Cellular architecture shapes the naïve T cell response.

After antigen stimulation, naïve T cells display reproducible population-level responses, which arise from individual T cells pursuing specific differentiation trajectories. However, cell-intrinsic predeterminants controlling these single-cell decisions remain enigmatic. We found that the subcellular architectures of naïve CD8 T cells, defined by the presence (TØ) or absence (TO) of nuclear envelope invaginations, changed with maturation, activation, and differentiation. Upon T cell receptor (TCR) stimulation, naïve TØ cells displayed increased expression of the early-response gene Nr4a1, dependent upon heightened calcium entry. Subsequently, in vitro differentiation revealed that TØ cells generated effector-like cells more so compared with TO cells, which proliferated less and preferentially adopted a memory-precursor phenotype. These data suggest that cellular architecture may be a predeterminant of naïve CD8 T cell fate.

Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis.

Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either JAK2 or Calreticulin (CALR) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First, CALR mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous CALR mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment.

Ex vivo drug response heterogeneity reveals personalized therapeutic strategies for patients with multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy defined by complex genetics and extensive patient heterogeneity. Despite a growing arsenal of approved therapies, MM remains incurable and in need of guidelines to identify effective personalized treatments. Here, we survey the ex vivo drug and immunotherapy sensitivities across 101 bone marrow samples from 70 patients with MM using multiplexed immunofluorescence, automated microscopy and deep-learning-based single-cell phenotyping. Combined with sample-matched genetics, proteotyping and cytokine profiling, we map the molecular regulatory network of drug sensitivity, implicating the DNA repair pathway and EYA3 expression in proteasome inhibitor sensitivity and major histocompatibility complex class II expression in the response to elotuzumab. Globally, ex vivo drug sensitivity associated with bone marrow microenvironmental signatures reflecting treatment stage, clonality and inflammation. Furthermore, ex vivo drug sensitivity significantly stratified clinical treatment responses, including to immunotherapy. Taken together, our study provides molecular and actionable insights into diverse treatment strategies for patients with MM.