Chimeric antigen receptor (CAR) T-cell efficacy depends critically on the costimulatory domain, which shapes downstream signaling and the immunophenotype of manufactured products. Despite mechanistic evidence that immune receptors function as motif-based signaling scaffolds, CAR engineering has focused on a narrow set of costimulatory domains-principally CD28 and 4-1BB-leaving much of the available signaling design space unexplored. Here, we screened 1,243 naturally occurring intracellular domains as costimulatory modules in an anti-CD20 CAR backbone in primary human CD8+ T cells and quantified construct enrichment across memory-differentiation and PD-1-defined immunophenotypic compartments. Using Eukaryotic Linear Motif (ELM) annotations, we analyzed motif-phenotype associations via complementary statistical approaches: Mann-Whitney screening and negative binomial regression identified ELM features associated with differential construct representation, while Dirichlet-Multinomial modeling-which properly accounts for the compositional structure of FACS-partitioned data-revealed that individual ELMs do not significantly alter phenotype distributions. This discrepancy indicates that single motifs primarily affectproliferation orsurvival ratherthandifferentiation fate. In contrast, construct-level analysis using a leave-one-out compositional test identified specific costimulatory domains with significant phenotype-shifting effects, demonstrating that particular combinations of ELMs-rather than individual motifs-determine immunophenotype. These results suggest that CAR T-cell differentiation state is governed by the integrated output of multiple signaling motifs and provide a combinatorial framework for rational costimulatory domain engineering.

Monoclonal antibodies (mAbs) improve survival of patients with mature B-cell malignancies. Fcγ-receptor dependent effector mechanisms kill tumor cells but can promote antigen loss through trogocytosis, contributing to treatment failures. Cell-bound mAbs trigger the complement cascade to deposit C3 activation fragments and lyse cells. Within 24 hours after ofatumumab administration to patients with chronic lymphocytic leukemia (CLL), circulating tumor cells had lost CD20 and were opsonized with C3d. We hypothesized that C3d provides a target to eliminate residual CD20 negative tumor cells. To test this hypothesis, we generated C8xi, a mouse/human chimeric IgG1 that reacts with human but not mouse C3d. C8xi was effective in a patient-derived xenograft model against CD20 negative, C3d opsonized CLL cells from patients treated with ofatumumab. We also generated rabbit mAbs, two of which were chosen because they bound mouse and human C3d with low nanomolar affinity but were minimally cross-reactive with full-length C3. Anti-C3d rabbit/human chimeric IgG1 in combination with ofatumumab or rituximab prolonged survival of xenografted mice that model three different types of non-Hodgkin lymphoma (NHL). For example, in a diffuse large B-cell lymphoma model (SU-DHL-6), median survival with single-agent CD20 mAb was 114 days but was not reached for mAb combination treatment (P=.008). In another NHL model (SU-DHL-4), single-agent and combination mAb therapy eradicated lymphoma in most mice. In long-term survivors from both cohorts, there was no evidence of adverse effects. We propose that C3d mAbs combined with complement fixing CD20 mAbs can deliver a one-two punch and increase efficacy of mAb-based therapy.

Immune checkpoint blockade has been shown to restore anti-tumor T-cell function and elicit durable responses in select solid and hematopoietic malignancies. However, single-agent anti-programmed death 1 (PD-1) antibodies proved less efficacious in patients with chronic lymphocytic leukemia (CLL). In patients with high-risk or relapsed/refractory CLL, we conducted a phase 2 study testing the combination of lead-in ibrutinib and up to 2 cycles of fludarabine, followed by continuous therapy with ibrutinib and 17 cycles of pembrolizumab administered every 3 weeks. A total of 15 patients were enrolled. In 10 patients evaluable for response, we observed 1 complete response and 9 partial responses. There was no discernible benefit of the combination beyond what is expected from ibrutinib monotherapy. However, 3 weeks after the first dose of pembrolizumab, we detected CD8 T-cell proliferation in a subset of patients, whom we called “immune responders.” In the responders, CD27-expressing CD8 T cells were relatively increased over immune nonresponders. Paired single-cell RNA and TCR sequencing revealed clonal expansion of activated GZMK+ CD8 effector memory and terminally differentiated effector cells. After 6 months of pembrolizumab treatment, the proportion of activated and proliferating CD8 T cells returned to baseline levels. Similarly, most novel clonotypes identified after 1 cycle of pembrolizumab decreased in frequency on long-term treatment. In summary, pembrolizumab did not improve the clinical response over ibrutinib monotherapy but transiently activated distinct clonotypes of CD8 T cells in a subset of CLL patients.

Gene and cellular therapies extend the hope of cure to wider range of human diseases. Durability of these therapies is correlated to persistence of the adoptively conferred cells; while mature effector functions are key to effectiveness. Physiologically, cells are rarely simultaneously endowed with indefinite self-renewal and mature effector functions. Cellular therapies must, in response to environmental signals, dynamically transition between self-renewing and mature effector states while avoiding dysfunctional states. Chimeric Antigen Receptor (CAR) T cells have emerged as effective therapies for B cell and plasma cell malignancies. While they induce prolonged remission in most patients, fewer than half achieve durable control of their disease. Factors associated with durable remissions can be broadly categorized into CAR T cell extrinsic, encompassing malignancy type, tumor burden, antigen escape and the tumor microenvironment, and intrinsic factors, pertaining to the dynamics of transition between the persistent and mature effector states. Strategies to design of CAR T therapies to surmount intrinsic mechanisms of failure are essential. We carried out CRISPR activation screening targeting all human transcription factors and epigenetic modifiers in anti-CD20 CAR T cells cultured in vitro under conditions designed to provoke exhaustion. We observed statistically significant depletion in sgRNA targeting the TGFb (including KLF10) and TNFa (including NFATC4) signaling pathways in CAR with immunophenotypes associated with durability and effectiveness. Model-based Analysis of Genome-Wide CRISPR-Cas9 Knockout (MAGeCK) for the central memory population revealed enrichment of MFNG, a regulator of NOTCH signaling; NFIB, a master regulator of cell differentiation; SMYD2, an epigenetic regulator controlling Treg development; and TRIAP1, a TP53 regulated inhibitor of apoptosis. Although biologically plausible, these targets play important roles in regulating proliferation and cytotoxicity and entirely disabling these host-protective mechanisms raises important safety concerns. Transcription factors and epigenetic modifiers have broad effects on cellular states and regulatory mechanisms. By contrast, cis-regulatory elements (CRE) directly affect only the transcription of genes in their vicinity. Perturbating CRE alters T cell regulation with a finer degree of control and is incompletely explored. Systematically screening the large number of candidate CRE is not feasible via current experimental approaches. We developed a model, trained on single cell multi-omics data, to predict the time evolution of transcriptomes and regulomes. The introduction of chromatin state in the description of cellular dynamics, permits prediction of cellular dynamics after enhancing or disrupting specific and computes therapeutically salient metrics such as overall proliferation rate and proportions of CAR in memory and functional effector subsets. Our model predicts that the conditional knock out of the trans-acting factor TCF7 will increase the transition rates from naïve and memory T cell subsets into effector states; a prediction that agrees with the established biological roles for TCF7. By contrast, targeting the disruption of a candidate cis-regulatory element (cCRE) in the vicinity of KLF10 (chr8:101,790,788-101,791,270) is predicted to strongly increase transition probabilities into the Naïve and Memory subsets. Our model casts the problem of optimizing cellular therapies into the framework of dynamical systems theory and provides a rigorous approach for prioritizing the experimentally intractable number of perturbations by metrics directly relevant to persistence of efficacious adoptive cells. Future directions include the incorporation of additional single cell multiomic modalities to better characterize regulatory dynamics.

Chimeric Antigen Receptors (CARs) are engineered into immune cells to bestow specificity towards identified targets on cancer cells. CAR T cells have established themselves as therapies for B cell and plasma cell malignancies, where they induce prolonged remission in most patients, though fewer than half achieve durable control of their disease. CARs follow a modular design, with an extracellular portion conferring target-specificity, a transmembrane hinge, and two or more intracellular signaling domains. One of the signaling components - typically CD3z - initiates the signaling cascades crucial for T-cell activation, proliferation, attainment of cytotoxic function, and ultimately immune memory. The other signaling component, called the costimulatory domain, is crucial for ensuring proper and controlled immune responses. Absence of this domain results in T cell anergy, but the optimal costimulatory domains remain unknown. Intracellular immune receptor signaling domains often comprise disordered regions lacking fixed tertiary protein structure. Their primary function is fulfilled by their primary structure consisting of one or more short (3 - 10 amino acid long) conserved sequences called Eukaryotic Linear Motifs. Adhering to this design principle, we systematically identified all transmembrane proteins within the Gene Ontology database associated with immune cell differentiation, proliferation, regulation or signaling. We specifically focused on proteins with known topologies in UniProt, extracting the primary sequences of the cytosolic portions. Through this process we curated a pool of over 1,200 distinct costimulatory domains, encompassing receptors not native to T cells, including cytokine/chemokine and inhibitory receptors. We lentivirally transduced anti-CD20 CAR with this pool of costimulatory domains into CD8+ T cells from 2 healthy volunteers. We cultured the resulting CAR T in triplicate, alone, activated by CD2/CD28 beads, in the presence of Raji (target) cells or K562 (negative control) cells over a 14-day period. At the conclusion of the experiment, we immunophenotypically sorted the cells into 6 sets: distinguishing central memory, effector and naïve T cell subsets each characterized by low or high PD1 expression. We prepared targeted sequencing libraries from the sorted cells to identify the costimulatory domains. Our screen yielded several promising novel ‘hits’ that we are evaluating through arrayed experiments. Notably, CD74 displayed enrichment in both the non-exhausted memory and effector subsets. Recognized as the Major Histocompatibility Complex (MHC) class II-associated invariant chain, CD74 is primarily known for its role in facilitating the assembly and trafficking of MHC class II molecules within cells. Beyond this canonical function, CD74 also harbors intracellular signaling capabilities that contribute to immune responses, engaging the ERK1/2, PI3K/AKT, and NF-κB pathways. By contrast, 4-1BB, which is used in most commercial CAR, activates a superset of these pathways, MAPK, PI3K/AKT and NF-κB. ERK1/2 is a subset of the MAPK pathway that primarily responds to growth factors and mitogens. Large language models have found powerful application in biology. ESM-2 (Evolutionary Scale Modeling) is a foundation model developed by Meta AI, that maps primary sequences to a representation (‘embedding‘) that captures evolutionary and structural information. To gain a predictive understanding of proportions of immunophenotypic subsets resulting from different costimulatory domains, we trained a contrastive learning model to learn a shared embedding between ESM-2 representations (a 5,120-dimensional space) and our experimentally determined proportions of immunophenotypic proportions under different co-culture conditions (a 24-dimensional space). We trained two dense neural networks with 2 hidden layers to project to a 20-dimensional shared embedding using 90% of our data, we achieved a moderate Fraction of Samples Closer than the True Match (FOSCTTM) of 0.45 in our validation sample. We further refined this shared embedding with fused Gromov-Wasserstein optimal transport to achieve a respectable FOSCTTM of 0.13. Our model predicts proportions of immunophenotypic subsets for different costimulatory domains in the context of our anti-CD20 CAR. We plan arrayed testing of domains predicted to improve those in our previous pool.

Histogram of PD-1 expression in total CD3+, total CD8+, and the most abundant CD8+ clonotype in each patient after expansion with beads or autologous CLL cells.

<p>(A) Frequency of predominant clonotypes as detected by NGS and of the corresponding CD8+ TRBV families as measured by flow cytometry. Grey bars indicate TRBV families that did not have commercially available fluorescent-labelled antibodies. (B) Pie chart of the most expanded clonotypes and expression of granzyme B (GrB). CD8+ GrB+ clonotypes comprising >2% of the repertoire at the time of response are shown in color.-</p>

Chediak–Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients’ induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype.

<p>(A) Frequency of predominant clonotypes as detected by NGS and of the corresponding CD8+ TRBV families as measured by flow cytometry. Grey bars indicate TRBV families that did not have commercially available fluorescent-labelled antibodies. (B) Pie chart of the most expanded clonotypes and expression of granzyme B (GrB). CD8+ GrB+ clonotypes comprising >2% of the repertoire at the time of response are shown in color.-</p>

<p>Scatter plot of the TRBV frequency as detected by next generation sequencing (NGS) and flow cytometry. Spearman rho correlation coefficient and two tail p-value are shown.</p>

<p>T cell counts at baseline, time of ibrutinib response, and progressive disease (PD). Individual values, median and interquartile range are shown. Dashed lines indicate the lower and upper limits of the normal reference range.</p>

<p>T cell counts at baseline, time of ibrutinib response, and progressive disease (PD). Individual values, median and interquartile range are shown. Dashed lines indicate the lower and upper limits of the normal reference range.</p>

<p>Scatter plot of the TRBV frequency as detected by next generation sequencing (NGS) and flow cytometry. Spearman rho correlation coefficient and two tail p-value are shown.</p>

Expansion of T cells under different conditions. Total CD3+, CD8+, CD4+ counts at Day 0 and Day 7 of expansion in the presence of anti-CD3/CD28/CD137 beads or autologous CLL cells.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the β2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

Transplantation of genetically modified autologous hematopoietic stem and progenitor cells (HSPCs) holds a curative potential for subjects with inherited blood disorders. In recent years, transfer of a therapeutic gene to HSPCs has been successfully achieved using replication-incompetent integrating lentiviral vectors. More recently, advances have emerged to more precisely edit cellular genomes by specific correction of mutations or targeted gene addition at endogenous genomic loci. However, cellular processes triggered in HSPCs by the programmable nucleases utilized in these gene editing approaches may negatively impact their ability to reconstitute and maintain hematopoiesis long-term in recipient hosts. Granulocyte colony-stimulating factor (G-CSF) use after autologous HSPC transplantation is generally recommended to shorten the duration of severe neutropenia. However, little is known about the safety and efficacy of G-CSF use after transplantation of genetically modified autologous HSPCs. G-CSF is the principal cytokine regulating granulopoiesis, but also plays an important role in regulating hematopoietic stem cell (HSC) function (Schuttpelz, Leukemia 2014). Studies have suggested that G-CSF can exacerbate HSC damage caused by chemotherapeutic agents and irradiation by promoting differentiation at the expense of self-renewal and by inducing cellular senescence (van Os, Stem Cells 2000; Li, Cell Biosci 2015). Here, we asked whether G-CSF use after transplantation of gene edited HSPCs may negatively affect their long-term repopulating (LTR) and self-renewal capacities.

PURPOSE:In chronic lymphocytic leukemia (CLL), the T-cell receptor (TCR) repertoire is skewed and tumor-derived antigens are hypothesized as drivers of oligoclonal expansion. Ibrutinib, a standard treatment for CLL, inhibits not only Bruton tyrosine kinase of the B-cell receptor signaling pathway, but also IL2-inducible kinase of the TCR signaling pathway. T-cell polarization and activation are affected by ibrutinib, but it is unknown whether T cells contribute to clinical response.EXPERIMENTAL DESIGN:High-throughput TCRβ sequencing was performed in 77 longitudinal samples from 26 patients with CLL treated with ibrutinib. TCRβ usage in CD4+ and CD8+ T cells and granzyme B expression were assessed by flow cytometric analysis. Antitumor cytotoxicity of T cells expanded with autologous CLL cells or with antigen-independent anti-CD3/CD28/CD137 beads was tested.RESULTS:The clonality of the TCR repertoire increased at the time of response. With extended treatment, TCR clonality remained stable in patients with sustained remission and decreased in patients with disease progression. Expanded clonotypes were rarely shared between patients, indicating specificity for private antigens. Flow cytometry demonstrated a predominance of CD8+ cells among expanded clonotypes. Importantly, bulk T cells from responding patients were cytotoxic against autologous CLL cells in vitro and selective depletion of major expanded clonotypes reduced CLL cell killing.CONCLUSIONS:In patients with CLL, established T-cell responses directed against tumor are suppressed by disease and reactivated by ibrutinib.See related commentary by Zent, p. 4465.

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.