Restricted T-cell Receptor Research Articles

AFNT-211: A phase 1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR, a CD8α/β coreceptor, and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors.

TPS8650 Background: Activating mutations in KRAS (including KRAS G12V) are well-described oncogenic drivers in solid tumors. Patients with KRAS driver mutations have a poor prognosis, and most KRAS-mutated cancers lack effective therapies. T cell receptor (TCR)-T cell therapies targeting mutant KRAS have demonstrated proof of concept in the clinic, but duration of response remains a challenge. AFNT-211 represents a novel strategy to address the immunosuppressive tumor microenvironment and improve response rate and duration in solid tumors. AFNT-211 autologous CD4+ and CD8+ T cells are engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; the wildtype CD8α/β coreceptor; and a FAS-41BB switch receptor. The intended mechanism of action is recognition and elimination of KRAS G12V-mutated tumor cells by AFNT-211. AFNT-211 is designed to enhance anti-tumor activity via the following mechanisms of action as shown in preclinical models: (1) the CD8α/β coreceptor enables MHC-I– restricted TCR recognition of the KRAS G12V target by CD4+ T cells; (2) engaging CD4+ helper T cell responses drives enhanced CD8+ T cell cytotoxic activity and prevents T cell exhaustion; (3) the FAS-41BB chimeric switch receptor drives increased T cell activity, circumventing native FAS apoptotic signaling. Methods: This ongoing Phase 1, first-in-human, multicenter, open-label study of AFNT-211 (1) evaluates safety/tolerability, (2) determines the optimal biological dose (OBD) and recommended Phase 2 dose (RP2D), and (3) assesses preliminary antitumor activity. The study consists of 2 parts: dose escalation and dose expansion. Dose escalation is guided by the Bayesian optimal interval Phase 1/2 (BOIN12) design to determine the OBD. The BOIN12 design quantifies the desirability of a dose in terms of toxicity-efficacy tradeoff and adaptively allocates patients to the dose with the highest estimated desirability. The RP2D will be selected based on the BOIN12 design and the totality of benefit/risk assessment. Patients enrolling in dose expansion will be treated at the OBD/RP2D in indication-specific cohorts. This study is conducted in patients aged ≥ 18 years who are HLA-A*11:01-positive with advanced/metastatic solid tumors harboring a KRAS G12V mutation, and intolerance of/progression on at least 1 prior systemic therapy. Patients undergo leukapheresis to collect T cells for the manufacturing of AFNT-211 and receive lymphodepleting chemotherapy prior to AFNT-211 infusion. This is followed by a 28-day dose-limiting toxicity observation period (dose escalation only) and a post-treatment follow-up period for 24 months/ until disease progression. The study is open for recruitment in the United States. Clinical trial information: NCT06105021 .

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes.

The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Abstract P65: Single Cell Profiling Reveals Unique TCR Repertoire and Transcriptomic Signatures of Epstein-Barr Virus-Specific T cells (EBVSTs) Armoured with Chimeric Antigen Receptors (CARs)

Abstract Allogeneic Epstein-Barr Virus-specific T cells (EBVSTs) have thus far been a safe immunotherapy product in the clinic, with little graft-versus-host-disease reported in over 300 recipients to date. Consequently, EBVSTs armoured with chimeric antigen receptors (CARs) have emerged as a promising platform for allogeneic T cell therapies. Believing that a deep characterisation of CAR EBVST cell products can enhance insights into their clinical safety, we performed single cell T cell receptor (TCR) sequencing on CAR EBVSTs, taking CD30.CAR EBVSTs as a prototype. Non-transduced (NT) EBVSTs and CD30.CAR EBVSTs generated from three healthy donors exhibited a narrower, more focused TCR repertoire by diversity indices, compared to resting T cells and CD30.CAR-transduced activated T cells (ATCs) from the same donors. EBVSTs harboured a lower frequency of unique TCR clonotypes and a higher proportion of expanded clones, indicating a tendency toward clonal dominance. Narrower TCR repertoires corresponded to greater IFNγ and TNFα secretion in response to stimulation with EBV peptides, with no cross-reactivity toward other common viruses, and were associated with lower allogeneic reactivity in mixed lymphocyte reactions. To investigate whether the EBVST nature of the product might contribute to better CAR activity, we concurrently performed single cell transcriptomic sequencing on NT EBVSTs, CD30.CAR EBVSTs, and CD30.CAR ATCs. Transcriptomic signatures revealed that each sample type contained two major, distinct populations of CD4+ T cells: proliferative and activated. While the proliferative subsets appeared to be closely related, the activated populations had distinct transcriptional profiles. In particular, CD30.CAR EBVSTs appeared to be more polyfunctional and metabolically poised than CD30.CAR ATCs. The restricted TCR repertoire and apparent functional superiority of CD30.CAR EBVSTs may underlie their safety and efficacy observed in early-stage clinical trials and preclinical models, with the larger implication that these qualities extend to CAR EBVSTs targeting antigens beyond CD30. Citation Format: Lindsay Kua, Ting Wei Lim, Fiona Wong, Pei Yun Teo, Lionel Low, Ivan Horak, Jinmiao Chen, Kar Wai Tan. Single Cell Profiling Reveals Unique TCR Repertoire and Transcriptomic Signatures of Epstein-Barr Virus-Specific T cells (EBVSTs) Armoured with Chimeric Antigen Receptors (CARs) [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P65.

HLA-class II restricted TCR targeting human papillomavirus type 18 E7 induces solid tumor remission in mice

T cell receptor (TCR)-engineered T cell therapy is a promising potential treatment for solid tumors, with preliminary efficacy demonstrated in clinical trials. However, obtaining clinically effective TCR molecules remains a major challenge. We have developed a strategy for cloning tumor-specific TCRs from long-term surviving patients who have responded to immunotherapy. Here, we report the identification of a TCR (10F04), which is human leukocyte antigen (HLA)-DRA/DRB1*09:01 restricted and human papillomavirus type 18 (HPV18) E784-98 specific, from a multiple antigens stimulating cellular therapy (MASCT) benefited metastatic cervical cancer patient. Upon transduction into human T cells, the 10F04 TCR demonstrated robust antitumor activity in both in vitro and in vivo models. Notably, the TCR effectively redirected both CD4+ and CD8+ T cells to specifically recognize tumor cells and induced multiple cytokine secretion along with durable antitumor activity and outstanding safety profiles. As a result, this TCR is currently being investigated in a phase I clinical trial for treating HPV18-positive cancers. This study provides an approach for developing safe and effective TCR-T therapies, while underscoring the potential of HLA class II-restricted TCR-T therapy as a cancer treatment.

Intraepithelial Lymphocytes of the Intestine.

The intestinal epithelium, which segregates the highly stimulatory lumen from the underlying tissue, harbors one of the largest lymphocyte populations in the body, intestinal intraepithelial lymphocytes (IELs). IELs must balance tolerance, resistance, and tissue protection to maintain epithelial homeostasis and barrier integrity. This review discusses the ontogeny, environmental imprinting, Tcell receptor (TCR) repertoire, and function of intestinal IELs. Despite distinct developmental pathways, IEL subsets share core traits including an epithelium-adapted profile, innate-like properties, cytotoxic potential, and limited TCR diversity. IELs also receive important developmental and functional cues through interactions with epithelial cells, microbiota, and dietary components. The restricted TCR diversity of IELs suggests that a limited set of intestinal antigens drives IEL responses, with potential functional consequences. Finally, IELs play a key role in promoting homeostatic immunity and epithelial barrier integrity but can become pathogenic upon dysregulation. Therefore, IELs represent intriguing but underexamined therapeutic targets for inflammatory diseases and cancer. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Integrative Genomic and Transcriptomic Analysis Reveals Targetable Vulnerabilities in Angioimmunoblastic T-Cell Lymphoma

Follicular helper T-cell lymphoma of the angioimmunoblastic type (AITL) is associated with dismal prognosis. We performed functional genomic approaches including whole-exome sequencing (WES; n=119), transcriptomic (n=78) and methylation (n=40) analysis. We identified recurrent mutations in known epigenetic drivers ( TET2, DNMT3A, IDH2 R172), and also identified novel ones (TET3, KMT2D). Somatic mutation of all three epigenetic drivers ( TET2, IDH2, and DNMT3A) was associated with poor prognosis (p<.001). Mutations in genes regulating T-cell receptor (TCR) signaling ( CD28 VAV1, FYN, PLCG1) or activation ( RHOA G17V), and regulators of the PI3K pathway (PIK(3)C members, PTEN,PHLPP-1/-2) were also found. Genome-wide DNA-methylation analysis integrated with mRNA expression profiling also revealed epigenetic alterations in genes regulating TCR-RHOA/B/C or PI3K-signaling. TET2 loss was noted in 85% AITLs and was significantly associated with RHOA G17V, CD28 and IDH2 R172mutations. AITLs lacking RHOA G17V tended to have mutations regulating the JAK-STAT pathway ( JAK2, JAK3, STAT1, STAT3, SOCS1). RNA-seq analysis identified fusion transcripts in genes regulating TCR activation (8%), revealed a restricted TCR repertoire in the majority of cases (a=87%, b=72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated association of B-cells in the tumor-milieu with better prognosis (p=.006), while dendritic cells were associated with worse prognosis (p=.001), which was further validated by immunohistochemistry using CD20, CD68, and CD163 antibodies. RNA-seq and corresponding WES analysis of 12 AITL patient-derived-xenografts (PDX) showed that bi-allelic TET2 mutations, DNMT3A mutations or sub-clonal mutations ( PLCG1 PHLPP2) werepropagated in sequential passages. Gene signatures related to T FH (follicular helper) and T CM (central memory) were also well-maintained in secondary passages in PDX models. Gene signatures of late PDX passages (3 rd-5 th) were enriched with genes related to proliferation and metabolic reprogramming, and in an independent cohort of AITLs, high expression of T3/T5 related signatures was associated with worse outcome (p=0.02/p=0.009). Low mRNA expression of PHLPP2 predicted poor prognosis (p=.03) and engineered PHLPP2 loss showed enhanced PI(3)K activation and FOXO1 inactivation in CD4+ T-cells in-vitro. Thus, we defined the genomic landscape for AITL, which is largely characterized by epigenetic alterations, TCR signaling and PI3K/AKT dysregulation, which may be amenable for therapeutic targeting.

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR–LLL–HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

P06.12.A NLGN4X-SPECIFIC TCR T CELLS TARGETING EXPERIMENTAL GLIOMAS

Abstract BACKGROUND A human leukocyte antigen (HLA)-A*02-restricted tumor associated antigen in the neuroligin 4 X-linked (NLGN4X) protein was found to be specifically overexpressed in human gliomas. Individualized multipeptide vaccination targeted NLGN4X and induced specific cytotoxic T cells responses in patients with newly diagnosed glioblastoma. MATERIAL AND METHODS Post vaccination NLGN4X-tetramer-sorted T cells were subjected to single cell T cell receptor (TCR) sequencing for TCR discovery. The identified TCR was delivered to human T cells (NLGN4X-TCR-T) and functional profiling was performed by flow cytometry and in vitro cytotoxicity assays. NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice were challenged with NLGN4X-expressing experimental gliomas and treated with intracerebroventricular injection of NLGN4X-TCR-T to assess its therapeutic potential. RESULTS We apply for the first time for therapeutic use of an HLA-A*02 restricted vaccine-induced TCR that binds to the NLGN4X antigen. We show the cytotoxic and polyfunctional phenotype of NLGN4X-TCR-T in various cellular models. Intracerebroventricular delivery of NLGN4X-TCR-T prolongs survival and leads to objective radiographic responses in experimental gliomas-bearing NSG MHC I/II KO mice. CONCLUSION NLGN4X-TCR-T demonstrates efficacy in a preclinical experimental glioblastoma model. On a global scale, we provide first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.

Analysis of tumor infiltrating CD4+ and CD8+ CDR3 sequences reveals shared features putatively associated to the anti-tumor immune response.

Tumor-infiltrating lymphocytes (TILs) have predictive and prognostic value in breast cancer (BC) and exert a protective function against tumor growth, indicating that it is susceptible to treatment using adoptive cell transfer of TILs or T cell receptor (TCR)-based therapies. TCR can be used to identify naturally tumor-reactive T cells, but little is known about the differences in the TCR repertoires of CD4+ and CD8+ TILs. TCR high-throughput sequencing was performed using TILs derived from the initial cultures of 11 BC biopsies and expanded and sorted CD4+ and CD8+ TILs as well as using PBMCs from healthy donors expanded and sorted using the same methodology. Physicochemical TCR differences between T cell subsets were observed, as CD4+ TILs presented larger N(D)Nnt TRB sequences and with a higher usage of positively charged residues, although only the latest was also observed in peripheral T cells from healthy individuals. Moreover, in CD4+ TILs, a more restricted TCR repertoire with a higher abundance of similar sequences containing certain amino acid motifs was observed. Some differences between CD4+ and CD8+ TCRs were intrinsic to T cell subsets as can also be observed in peripheral T cells from healthy individuals, while other were only found in TILs samples and therefore may be tumor-driven. Notably, the higher similarity among CD4+ TCRs suggests a higher TCR promiscuity in this subset.

PRAME and CTCFL-reactive TCRs for the treatment of ovarian cancer.

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2'-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.

Examination of resistance to nivolumab monotherapy through single-cell analysis of tumors from patients enrolled in the HCRN GU16-260 study of nivolumab monotherapy.

698 Background: Nivolumab (nivo) monotherapy demonstrated anti-tumor activity in treatment-naïve renal cell carcinoma (RCC) in Part A of HCRN GU16-260 across all IMDC groups and in multiple histologies. Patient tumor samples were collected to characterize the tumor-immune microenvironmental (TME) determinants of effective anti-tumor immunity with nivo. Methods: Eligible patients (with clear cell or non-clear cell histology) underwent tumor biopsy prior to and/or at resistance to nivo monotherapy. Fresh tissue fragments were cryopreserved locally and centrally processed to extract viable single-cells from the RCC TME. Single-cell RNA-sequencing (scRNA-seq) and T cell receptor (TCR)-sequencing (scTCR-seq) was performed on all tumor samples. Gene expression signatures discovered through scRNA-seq were used to interrogate previously published bulk transcriptomic data from the CheckMate-009/010/025 trials of nivo in the treatment-refractory setting. Results: In total, 72,730 viable single-cells (56,900 immune and 15,830 tumor or stromal cells) were sequenced from 17 patients (8 with baseline only, 7 with progression only, and 2 with paired baseline and progression samples) across 7 trial sites. Trajectory inference analysis of tumor-infiltrating T cells revealed a bifurcating trajectory, starting with naïve T cells and ending either in PMCH+ terminally exhausted CD8+ T cells or SLAMF7+ CD8+ T cells. Notably, the SLAMF7+ T cell population expressed high levels of cytotoxic genes (including GZMA, GZMB, GNLY) and markers of tissue residency ( ZNF683/HOBIT and ITGAE/CD103), and had a restricted TCR diversity (normalized Shannon index = 0.57). Among patients with at least 100 sequenced T cells (n = 14), a higher percentage of SLAMF7+ CD8+ T cells (relative to total T cells) was associated with primary resistance (progressive disease [PD] as best response) to nivo (mean percentage in PD [n = 4] patients 32.7%; stable disease [n = 4] patients, 9.1%; complete or partial response [CR/PR; n = 6] patients, 2.2%; p = 0.019 for CR/PR vs PD). A signature derived using genes expressed in the T cell trajectory branch containing the SLAMF7+ CD8+ T cell population was used to interrogate bulk RNA-seq data from 172 pre-treatment tumors from the nivo arms of the CheckMate-009/010/025 trials. The signature score was enriched in patients with PD compared to CR/PR as best response (p = 0.032). Analysis of bulk whole exome sequencing and RNA-seq from patients enrolled in the HCRN GU16-260 is pending. Conclusions: Single-cell transcriptomic analysis uncovered a SLAMF7+ CD8 + T cell population with markers of cytotoxicity and tissue residency that was associated with resistance to nivo monotherapy in RCC. Further, the study highlights that scRNA-seq is a viable scientific strategy for deep correlative analysis in multicenter clinical trials.

A Sampling of Highlights from the Literature.

What We're Reading| January 03 2023 A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors Author & Article Information Online Issn: 2326-6074 Print Issn: 2326-6066 ©2022 American Association for Cancer Research2022American Association for Cancer Research Cancer Immunol Res (2023) 11 (1): 1. https://doi.org/10.1158/2326-6066.CIR-11-1-WWR Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record January 3 2023 Citation A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors. Cancer Immunol Res 1 January 2023; 11 (1): 1. https://doi.org/10.1158/2326-6066.CIR-11-1-WWR Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Myocarditis is the most lethal ICI-associated adverse event (from OpenStax Anatomy and Physiology via Wikimedia Commons) Myocarditis is a rare, often fatal, and poorly mechanistically understood adverse event associated with immune checkpoint inhibitor (ICI) therapy. Axelrod et al. find that in a mouse model of ICI-associated myocarditis—Pdcd1–/–Ctla4+/–mice—CD8+ T cells are necessary for fatal myocarditis to develop. The cardiac-infiltrating CD8+ T cells are highly clonal, with the T-cell receptors (TCR) from three clones recognizing MHC class I–presented α-myosin peptides. The clinical relevance of the data is supported by the detection of α-myosin–specific MHC class I–restricted TCRs in cardiac and skeletal muscle of patients with ICI-associated myocarditis. Axelrod ML, …, Balko JM. Nature 2022 November 16;611:818–26. Research is identifying new ways to enhance the antitumor activity of CAR T cells (from Alex Ritter, Jennifer Lippincott Schwartz, and Gillian Griffiths via NIH Flickr) Many research... You do not currently have access to this content.

Immunotherapy By Targeting CD45 in Mismatched HLA-DP to Treat Patients after Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation (alloSCT) is a curative treatment for patients with hematological malignancies. The anti-tumor or Graft-versus-Leukemia (GvL) effect of alloSCT is mediated by donor T cells targeting patient hematopoietic cells. Donor T cells may, however, also target non-hematopoietic tissues of the patient, thereby inducing Graft-versus-Host Disease (GvHD). Patients without suitable related donors are transplanted with unrelated donors matched for HLA-A, -B, -C, -DRB1, and -DQB1 (10/10 match). HLA-DP alleles are mismatched in most patients transplanted with unrelated donors. HLA class II molecules are mainly expressed on hematopoietic cells, but under inflammatory conditions the expression of HLA class II on non-hematopoietic tissues is upregulated, thereby increasing the risk of GvHD. To reduce this risk and retain the anti-tumor effect, patients transplanted with unrelated donors may be treated with immunotherapy specifically targeting hematopoietic peptides presented by mismatched HLA-DP. CD45 is an attractive source protein for these peptides because of its broad expression in all hematopoietic lineages. Despite CD45 expression, healthy hematopoietic cells after alloSCT are of donor origin and will therefore be spared. To investigate whether CD45 peptides are presented by HLA-DP on hematopoietic cells, peptides were eluted from HLA-DP and measured by tandem mass spectrometry. HLA-DP peptidome data were generated from acute myeloid leukemia (AML) cases as well as hematopoietic (CD45pos) K562 cells separately transduced with 13 different HLA-DP alleles. Peptidome searching revealed one CD45 peptide on K562 cells transduced with HLA-DP2 (DPA1*01:03 & DPB1*02:01) or HLA-DP4 (DPA1*01:03 & DPB1*04:01 or DPB1*04:02) as well as AML cases expressing these HLA-DP alleles. Next, (CD45pos) K562 cells transduced with HLA-DP2 or HLA-DP4 and costimulatory molecules (CD40, CD80, CD86) were used to stimulate CD4 T cells from healthy individuals negative for these HLA-DP alleles. After 14 days of stimulation, CD4 T cells were restimulated and activated CD137+ and CD154+ CD4 T cells were single cell sorted. Growing T cell clones were screened for reactivity against CD45 peptide loaded on non-hematopoietic (CD45neg) HeLa cells transduced with HLA-DP2 or HLA-DP4. Three clones were able to specifically recognize CD45 in HLA-DP2 or HLA-DP4 (Figure 1A). T cell clones M6-5 and M6-6 were specific for CD45 in HLA-DP2, and clone M6-7 recognized the CD45 peptide in HLA-DP4. No cross-reactivity was observed against other HLA-DP alleles. In addition to HLA-DP transduced K562 cells, all T cell clones specifically reacted against HLA-DP2 or HLA-DP4 positive AML cases. We next investigated whether the T cell receptors (TCRs) of the T cell clones could be used to engineer and redirect CD4 T cells to CD45-derived peptides in mismatched HLA-DP. The HLA-DP2 restricted TCRs of M6-5 and M6-6 and HLA-DP4 restricted TCR of M6-7 were sequenced, cloned and transferred to CD4 T cells from three HLA-DP2 and HLA-DP4 negative healthy donors. TCR-T cells showed specific reactivity against K562 transduced with HLA-DP2 or HLA-DP4, whereas HeLa cells with these HLA-DP alleles were not recognized (Figure 1B, example of one donor). In conclusion, we identified three TCRs for a CD45 peptide presented by HLA-DP2 or HLA-DP4 that are able to target hematopoietic cells. The TCRs can be used for CD4 T cell immunotherapy to treat patients with hematological malignancies after HLA-DP mismatched alloSCT. Figure 1. CD45 as a target for specific CD4 T cells. (A) CD4 T cell clones recognizing CD45 in HLA-DP2 or HLA-DP4 on AML. (B) M6-5, M6-6 and M6-7-TCR transduced CD4 T cells recognize HLA-DP2 or -DP4 transduced K562 (CD45pos) but not HeLa (CD45neg) cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of a distinct NK-like hepatic T-cell population activated by NKG2C in a TCR-independent manner.

The liver provides a unique niche of lymphocytes enriched with a large proportion of innate-like T cells. However, the heterogeneity and functional characteristics of the hepatic T-cell population remain to be fully elucidated. We obtained liver sinusoidal mononuclear cells from the liver perfusate of healthy donors and recipients with HBV-associated chronic liver disease (CLD) during liver transplantation. We performed a CITE-seq analysis of liver sinusoidal CD45+ cells in combination with T cell receptor (TCR)-seq and flow cytometry to examine the phenotypes and functions of liver sinusoidal CD8+ T cells. We identified a distinct CD56hiCD161-CD8+ T-cell population characterized by natural killer (NK)-related gene expression and a uniquely restricted TCR repertoire. The frequency of these cells among the liver sinusoidal CD8+ T-cell population was significantly increased in patients with HBV-associated CLD. Although CD56hiCD161-CD8+ T cells exhibit weak responsiveness to TCR stimulation, CD56hiCD161-CD8+ T cells highly expressed various NK receptors, including CD94, killer immunoglobulin-like receptors, and NKG2C, and exerted NKG2C-mediated NK-like effector functions even in the absence of TCR stimulation. In addition, CD56hiCD161-CD8+ T cells highly respond to innate cytokines, such as IL-12/18 and IL-15, in the absence of TCR stimulation. We validated the results from liver sinusoidal CD8+ T cells using intrahepatic CD8+ T cells obtained from liver tissues. In summary, the current study found a distinct CD56hiCD161-CD8+ T-cell population characterized by NK-like activation via TCR-independent NKG2C ligation. Further studies are required to elucidate the roles of liver sinusoidal CD56hiCD161-CD8+ T cells in immune responses to microbial pathogens or liver immunopathology. The role of different immune cell populations in the liver is becoming an area of increasing interest. Herein, we identified a distinct T-cell population that had features similar to those of natural killer (NK) cells - a type of innate immune cell. This distinct population was expanded in the livers of patients with chronic liver disease and could thus have pathogenic relevance.

Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer

SummaryA patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×109 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02–restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.)

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.

A Sampling of Highlights from the Literature

T cells expressing WT137–45-specific TCRs kill AML cells (by BruceBlaus via Wikimedia Commons)T cells engineered to express T-cell receptors (TCR) targeting intracellular tumor antigens have potential for treating cancer. Two studies identify HLA-A*02:01–restricted TCRs specific for Wilms’ tumor antigen 1 (WT1) peptide WT137–45 as candidates for clinical translation. Ruggiero et al. show that T cells engineered to express such TCRs significantly reduce tumor growth in xenograft models of acute myeloid leukemia (AML), acute lymphoblastic leukemia, and glioblastoma. Lahman et al. show that generation of WT137–45 is immunoproteasome independent, which is important because AML recurrence in one patient they treated with T cells expressing an HLA-A*02:01–restricted, WT126–134-specific TCR occurred because of loss of immunoproteasome processing of WT1.Ruggiero E, …, Bonini C. Sci Trans Med 2022 February 9;14. DOI:10.1126/scitranslmed.abg8027.Lahman MC, …, Greenberg PD. Sci Trans Med 2022 February 9;14. DOI:10.1126/scitranslmed.abg8070.PERK signaling promotes M2 macrophage immunosuppressive function (by Noahtsmith via Wikimedia Commons)Intratumoral macrophage phenotype is determined by the signals the cells receive from the tumor microenvironment. Raines et al. identify new molecular pathways that promote the activation and immunosuppressive function of M2 macrophages. They find that tumor-associated macrophages from patients with lung carcinoma are characterized by increased activity of protein kinase RNA-like ER kinase (PERK) signaling. This promotes an immunosuppressive phenotype in macrophages through metabolic reprograming and histone demethylation. Inhibition of PERK signaling reduces growth of B16-F10 tumors. This is associated with reduced numbers and immunosuppressive activity of intratumoral macrophages, suggesting PERK signaling inhibition as a potential therapeutic approach to target macrophages.Raines LN, …, Huang SCC. Nature Immunol 2022;23:431–445.CRISPR technology allows for characterization of T-cell functional regulators (by Clipart Panda via Clipart Panda)Identifying regulators of cytokine production upon cellular activation has had its limitations. Using novel CRISPR screens, Schmidt et al. identify regulators of IL2 and IFNγ production by T cells. Distinct cytokine profiles are identified and characterize T cells during various states of activation. The data highlight screening that can be performed to determine how T cells can be reprogramed and could be used to better design immunotherapy for the treatment of cancer.Schmidt R, …, Marson A. Science 2022 February 4;375. DOI:10.1126/science.abj4008.Prediction modeling could be used to identify patients who will respond to immune checkpoint blockade (by Eva K. via Wikimedia Commons)Identifying markers of therapy response and patient outcome is an unmet clinical need. Patil et al. find that plasma cells in tumoral tertiary lymphoid structures are a biomarker of overall survival in patients treated with immune checkpoint blockade (ICB), an effect independent of tumor CD8+ T cells and PD-L1 expression. Asrir et al. identify tumor-associated high endothelial venules as key players for lymphocyte entry into tumors and associated with survival and response to combination ICB. These two studies highlight key immune mechanisms and biomarkers that could be potentially used to improve treatment strategies for patients with cancer.Asrir A, …, Girard JP. Cancer Cell 2022 March 14;40:318–334.e9.Patil NS, …, Shames DS. Cancer Cell 2022 March 14;40:289–300.e4.CAR T cells are being developed for many types of cancer (from Fig. 7B of Kantari-Mimoun et al., Cancer Immunol Res 2021)Patient data following chimeric-antigen receptor (CAR) T-cell therapy are critical to ongoing clinical development. Through longitudinal analysis of two patients with chronic lymphocytic leukemia who achieved lasting complete remission after CD19-specific CAR T-cell therapy in 2010, Melenhorst et al. find that CAR T cells persist for more than a decade, with a population of highly activated CD4+ CAR T cells dominating in both patients. Majzner et al. report that three of four patients with H3K27M-mutant diffuse midline glioma who received GD2-specific CAR T cells intravenously and then intracerebroventricularly had clinical and radiographic improvement. These studies provide new insights that will inform continued CAR T-cell development for hematological malignancies and solid tumors.Melenhorst JJ, …, June CH. Nature 2022;602:503–509.Majzner RG, …, Monje M. Nature 2022 February 7;14. DOI:10.1038/s41586-022-04489-4.The mycobiome facilitates pancreatic tumor progression (Pancreas, in Opera Omnia via Wellcome Collection)T helper 2 cells (TH2) and innate lymphoid cells 2 (ILC2) infiltrate pancreatic ductal adenocarcinomas (PDAC) and secrete cytokines that promote tumorigenesis. Alam et al. identify KrasG12D-regulated IL33 as a mediator of TH2 and ILC2 recruitment in mouse models of PDAC. Fungal species found in the gut and tumor microenvironment induce tumor cell IL33 secretion by activating the dectin-1 receptor and downstream Src–Syk–CARD9 pathway. Anti-fungal treatment reduces TH2 and ILC2 infiltration and reduces tumor growth, highlighting a novel mechanism by which the PDAC tumor mycobiome can regulate the pro-tumorigenic inflammatory response and suggesting a potential therapeutic strategy.Alam A, …, Dey P. Cancer Cell 2022 February 14;40:153–167.E11.

HLA-A∗02:01 restricted Tcell receptors against the highly conserved SARS-CoV-2 polymerase cross-react with human coronaviruses.

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαβ sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαβ constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.

Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL.Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure)Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively.Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data.To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens.To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response.Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes.Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. [Display omitted] DisclosuresLoughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

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