminal differentiation, likely increasing persistence. Another patient exhibited blasts with a myeloid derived suppressor cells transcriptional profile which became treatment-resistant, consistent with previous findings (Van Galen et al., Cell 2019). Killing assays showed that using a higher affinity TCR and co-targeting WT-1 with CD4+ and CD8+ T cells, can overcome this immune escape. In conclusion, we demonstrate that AML-specific T cell dysfunction is driven by NKL skewing rather than T cell exhaustion. Our findings inform strategies that can be developed to target T cell dysfunction and overcome immune escape in TCR-T cell therapies for AML patients.

TCR-T cell therapy using T cells engineered to express high-affinity receptors targeting Wilms Tumor Antigen1 (WT1), known as TTCR-C4, has shown promising results in the treatment of AML (Chapuis A et al., Nat Med 2019). However, immune-escape of tumor cells remains one of the biggest obstacles to successful cell therapy. We previously described, a mechanism of immune escape characterized by a change in the proteosomal machinery compromising antigen presentation (Lahman M et al., Sci Transl Med 2022). Here, we used single-cell RNA sequencing to study a patient exhibiting long-term (> 100 days post-infusion) TTCR-C4 persistence who experienced relapse post-infusion. Leveraging the sex mismatch between the male donor and female recipient, we tracked the transcriptome of blasts before infusion and at relapse. Specifically, we used the female-specific gene XIST and the male-specific gene RPS4Y1 and employed the marker-based purification algorithm scGate (Andreatta M et al., Bioinformatics 2022) to precisely identify the blasts. Furthermore, we identified healthy myeloid cells, endogenous CD8+ T cells, and TTCR-C4 cells. In line with clinical reports, blasts were detected before TTCR-C4 infusion and at day 178 post-infusion. Notably, the number of TTCR-C4 progressively declined from day 21 to day 178. Further analysis of TTCR-C4 transcriptional changes revealed the expression of genes associated with mitochondrial fitness and energy production at day 178, suggesting activation of the mitochondrial machinery in response to the presence of blasts. TTCR-C4 cell gene-set variation analysis showed persistence of immune-response (interferon-gamma response) and mitochondrial activity (oxidative phosphorylation) but also revealed enriched signatures of apoptosis, hypoxia, and inflammation at day 178. These findings suggested that although TTCR-C4 could mount an immune response against the blasts, a potential change in leukemia hampered their response and induced TTCR-C4 apoptosis and decline. Further examination of the blasts' transcriptomes before infusion and at day 178 showed that blasts skewed towards a monocytic phenotype, expressing markers such as CD14, CCR2, and ITGAM. Prior studies (Van Galen et al., Cell 2019) have indicated that AML monocytic myeloid-derived suppressor cell (MDSC) skewing can impair T-cell responses and promote leukemic cell survival. Our analysis revealed higher expression of most MDSC genes at day 178 compared to the pre-infusion timepoint, indicating a skewing of blasts towards an MDSC phenotype. To further support these findings, we analyzed an independent bulk-RNAseq dataset from diagnostic samples of the BeatAML2 cohort (Bottomly et al., Cancer Cell 2022). Using a manually curated gene signature of monocytic MDSCs, we employed a Cox's proportional hazard model with lasso penalty to select informative genes. The resulting gene signature score, consisting of nine genes involved in mechanisms of immunosuppression and tumor survival, predicted overall survival, consistent with previous data indicating that MDSC genes adversely affect overall survival in AML patients. Finally, we explored in-vitro strategies to control this mechanism of escape using a TCR with higher affinity for WT1 compared to TTCR-C4, and using engineered CD4 and CD8 against the same target (WT1). In conclusion, this case report highlights the skewing of AML blasts towards a monocytic myeloid-derived suppressor cell phenotype as a potential mechanism of immune escape. The findings underscore the need for more potent cell therapies able to prevent immune-escape and address blast heterogeneity.

HIV causes susceptibility to respiratory pathogens, including tuberculosis (TB), but the underlying immunological mechanisms remain incompletely understood. We obtained whole blood and bronchoalveolar lavage (BAL) from TB-exposed people in the presence or absence of antiretroviral-naïve HIV co-infection. Bulk transcriptional profiling demonstrated compartment-specific enrichment of immunological processes. Systems-level deconvolution of whole blood from people living with HIV identified elevated type I and type II interferon cytokine activity and T cell proliferation. Transcriptional modules derived from both peripheral blood and sorted BAL immune cells demonstrated an increased frequency of effector memory CD8 T cells in whole BAL samples. Both compartments displayed reduced induction of CD8 T-cell-derived interleukin-17A (IL-17A) in people with HIV, associated with elevated T cell regulatory molecule expression. The data suggest that dysfunctional CD8 T cell responses in uncontrolled HIV may contribute to compromised respiratory immunity to pathogens, a process that could be modulated by host-directed therapies that target CD8 T cell effector functions.

Cutaneous mycobacterial infections cause substantial morbidity and are challenging to diagnose and treat. An improved understanding of the dermal immune response to mycobacteria may inspire new therapeutic approaches. We conducted a controlled human infection study with 10 participants who received 2 × 106 CFUs of Mycobacterium bovis bacillus Calmette-Guérin (Tice strain) intradermally and were randomized to receive isoniazid or no treatment. Peripheral blood was collected at multiple time points for flow cytometry, bulk RNA sequencing (RNA-seq), and serum Ab assessments. Systemic immune responses were detected as early as 8 d postchallenge in this M. bovis bacillus Calmette-Guérin-naive population. Injection-site skin biopsies were performed at days 3 and 15 postchallenge and underwent immune profiling using mass cytometry and single-cell RNA-seq, as well as quantitative assessments of bacterial viability and burden. Molecular viability testing and standard culture results correlated well, although no differences were observed between treatment arms. Single-cell RNA-seq revealed various immune and nonimmune cell types in the skin, and communication between them was inferred by ligand-receptor gene expression. Day 3 communication was predominantly directed toward monocytes from keratinocyte, muscle, epithelial, and endothelial cells, largely via the migration inhibitory factor pathway and HLA-E-KLRK1 interaction. At day 15, communication was more balanced between cell types. These data reveal the potential role of nonimmune cells in the dermal immune response to mycobacteria and the utility of human challenge studies to augment our understanding of mycobacterial infections.

A recent clinical trial demonstrated that Bacille Calmette-Guérin (BCG) revaccination of adolescents reduced the risk of sustained infection with Mycobacterium tuberculosis (M.tb). In a companion phase 1b trial, HVTN 602/Aeras A-042, we characterize in-depth the cellular responses to BCG revaccination or to a H4:IC31 vaccine boost to identify T cell subsets that could be responsible for the protection observed. High-dimensional clustering analysis of cells profiled using a 26-color flow cytometric panel show marked increases in five effector memory CD4⁺ T cell subpopulations (TEM) after BCG revaccination, two of which are highly polyfunctional. CITE-Seq single-cell analysis shows that the activated subsets include an abundant cluster of Th1 cells with migratory potential. Additionally, a small cluster of Th17 TEM cells induced by BCG revaccination expresses high levels of CD103; these may represent recirculating tissue-resident memory cells that could provide pulmonary immune protection. Together, these results identify unique populations of CD4⁺ T cells with potential to be immune correlates of protection conferred by BCG revaccination.

Chimeric antigen receptor (CAR) T-cell therapy produces high response rates in refractory B-cell non–Hodgkin lymphoma, but long-term data are minimal to date. In this study, we present long-term follow-up of a pilot trial testing a CD20-targeting third-generation CAR in patients with relapsed B-cell lymphomas following cyclophosphamide-only lymphodepletion. Two of the three patients in the trial, with mantle cell lymphoma and follicular lymphoma, had remissions lasting more than 7 years, though they ultimately relapsed. The absence of B-cell aplasia in both patients suggested a lack of functional CAR T-cell persistence, leading to the hypothesis that endogenous immune responses were responsible for these long-term remissions. Correlative immunologic analyses supported this hypothesis, with evidence of new humoral and cellular antitumor immune responses proximal to clinical response time points. Collectively, our results suggest that CAR T-cell therapy may facilitate epitope spreading and endogenous immune response formation in lymphomas. Significance: Two of three patients treated with CD20-targeted CAR T-cell therapy had long-term remissions, with evidence of endogenous antitumor immune response formation. Further investigation is warranted to develop conditions that promote epitope spreading in lymphomas.

Background: Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 30 years, highlighting the need for therapy innovation. Although immunotherapy has revolutionized cancer treatment, efforts to harness endogenous patient immune responses have yielded limited therapeutic activity in ovarian cancer patients. T cells engineered to express a T cell receptor (TCR) targeting proteins uniquely overexpressed in tumors have the potential to control tumor growth without toxicity. Mesothelin (Msln) is over-expressed in ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill ovarian cancer. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies alone or in combination, ultimately targeting up to three inhibitory receptors simultaneously. Single-cell RNA-sequencing (scRNAseq) was used to profile the impact of combination checkpoint blockade on engineered T cells and the tumor microenvironment (TME). Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated in established tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, elements in the TME limited engineered T cell persistence and cytolytic function. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased antitumor function by intratumoral TCRMsln T cells. scRNAseq of tumor-infiltrating cells revealed distinct transcriptome changes in engineered and endogenous T cells and myeloid-derived cells. Engineered T cells, when combined with triple checkpoint blockade, increased expression of genes associated with effector and memory gene signatures, including proliferation and metabolic function, and reduced expression of genes associated with exhaustion. Moreover, combining adoptive immunotherapy with triple checkpoint blockade significantly prolonged survival in the cohort of treated tumor-bearing mice, relative to mice that received TCRMsln T cells alone or with anti-PD1 or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the TME can dramatically reduce T cell function, suggesting tumor cells may upregulate the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In an advanced ovarian cancer model, triple checkpoint blockade significantly improved engineered T cell function and outcomes in mice in a setting where single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory pathways simultaneously, which can be more safely pursed in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Valentin Voillet, Raphael Gottardo, Philip D. Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B084.

Approximately 50% of Merkel cell carcinoma (MCC) patients facing this highly aggressive skin cancer initially respond positively to PD-1-based immunotherapy. Nevertheless, the recurrence of MCC post-immunotherapy emphasizes the pressing need for more effective treatments. Recent research has highlighted Cyclin-dependent kinases 4 and 6 (CDK4/6) as pivotal cell cycle regulators gaining prominence in cancer studies. This study reveals that the CDK4/6 inhibitor, palbociclib can enhance PD-L1 gene transcription and surface expression in MCC cells by activating HIF2α. Inhibiting HIF2α with TC-S7009 effectively counteracts palbociclib-induced PD-L1 transcription and significantly intensifies cell death in MCC. Simultaneously, co-targeting CDK4/6 and HIF2α boosts ROS levels while suppressing SLC7A11, a key regulator of cellular redox balance, promoting ferroptosis- a form of immunogenic cell death linked to iron. Considering the rising importance of immunogenic cell death in immunotherapy, this strategy holds promise for improving future MCC treatments, markedly increasing immunogenic cell death various across various MCC cell lines, thus advancing cancer immunotherapy.