TIM3 blockade with hypomethylating therapy restores NK-cell and cytotoxic CD4+ T-cell activity in patients with AML or MDS.

Peripheral CD8⁺ T-cell clonality and NK-cell expansion mark early response to mosunetuzumab in indolent B-cell lymphoma.

Mosunetuzumab is highly effective against many B-cell lymphomas, but longitudinal immune changes induced by intermittently dosed CD20×CD3 bispecific antibodies remain incompletely characterized. We performed immune profiling of peripheral blood samples from patients receiving fixed-duration (6 months) subcutaneous mosunetuzumab for previously untreated follicular or marginal zone lymphoma on a phase 2 clinical trial (NCT04792502). Analyses included flow cytometry, cytokine profiling, and RNA-seq of isolated CD8+ T-cells with TCR repertoire inference. Early (at 3 weeks) on-treatment samples showed broad cytokine induction, CD8+ T-cell redistribution with decreased TEMRA cells and increased CD27+CD62L+ transitional effector memory-like cells, increased PD-1 and LAG-3 expression, and induction of CD8+ transcriptional programs consistent with activation, proliferation, exhaustion priming, and SREBF2-regulated cholesterol metabolism. By mid-treatment (at 3 months), activation-associated features contracted toward baseline, with no evidence of phenotypic exhaustion, though with persistent overexpression of LAG3, HAVCR2, and LAYN on RNA-seq, as well as sustained SREBF2 activation. CD8+ T-cell clonality increased at mid-treatment compared with baseline and higher clonality was associated with early clinical complete response. Mosunetuzumab also induced indirect NK-cell activation and expansion, including skewing toward CD56dimCD16bright effector state. HLA-DR upregulation on NK cells correlated with early increases in NK-activating plasma cytokines, and higher NK-cell count at mid-treatment was associated with complete response. Together, these findings define a CD8+ effector-remodeling state on mosunetuzumab therapy, characterized by clonal expansion, selective inhibitory receptor expression, sustained activation of cholesterol biosynthesis, and cytokine-linked engagement of innate immune mechanisms, providing a framework for potential strategies to support T-cell function and enhance bispecific antibody efficacy.

Targeting CD38 with bispecific antibody XmAb 18968 in patients with relapsed/refractory acute myeloid leukemia and T-cell acute lymphoblastic leukemia.

Epigenetic plus cytokine therapy induces stem-like CD8+ T-cell reservoirs and intratumoral immune niches to overcome anti-PD-1 resistance

Abstract Background Checkpoint blockade (CPB) has limited efficacy in patients with colorectal cancer (CRC), warranting improved therapeutic strategies capable of remodeling the tumor microenvironment. Stem-like TCF1+CD8+ T cells, cytotoxic CD8+ T cells, and lymphoid aggregates have been associated with CPB responsiveness, either individually or as coordinated immune hubs. Methods We evaluated the combination of αPD-1, the class I histone deacetylase inhibitor entinostat, and the IL-15 superagonist N-803 in CPB-resistant murine CRC and breast cancer models with varying antigen presentation deficiencies. Proteomic, transcriptomic, spatial, and functional analyses were completed to identify treatment-specific immune remodeling. Translational relevance was assessed using publicly available CPB-treated cancer genomic datasets and functional assays with patient-derived peripheral blood mononuclear cells (PBMCs). Results Triple therapy increased stem-like TCF1+CD8+ T cells in tumor-draining lymph nodes and tumors, concomitant with increased intratumoral cytotoxic GZMB+CD8+ T cells. Therapeutic efficacy was associated with remodeling of the tumor microenvironment, uniquely marked by enrichment of intratumoral immune niches comprising TCF1+ and cytotoxic CD8+ T cells, type 1 conventional dendritic cells, and B cells, along with concerted cytokine and chemokine production. A transcriptional signature derived from triple therapy-induced immune niches predicted favorable clinical responses across multiple CPB-experienced cancer cohorts. These responses were functionally corroborated by increased markers of cytotoxicity in patient-derived PBMCs in vitro. Conclusions Treatment with entinostat, N-803, and αPD-1 boosts anti-tumor intratumoral immune niches comprising TCF1+CD8+ T cells. Collectively, these findings suggest that TCF1+CD8+ T cell immune niches previously associated with αPD-1 responsiveness and lacking in non-responders, including most CRC patients, may be restored by the addition of entinostat and N-803 to αPD-1 therapy to treat αPD-1-refractory patients.

Double-Negative T Cells Induce Ferroptosis-Dependent, MHC-Independent Cytotoxicity of CD8+ T Cells Against AML 2256323

Acute myeloid leukemia (AML) is a blood cancer originating from myeloid cells in the bone marrow with poor prognosis. CD3+CD4-CD8-double negative T cells (DNTs) is a rare subset of immune cells that have shown strong anti-AML properties. Our previous work demonstrated that allogeneic DNTs from healthy donors effectively targeted AML in vitro and in vivo without off-tumor toxicity in preclinical models and early phase clinical trials. However, the mechanisms by which DNTs influence host immune cells’ anti-leukemic activity remain unclear. We cultured patient or healthy donor CD8+ T cells with autologous AML blasts or AML cell lines and characterized their immunophenotype and anti-leukemic activity. We performed proteomic analysis comparing CD8+ T cells activated by DNT+AML, AML alone, or CD3/CD28 beads, and differentially expressed proteins and pathways were identified and validated using functional assays. We conducted blocking experiments targeting ferroptosis and generated MHC-I knockout AML cells to test whether DNT-activated CD8+ T cells can kill AML cells in an MHC-independent manner. CD8+ T cells co-cultured with DNTs mediated cytotoxicity toward AML cells. Proteomic profiling revealed that CD8+ T cells activated by DNT+AML upregulated innate immune response and ferroptosis-pathways. Blocking ferroptosis markedly inhibited their killing capacity, indicating that CD8+ T cell cytotoxicity depends on ferroptosis. Notably, these CD8+ T cells could kill MHC-I—deficient AML cells, suggesting an antigen-independent mechanism. These findings show that, beyond direct cytotoxicity, DNTs promote a non-conventional anti-leukemic activity of CD8+ T cells through ferroptosis-dependent mechanisms. This synergy between DNTs and CD8+ T cells represents a novel immune axis that could be leveraged to enhance adoptive cell therapy efficacy against AML. Alberta Cancer Foundation, Kids Cancer Care, CIHR Project Grant, Cancer Research Society Operating Grant Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

T-cell dynamics, CD8/NK ratio and tumor survival hallmarks determine response to T-cell bispecific forimtamig in myeloma.

T-cell bispecific (TCB) therapies have demonstrated promising therapeutic efficacies for patients with relapsed/refractory multiple myeloma. However, responses are transient and essentially all patients eventually relapse. Cytokine release syndrome and other on-target off-tumor toxicities are common. Together, these challenges ask for new approaches to optimize treatment and to eventually understand resistance mechanisms. This phase 1 study (NCT04557150) investigated mode of action and resistance mechanisms of forimtamig, a novel GPRC5DxCD3 TCB. Biomarker analyses included deep phenotyping of patients' tumor and immune cells, functional T cell characterization, cytokine release, soluble B-cell maturation antigen kinetics and minimal residual disease evaluation. Biomarker data analysis assessed the impact of different TCB treatment approaches on T cell-mediated tumor killing dynamics: subcutaneous vs intravenous administration, weekly vs every 4 days step-up dosing, and low vs high target doses. A machine learning model revealed T-to-natural killer (NK)cell ratio in blood and bone marrow as a strong predictor of response. Single cell transcriptomics showed increased DNA repair and proliferation as major tumor hallmarks associated with resistance, while T cell dysfunction signatures were not predictive of response. T cells from non-responding patients showed an increased cytokine release when exposed to higher forimtamig exposure in an ex vivo setting. T cells from responding and non-responding patients were equally fit in killing tumor cells ex vivo. Our results highlight that forimtamig treatment optimization may be possible by boosting T cell numbers over NK cells, targeting tumor intrinsic resistance mechanisms or individualized dosing approach.

T lymphocytes and natural killer cells in myelodysplastic syndromes: function, dysfunction, and therapeutic potential

Myelodysplastic syndrome (MDS) are clonal myeloid neoplasms that cause cytopenias and can progress to acute myeloid leukemia (AML). Hypomethylating agents (HMA) are the mainstay of treatment for higher risk disease, but they achieve responses in only half of treated patients and complete remission rates are low. Several scientifically based combinatorial regimens have been tested in clinical trials but none has demonstrated a survival benefit over HMA monotherapy. Allogeneic stem cell transplant remains the only curative therapy and is dependent on effective donor lymphocytes for its efficacy. However, access is limited by its toxicity, so alternative approaches are sorely needed. Recent clinical and translational studies have shown that MDS is not only a clonal myeloid disorder, but also associated with immune dysregulation, inflammatory signaling, T-cell repertoire restriction, immune exhaustion, and immune mediated suppression of hematopoiesis. These findings suggest that there is potential for unlocking a novel approach to the treatment of MDS by restoring and/or enhancing the lymphoid immune response. In this review, we discuss the current understanding of the role of normal T lymphocytes in MDS, the causes and manifestations of dysfunctional T lymphocytes as well as the role and dysfunction of natural killer (NK) cells. The role of therapeutic immunosuppression in lower risk MDS is reviewed, as is the impact of HMAs on dysregulated T lymphocytes and NK cells in higher risk disease. We propose that there is tremendous potential for more targeted approaches to engage the lymphoid compartment in addressing the unmet therapeutic need in MDS.