CD4 T-cell cytokines synergize to induce proliferation of malignant and nonmalignant innate intraepithelial lymphocytes.

Refractory celiac disease type II (RCDII) is a severe complication of celiac disease (CD) characterized by the presence of an enlarged clonal population of innate intraepithelial lymphocytes (IELs) lacking classical B-, T-, and natural killer (NK)-cell lineage markers (Lin-IELs) in the duodenum. In ∼50% of patients with RCDII, these Lin-IELs develop into a lymphoma for which no effective treatment is available. Current evidence indicates that the survival and expansion of these malignant Lin-IELs is driven by epithelial cell-derived IL-15. Like CD, RCDII is strongly associated with HLA-DQ2, suggesting the involvement of HLA-DQ2-restricted gluten-specific CD4+ T cells. We now show that gluten-specific CD4+ T cells isolated from CD duodenal biopsy specimens produce cytokines able to trigger proliferation of malignant Lin-IEL lines as powerfully as IL-15. Furthermore, we identify TNF, IL-2, and IL-21 as CD4+ T-cell cytokines that synergistically mediate this effect. Like IL-15, these cytokines were found to increase the phosphorylation of STAT5 and Akt and transcription of antiapoptotic mediator bcl-xL Several small-molecule inhibitors targeting the JAK/STAT pathway blocked proliferation elicited by IL-2 and IL-15, but only an inhibitor targeting the PI3K/Akt/mTOR pathway blocked proliferation induced by IL-15 as well as the CD4+ T-cell cytokines. Confirming and extending these findings, TNF, IL-2, and IL-21 also synergistically triggered the proliferation of freshly isolated Lin-IELs and CD3-CD56+ IELs (NK-IELs) from RCDII as well as non-RCDII duodenal biopsy specimens. These data provide evidence implicating CD4+ T-cell cytokines in the pathogenesis of RCDII. More broadly, they suggest that adaptive immune responses can contribute to innate IEL activation during mucosal inflammation.

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.

Development of a diverse human T-cell repertoire despite stringent restriction of hematopoietic clonality in the thymus.

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.

Somatic mutations found in the healthy blood compartment of a 115-yr-old woman demonstrate oligoclonal hematopoiesis.

The somatic mutation burden in healthy white blood cells (WBCs) is not well known. Based on deep whole-genome sequencing, we estimate that approximately 450 somatic mutations accumulated in the nonrepetitive genome within the healthy blood compartment of a 115-yr-old woman. The detected mutations appear to have been harmless passenger mutations: They were enriched in noncoding, AT-rich regions that are not evolutionarily conserved, and they were depleted for genomic elements where mutations might have favorable or adverse effects on cellular fitness, such as regions with actively transcribed genes. The distribution of variant allele frequencies of these mutations suggests that the majority of the peripheral white blood cells were offspring of two related hematopoietic stem cell (HSC) clones. Moreover, telomere lengths of the WBCs were significantly shorter than telomere lengths from other tissues. Together, this suggests that the finite lifespan of HSCs, rather than somatic mutation effects, may lead to hematopoietic clonal evolution at extreme ages.

Identification of checkpoints in human T-cell development using severe combined immunodeficiency stem cells.

BACKGROUND: Severe combined immunodeficiency (SCID) represents congenital disorders characterized by a deficiency of T cells caused by arrested development in the thymus. Yet the nature of these developmental blocks has remained elusive because of the difficulty of taking thymic biopsy specimens from affected children. OBJECTIVE: We sought to identify the stages of arrest in human T-cell development caused by various major types of SCID. METHODS: We performed transplantation of SCID CD34(+) bone marrow stem/progenitor cells into an optimized NSG xenograft mouse model, followed by detailed phenotypic and molecular characterization using flow cytometry, immunoglobulin and T-cell receptor spectratyping, and deep sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor δ (TRD) loci. RESULTS: Arrests in T-cell development caused by mutations in IL-7 receptor α (IL7RA) and IL-2 receptor γ (IL2RG) were observed at the most immature thymocytes much earlier than expected based on gene expression profiling of human thymocyte subsets and studies with corresponding mouse mutants. T-cell receptor rearrangements were functionally required at the CD4(-)CD8(-)CD7(+)CD5(+) stage given the developmental block and extent of rearrangements in mice transplanted with Artemis-SCID cells. The xenograft model used is not informative for adenosine deaminase-SCID, whereas hypomorphic mutations lead to less severe arrests in development. CONCLUSION: Transplanting CD34(+) stem cells from patients with SCID into a xenograft mouse model provides previously unattainable insight into human T-cell development and functionally identifies the arrest in thymic development caused by several SCID mutations.

The composition and differentiation potential of the duodenal intraepithelial innate lymphocyte compartment is altered in coeliac disease.

OBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56(-)CD127(-), CD56(-)CD127(+), CD56(+)CD127(-) and CD56(+)CD127(+). Here we aimed to gain insight into the potential function of these innate IELs in health and disease. DESIGN: We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). RESULTS: Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127(-) branch expressing high levels of interleukin (IL) 2/15Rß but no IL-21R, and a CD127(+) branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56(-)CD127(-) and CD56(-)CD127(+) innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56(-)CD127(-) IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56(-)CD127(-) IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. CONCLUSIONS: Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII.

TRIF signaling drives homeostatic intestinal epithelial antimicrobial peptide expression.

Recent results indicate a significant contribution of innate immune signaling to maintain mucosal homeostasis, but the precise underlying signal transduction pathways are ill-defined. By comparative analysis of intestinal epithelial cells isolated from conventionally raised and germ-free mice, as well as animals deficient in the adaptor molecules MyD88 and TRIF, the TLR3 and TLR4, as well as the type I and III IFN receptors, we demonstrate significant TLR-mediated signaling under homeostatic conditions. Surprisingly, homeostatic expression of Reg3γ and Paneth cell enteric antimicrobial peptides critically relied on TRIF and, in part, TLR3 but was independent of IFN receptor signaling. Reduced antimicrobial peptide expression was associated with significantly lower numbers of Paneth cells and a reduced Paneth cell maturation and differentiation factor expression in TRIF mutant compared with wild-type epithelium. This phenotype was not transferred to TRIF-sufficient germ-free animals during cohousing. Low antimicrobial peptide expression in TRIF-deficient mice caused reduced immediate killing of orally administered bacteria but was not associated with significant alterations in the overall composition of the enteric microbiota. The phenotype was rapidly restored in a TRIF-independent fashion after transient epithelial damage. Our results identify TRIF signaling as a truly homeostatic pathway to maintain intestinal epithelial barrier function revealing fundamental differences in the innate immune signaling between mucosal homeostasis and tissue repair.

CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas.

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

Hepatic lentiviral gene transfer is associated with clonal selection, but not with tumor formation in serially transplanted rodents.

UNLABELLED: Lentiviral (LV) vectors are promising tools for long-term genetic correction of hereditary diseases. In hematopoietic stem cell gene therapies adverse events in patients due to vector integration-associated genotoxicity have been observed. Only a few studies have explored the potential risks of LV gene therapy targeting the liver. To analyze hepatic genotoxicity in vivo, we transferred the fumarylacetoacetate hydrolase (FAH) gene by LV vectors into FAH((-/-)) mice (n = 97) and performed serial hepatocyte transplantations (four generations). The integration profile (4,349 mapped insertions) of the LV vectors was assessed by ligation-mediated polymerase chain reaction and deep sequencing. We tested whether the polyclonality of vector insertions was maintained in serially transplanted mice, linked the integration sites to global hepatocyte gene expression, and investigated the effects of LV liver gene therapy on the survival of the animals. The lifespan of in vivo gene-corrected mice was increased compared to 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) control animals and unchanged in serially transplanted animals. The integration profile (4,349 mapped insertions) remained polyclonal through all mouse generations with only mild clonal expansion. Genes close to the integration sites of expanding clones may be associated with enhanced hepatocyte proliferation capacity. CONCLUSION: We did not find evidence for vector-induced tumors. LV hepatic gene therapy showed a favorable risk profile for stable and long-term therapeutic gene expression. Polyclonality of hepatocyte regeneration was maintained even in an environment of enforced proliferation.

Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages.

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4(+) CD25(hi) FoxP3(+) T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-ß1 mediates induction of Tregs. TGF-ß1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-ß1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-ß1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC.

Improving the Assessment of Risk Factors Relevant to Potential Carcinogenicity of Gene Therapies: A Consensus Article.

Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.

Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells.

Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in "expanded" groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity.

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery.

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB's catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage.

Next-generation sequencing for minimal residual disease monitoring in acute myeloid leukemia patients with FLT3-ITD or NPM1 mutations.

Systematic assessment of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has been hampered by lack of a reliable, uniform MRD marker applicable to all patients. We evaluated next-generation sequencing (NGS) for MRD assessment in AML patients (n = 80 samples). The ability of NGS technologies to generate thousands of clonal sequences makes it possible to determine the allelic ratio of sequence variants. Using NGS, we were able to determine the allelic ratio of different FLT3-internal tandem duplication (ITD) clones within one patient sample, in addition to resolution of FLT3-ITD insertion site, length, and sequence in a single analysis. Furthermore, NGS allowed us to study emergence of clonal dominance. Parallel assessment of MRD by NGS and quantitative real-time polymerase chain reaction in NPM1 mutated patients was concordant in 95% of analyzed samples (n = 38). The frequency of mutated alleles was linearly quantified by NGS. As NGS sensitivity is scalable depending on sequence coverage, it reflects a highly flexible and reliable tool to assess MRD in leukemia patients.

Identification of a small molecule for enhancing lentiviral transduction of T cells.

Genetic modification of cells using viral vectors has shown huge therapeutic benefit in multiple diseases. However, inefficient transduction contributes to the high cost of these therapies. Several transduction-enhancing small molecules have previously been identified; however, some may be toxic to the cells or patient, otherwise alter cellular characteristics, or further increase manufacturing complexity. In this study, we aimed to identify molecules capable of enhancing lentiviral transduction of T cells from available small-molecule libraries. We conducted a high-throughput flow-cytometry-based screen of 27,892 compounds, which subsequently was narrowed down to six transduction-enhancing small molecules for further testing with two therapeutic lentiviral vectors used to manufacture GSK's clinical T cell therapy products. We demonstrate enhanced transduction without a negative impact on other product attributes. Furthermore, we present results of transcriptomic analysis, suggesting alteration of ribosome biogenesis, resulting in reduced interferon response, as a potential mechanism of action for the transduction-enhancing activity of the lead compound. Finally, we demonstrate the ability of the lead transduction enhancer to produce a comparable T cell product using a 3-fold reduction in vector volume in our clinical manufacturing process, resulting in a predicted 15% reduction in the overall cost of goods.

Pre-treatment with systemic agents for advanced NSCLC elicits changes in the phenotype of autologous T cell therapy products.

The antitumor activity of adoptive T cell therapies (ACT) is highly dependent on the expansion, persistence, and continued activity of adoptively transferred cells. Clinical studies using ACTs have revealed that products that possess and maintain less differentiated phenotypes, including memory and precursor T cells, show increased antitumor efficacy and superior patient outcomes owing to their increased expansion, persistence, and ability to differentiate into effector progeny that elicit antitumor responses. Strategies that drive the differentiation into memory or precursor-type T cell subsets with high potential for persistence and self-renewal will enhance adoptively transferred T cell maintenance and promote durable antitumor efficacy. Because of the high costs associated with ACT manufacturing, ACTs are often only offered to patients after multiple rounds of systemic therapy. An essential factor to consider in producing autologous ACT medicinal products is the impact of the patient's initial T cell fitness and subtype composition, which will likely differ with age, disease history, and treatment with prior anti-cancer therapies. This study evaluated the impact of systemic anti-cancer therapy for non-small cell lung cancer treatment on the T cell phenotype of the patient at baseline and the quality and characteristics of the genetically modified autologous T cell therapy product after manufacturing.

Gene therapy: is IL2RG oncogenic in T-cell development?

The gene IL2RG encodes the gamma-chain of the interleukin-2 receptor and is mutated in patients with X-linked severe combined immune deficiency (X-SCID). Woods et al. report the development of thymus tumours in a mouse model of X-SCID after correction by lentiviral overexpression of IL2RG and claim that these were caused by IL2RG itself. Here we find that retroviral overexpression of IL2RG in human CD34+ cells has no effect on T-cell development, whereas overexpression of the T-cell acute lymphoblastic leukaemia (T-ALL) oncogene LMO2 leads to severe abnormalities. Retroviral expression of IL2RG may therefore not be directly oncogenic--rather, the restoration of normal signalling by the interleukin-7 receptor to X-SCID precursor cells allows progression of T-cell development to stages that are permissive for the pro-leukaemic effects of ectopic LMO2.