Citations

Abstract Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR-α and -β chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell–derived islet grafts (SC-islets) in vivo . This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.

Abstract Cytokine receptor–like factor 2 rearrangement (CRLF2r), an aberration causing a Philadelphia chromosome–like gene expression profile and a poor prognostic marker in B-cell acute lymphoblastic leukemia (B-ALL), endows relative chemotherapeutic resistance. Extramedullary disease (EMD), including both central nervous system (CNS) and non-CNS involvement, can also be associated with worse outcomes. The relationship between CRLF2r and EMD is unknown. Based on an initial observation of patients with relapsed/refractory (R/R) CRLF2r B-ALL and EMD, a single-center retrospective review was conducted to evaluate the incidence of EMD in patients with CRLF2r B-ALL. Across 177 children, adolescents, and young adults with R/R B-ALL, 21 (11.9%) harbored CRLF2r, of whom 19 (90.5%) had EMD at some point during their treatment course. In contrast, only 78 of the 156 patients (50%) without CRLF2r ever had EMD (P = .0003). In the CRLF2r cohort, 8 (38.1%) presented with EMD at diagnosis: 7 with CNS and 1 with non-CNS EMD. At relapse, 17 (80.9%) had EMD: 4 (19%) had CNS EMD, 9 (42.9%) had non-CNS EMD, and 4 (19%) had both. Isolated EMD was observed in 2 patients (11.8%) at first relapse and in 3 (17.6%) with multiple relapses. Given these findings, further study to evaluate this potential association between EMD and CRLF2r in R/R B-ALL is indicated.

Abstract Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics. Current classification incorporates clinical presentation and anaplastic lymphoma kinase (ALK) status but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities. We studied 689 patients with ALCL in the LLMPP (Lymphoma/Leukemia Molecular Profiling Project) and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple negative), and immunohistochemistry for phosphorylated STAT3Tyr705. RNA sequencing with unsupervised gene expression profiling in 393 patients identified 2 main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (type I) or absence (type II) of phosphorylated STAT3Y705 expression. Type I ALCLs included ALK+ ALCL and a subset of triple-negative ALCLs (TN-I); type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II). Type I ALCLs were enriched for JAK-STAT3, whereas type II ALCLs were enriched for non–tyrosine kinase pathways, particularly epigenetic regulators such as EZH2. Immunohistochemistry showed overexpression of EZH2 and its trimethylated substrate H3K27. Prognosis in systemic ALCL was favorable for DUSP22-rearranged ALCL (5-year overall survival, 95%) and ALK+ ALCL (88%), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%), and poor for TP63-rearranged ALCL (0%). We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies 4 molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II). This classification can be easily implemented on paraffin tissue in routine practice or clinical trials, and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants, and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdc scid Il2rg tm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1 + CD4 + peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh-like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. Tfh/Tph cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies, and lymphopenia-induced proliferation (LIP) have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

Abstract Lineage switch (LS), defined as the immunophenotypic transformation of acute leukemia, has emerged as a mechanism of relapse after antigen-targeted immunotherapy, which is associated with dismal outcomes. Through an international collaborative effort, we identified cases of LS after a host of antigen-targeted therapies (eg, CD19, CD22, CD38, and CD7), described how LS was diagnosed, reviewed treatment approaches, and analyzed overall outcomes for this form of postimmunotherapy relapse. Collectively, 75 cases of LS were evaluated, including 53 (70.7%) cases of B-cell acute lymphoblastic leukemia (B-ALL) transforming to acute myeloid leukemia (AML), 17 (22.7%) cases of B-ALL transforming to mixed phenotypic acute leukemia (MPAL)/acute leukemias of ambiguous lineage (ALAL), and 5 (6.7%) cases of rare LS presentation (ie, T-cell ALL to AML). An additional 10 cases with incomplete changes in immunophenotype, referred to as “lineage drift” were also described. With a primary focus on the 70 cases of LS from B-ALL to AML or MPAL/ALAL, LS emerged at a median of 1.5 months (range, 0-36.5) after immunotherapy, with 81.4% presenting with LS within the first 6 months from the most proximal immunotherapy. Although most involved KMT2A rearrangements (n = 45, 64.3%), other rare cytogenetic and/or molecular alterations were uniquely observed. Treatment outcomes were generally poor, with remission rates of <40%. The median overall survival after LS diagnosis was 4.8 months. Outcomes were similarly poor for those with rare immunophenotypes of LS or lineage drift. This global initiative robustly categorizes lineage changes after immunotherapy and, through enhanced understanding, establishes a foundation for improving outcomes of LS.

Abstract Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdcscid Il2rgtm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1+CD4+ peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh- like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. Tfh/Tph cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and lymphopenia-induced proliferation (LIP) have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

Human Immune System (HIS) mice constructed with mature human immune cells or with human hematopoietic stem cells and thymic tissue have provided an important tool for human immunological research. In this article, we first review the different types of HIS mice based on human tissues transplanted and sources of the tissues. We then focus on knowledge of human T cell development and responses obtained using HIS mouse models. These areas include the development of human T cell subsets, with a focus on αβ conventional T cells and regulatory T cells, and human T cell responses in the settings of infection, transplantation rejection and tolerance, autoimmune disease, cancer immunotherapy, and regulatory T cell therapy. We also discuss the limitations and potential future applications of HIS mouse models.

Background: Anaplastic large cell lymphomas (ALCLs) represent a heterogeneous group of T-cell lymphomas that currently are classified by the presence or absence of ALK tyrosine kinase (TK) fusion genes (ALK+ or ALK−) and clinical presentation (systemic, cutaneous, or breast implant-associated). Two overarching molecular types of ALCL recently were discovered, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. Fusions involving non-ALK TK genes occur in some ALK− cases, but the fusion landscape of ALCL remains incompletely characterized. Methods: Expert consensus pathology review was conducted in the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). RNAseq was performed and fusions were identified using FusionCatcher. Here, we focused on recurrent in-frame coding fusions. Previously unreported fusions were validated by RT-PCR. Type I/II was assigned using a previously validated gene expression-based model. Genes with adjusted (adj) P<0.05 were considered differentially expressed. Overall survival (OS) was assessed for systemic ALCL, when available. Results: We evaluated 379 ALCLs (229M/150F; mean age, 56 y). Of 199 candidate fusions (excluding reciprocal events), 28 were recurrent and passed quality metrics. At least 1 of these 28 fusions was present in 150 cases (40%). ALK fusions were present in 106 ALK+ ALCLs (all Type I; P<0.0001). Of these, NPM1::ALK was seen in 68/106 (64%). Alternate partners (X::ALK) included ATIC (N=24) and CTLC, COL1A2, MSN, MYH9, RNF213, SATB1, TFH, TPM3, and TRAF1 (1-3 cases each). X::ALK was associated with older age (mean, 52 y) than NPM1::ALK (32 y; P<0.0001) and showed relative overexpression of 49 genes, including multiple activators of small GTPases such as CGNL1 (FC, 5.6; Padj=2.4×10-6), SRGAP1, ALS2, DOCK1, and NCKAP1. ALK expression was similar in cases with NPM1::ALK and X::ALK and there was no significant difference in OS. Non-ALK TK fusions were seen in 17/273 ALK− cases (6%), including TYK2 (N=9); JAK2 (N=6); and ROS1 (N=2). All were Type I ALCLs (P<0.0001). ALK− cases with non-ALK TK fusions overexpressed 38 genes compared to ALK− cases without TK fusions, including cytokines involved in the IL17 signaling pathway such as CXCL6 (FC, 12.5; Padj=4.2×10-3), CXCL1, and CSF3. We then assessed the relationship between ALK− cases with non-ALK TK fusions and ALK+ cases by separately comparing each subset to ALK− cases without TK fusions. FC values for ALK− cases with non-ALK TK fusions were significantly correlated with FC values for ALK+ ALCLs over the set of expressed genes (R=0.54; P<0.0001). At a median follow-up of 32 months, 0/4 systemic ALCL patients with non-ALK TK fusions had died, while 30% of those with ALK− ALCL without TK fusions had died, but this was not statistically significant (P=0.26). The other 13 ALK− cases with non-ALK TK fusions were either localized ALCLs or did not have outcome data available. Of 20 TP63 fusions, 17 (85%) were TBL1XR1::TP63 and 3 were X::TP63. As previously reported, TP63 fusions were associated with poor OS (median OS, 10 months; not reached for other ALK− ALCLs; P=0.007). Four ALCLs had a novel NCL::UBTF fusion involving genes encoding the nucleolar proteins nucleolin and nucleolar (upstream binding) transcription factor-1. Four cases each had PARG::BMS1 and TNK1::GPS2 (the latter seen only in Type I ALCLs). Conclusions: This large consortium-based analysis of coding fusions further elucidates the molecular distinction between Type I and Type II ALCLs. Both ALK and non-ALK TK fusions were seen exclusively in Type I ALCLs, which we have shown previously are enriched for JAK-STAT3 pathway genes and correlate strongly with pSTAT3Y705 positivity by immunohistochemistry. These fusions were not identified in Type II ALCLs, which previous data suggest are associated with epigenetic alterations. ALK+ ALCLs with NPM1::ALK and X::ALK showed differences in gene expression, suggesting biological differences. Furthermore, ALCLs with non-ALK TK fusions shared gene expression features with ALK+ ALCL. Clinical fusion detection could enhance molecular classification of Type I and Type II ALCLs and may guide precision therapy.

Abstract Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdcscid Il2rgtm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1+CD4+ peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh-like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. T cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and LIP have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.