CD8 Coreceptor Research Articles

Anti-CD8/IL-15 (N72D)/sushi fusion protein: A promising strategy for improvement of cancer immunotherapy

BackgroundTo overcome the limitations of IL-15 and to improve the efficacy of IL-15 in immunotherapy, several strategies have been introduced. ObjectiveThe objective of this study was to generate and evaluate a novel anti-CD8/IL-15 (N72D)/Sushi fusion protein with the potential to target CD8+ T cells and enhance functionality of CD8+ T cells against tumor cells. MethodsIn this connection, a novel fusokine that contains IL-15(N72D), a Sushi domain, and anti-CD8 single-chain fragment variable (scFv) was designed. The size accuracy and binding potency of the isolated protein were assessed using western blotting and indirect surface staining. Following purification, the potential function of the anti-CD8/IL-15(N72D)/Sushi fusion protein in the induction of proliferation and cytotoxicity of CD8+ T cells was evaluated. ResultsIn-silico analysis revealed that fusokine is structurally stable, correctly folded and can interact with the CD8 co-receptor. Both fusokine and IL-15(N72D)/Sushi were produced in CHO-S cell line with a final concentration of 18.43 mg/l and 12.64 mg/l respectively. Fusokine bound to 97.6 % of CD8+ T cells and significantly induced T cell proliferation and cytotoxic potential in peripheral blood mononuclear cells (PBMCs) in a time dependent manner. Compared to both the control and the IL-15 (N72D)/sushi treated groups, fusokine showed superior potential in CD8+ T cell functionality. ConclusionAnti-CD8/IL-15(N72D)/Sushi has the ability to effectively target CD8+ T cells, promote lymphocyte proliferation and induce cytotoxicity against tumor cells. Due to its promising properties, it could be considered as a new potential immunotherapy approach.

Hyperlipidemia interferes with the behavior of variant NKT cells

Abstract Background NKT cells significantly influence atherosclerosis development. However, inconsistent findings on the function of the NKT cells and the invariant subset in atherosclerosis remains a challenge. Moreover, there is no clarity on the role and phenotype of the variant (vNKT) subset in atherosclerosis. Purpose To understand the role of the vNKT subsets in HFD-induced atherosclerosis. Methods We extracted the data for meta-analysis to understand the role of the whole NKT and iNKT cells in atherosclerosis. PRISMA guidelines were followed for the meta-analysis. In addition, C57BL/6 mice (WT) were fed with HFD for 20 weeks from 6-8 weeks of age. vNKTs were identified as CD3+NK1.1+CD1d-tetramer- within the CD3+NK1.1+ NKT cell population in both liver and spleen by flow cytometry. Intracellular cyotkines staining was performed. Statistical differences inthe means of two groups are calculated by two-tailed Student’s t-test with nonparametric Mann-Whitney tests. P-value<0.05 was considered significant. All data represented as mean±SEM. Results The NKT cell numbers increased significantly in the spleen, lymph nodes, blood, and liver in the HFD-fed mice fed from 1.5 to 24 weeks in Apoe-/-, Ldlr-/- in comparison to wild-type mice (SMD, 2.51 [95% CI, 1.42, 3.61]). Contrary to this, the iNKT cell numbers and the iNKT TCR gene expression levels, decreased significantly in the HFD-fed mice (SMD, -2.04 [95% CI, -3.34, -0.75]). The reduction in iNKT cell numbers is also supported by the clinical data from the AMI patients when compared to healthy controls [SMD = -1.81, 95% CI = -2.89, -0.74] (Figure 1). In HFD-fed C57BL/6 hyperlipidemic mice, significantly increased levels of total cholesterol (HFD vs Chow; 226.5±29.66 vs 118.5±10.56 mg/dL, n=4) and LDLc (44.56±11.96 vs 16.69±1.13 mg%, n=4) was observed. The number of vNKTs in the liver was significantly increased by 46% (HFD vs Chow 28.35±2.28 vs 19.41±1.36, n=6-7) (Figure 2A). However, the CD8+ vNKTs were significantly decreased by 68.6% in the liver (4.72±0.99 vs 15.06±0.96) and the CD4+ vNKTs decreased more than two times in the HFD-fed WT liver (7.39±0.76 vs 18.28±0.36) (Figure 2B). Also, the intracellular IFN-γ+ vNKT cells doubled in their numbers in the HFD-fed spleen (14.31±1.39 vs 6.367±0.87) compared to the chow-fed mice (Figure 2C). Conclusion(s) Meta-analysis revealed dual nature of the NKT cells and its invariant subsets in atherosclerosis, since the whole NKTs increase whereas the iNKTs decrease in number. The vNKT subset actively participates in immune modulation in the HFD-induced hyperlipidemia by increasing their number, as well as by differentially regulating CD8 and CD4 co-receptors between chow and HFD. HFD induced a pro-inflammatory nature in the splenic vNKTs but not in liver vNKTs, that in turn may aggravate other immune cells. This data confirms the dual nature of the NKT subsets and reveals a crucial systemic involvement of vNKT cells in the progression of dyslipidemia.

Natural TCRs targeting KRASG12V display fine specificity and sensitivity to human solid tumors.

BACKGROUNDNeoantigens derived from KRASMUT have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore KRASMUT immunogenicity and the properties of 4 T cell receptors (TCRs) specific for KRASG12V restricted to the HLA-A3 superfamily of class I alleles.METHODSA phase 1 clinical vaccine trial targeting KRASMUT was conducted. TCRs targeting KRASG12V restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics.RESULTSVaccination against KRASMUT resulted in the priming of CD8+ and CD4+ T cell responses. KRASG12V -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against KRASWT. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01-restricted TCR-redirected CD8+ T cells exhibited potent lytic activity against KRASG12V cancers, while only HLA-A*11:01-restricted TCR-T CD4+ T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All KRASG12V-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell.CONCLUSIONThis study identifies KRASG12V-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies.TRIAL REGISTRATIONClinicalTrials.gov NCT03592888.FUNDINGAACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.

Structural basis for mouse LAG3 interactions with the MHC class II molecule I-Ab

The immune checkpoint protein, Lymphocyte activation gene-3 (LAG3), binds Major Histocompatibility Complex Class II (MHC-II) and suppresses T cell activation. Despite the recent FDA approval of a LAG3 inhibitor for the treatment of melanoma, how LAG3 engages MHC-II on the cell surface remains poorly understood. Here, we determine the 3.84 Å-resolution structure of mouse LAG3 bound to the MHC-II molecule I-Ab, revealing that domain 1 (D1) of LAG3 binds a conserved, membrane-proximal region of MHC-II spanning both the α2 and β2 subdomains. LAG3 dimerization restricts the intermolecular spacing of MHC-II molecules, which may attenuate T cell activation by enforcing suboptimal signaling geometry. The LAG3-MHC-II interface overlaps with the MHC-II-binding site of the T cell coreceptor CD4, implicating disruption of CD4-MHC-II interactions as a mechanism for LAG3 immunosuppressive function. Lastly, antibody epitope analysis indicates that multiple LAG3 inhibitors do not recognize the MHC-II-binding interface of LAG3, suggesting a role for functionally distinct mechanisms of LAG3 antagonism in therapeutic development.

Structure, function, and immunomodulation of the CD8 co-receptor.

Expressed on the surface of CD8+ T cells, the CD8 co-receptor is a key component of the T cells that contributes to antigen recognition, immune cell maturation, and immune cell signaling. While CD8 is widely recognized as a co-stimulatory molecule for conventional CD8+ αβ T cells, recent reports highlight its multifaceted role in both adaptive and innate immune responses. In this review, we discuss the utility of CD8 in relation to its immunomodulatory properties. We outline the unique structure and function of different CD8 domains (ectodomain, hinge, transmembrane, cytoplasmic tail) in the context of the distinct properties of CD8αα homodimers and CD8αβ heterodimers. We discuss CD8 features commonly used to construct chimeric antigen receptors for immunotherapy. We describe the molecular interactions of CD8 with classical MHC-I, non-classical MHCs, and Lck partners involved in T cell signaling. Engineered and naturally occurring CD8 mutations that alter immune responses are discussed. The applications of anti-CD8 monoclonal antibodies (mABs) that target CD8 are summarized. Finally, we examine the unique structure and function of several CD8/mAB complexes. Collectively, these findings reveal the promising immunomodulatory properties of CD8 and CD8 binding partners, not only to uncover basic immune system function, but to advance efforts towards translational research for targeted immunotherapy.

The Structural Basis for Recognition of Human Leukocyte Antigen Class I Molecules by the Pan-HLA Antibody W6/32.

The central immunological role of HLA class I (HLA-I) in presenting peptide Ags to cellular components of the immune system has been the focus of intense study for >60 y. A confounding factor in the study of HLA-I has been the extreme polymorphism of these molecules. The mAb W6/32 has been a fundamental reagent bypassing the issue of polymorphism by recognizing an epitope that is conserved across diverse HLA-I allotypes. However, despite the widespread use of W6/32, the epitope of this Ab has not been definitively mapped. In this study, we present the crystal structure of the Fab fragment of W6/32 in complex with peptide-HLA-B*27:05. W6/32 bound to HLA-B*27:05 beneath the Ag-binding groove, recognizing a discontinuous epitope comprised of the α1, α2, and α3 domains of HLA-I and β2-microglobulin. The epitope comprises a region of low polymorphism reflecting the pan-HLA-I nature of the binding. Notably, the W6/32 epitope neither overlaps the HLA-I binding sites of either T cell Ag receptors or killer cell Ig-like receptors. However, it does coincide with the binding sites for leukocyte Ig-like receptors and CD8 coreceptors. Consistent with this, the use of W6/32 to block the interaction of NK cells with HLA-I only weakly impaired inhibition mediated by KIR3DL1, but impacted HLA-LILR recognition.

HA-1–targeted T-cell receptor T-cell therapy for recurrent leukemia after hematopoietic stem cell transplantation

AbstractRelapse is the leading cause of death after allogeneic hematopoietic stem cell transplantation (HCT) for leukemia. T cells engineered by gene transfer to express T cell receptors (TCR; TCR-T) specific for hematopoietic-restricted minor histocompatibility (H) antigens may provide a potent selective antileukemic effect post-HCT. We conducted a phase 1 clinical trial using a novel TCR-T product targeting the minor H antigen, HA-1, to treat or consolidate treatment of persistent or recurrent leukemia and myeloid neoplasms. The primary objective was to evaluate the feasibility and safety of administration of HA-1 TCR-T after HCT. CD8+ and CD4+ T cells expressing the HA-1 TCR and a CD8 coreceptor were successfully manufactured from HA-1–disparate HCT donors. One or more infusions of HA-1 TCR-T following lymphodepleting chemotherapy were administered to 9 HCT recipients who had developed disease recurrence after HCT. TCR-T cells expanded and persisted in vivo after adoptive transfer. No dose-limiting toxicities occurred. Although the study was not designed to assess efficacy, 4 patients achieved or maintained complete remissions following lymphodepletion and HA-1 TCR-T, with 1 patient still in remission at >2 years. Single-cell RNA sequencing of relapsing/progressive leukemia after TCR-T therapy identified upregulated molecules associated with T-cell dysfunction or cancer cell survival. HA-1 TCR-T therapy appears feasible and safe and shows preliminary signals of efficacy. This clinical trial was registered at ClinicalTrials.gov as #NCT03326921.

Thymocyte migration and emigration

Hematopoietic precursors (HPCs) entering into the thymus undergo a sequential process leading to the generation of a variety of T cell subsets. This developmental odyssey unfolds in distinct stages within the thymic cortex and medulla, shaping the landscape of T cell receptor (TCR) expression and guiding thymocytes through positive and negative selection. Initially, early thymic progenitors (ETPs) take residence in the thymic cortex, where thymocytes begin to express their TCR and undergo positive selection. Subsequently, thymocytes transition to the thymic medulla, where they undergo negative selection. Both murine and human thymocyte development can be broadly classified into distinct stages based on the expression of CD4 and CD8 coreceptors, resulting in categorizations as double negative (DN), double positive (DP) or single positive (SP) cells. Thymocyte migration to the appropriate thymic microenvironment at the right differentiation stage is pivotal for the development and the proper functioning of T cells, which is critical for adaptive immune responses. The journey of lymphoid progenitor cells into the T cell developmental pathway hinges on an ongoing dialogue between the differentiating cell and the signals emanating from the thymus niche. Herein, we review the contribution of the key factors mentioned above for the localization, migration and emigration of thymocytes.

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

Canonical α/β T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/β TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Abstract 3601: Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells

Abstract Patients with late-stage kidney cancer have poor long-term survival rates. Our lab isolated a low affinity CD8 dependent TCR named BZ-4 from CTLs of a patient who had immune-mediated regression of renal cell carcinoma (RCC) following an allogeneic stem cell transplant and found this TCR recognized a human endogenous retrovirus type E (HERV-E) derived 10-mer peptide, CT-RCC-1. The BZ-4 clone belongs to the Vβ7.1 family and binds to CT-RCC-1 in an HLA-A11 dependent manner. Adoptive infusion of CD8+ BZ-4 transduced T cells is currently being explored in an NIH phase I trial (NCT03354390). Because CD4+ T cells have been shown to augment the anti-tumor effects of TCR-modified CD8+ T cells, we hypothesized that CD4+ T cells genetically engineered to co-express BZ-4 and CD8 would recognize and kill RCC tumors expressing the CT-RCC-1 and would improve the in vivo proliferation and tumor cytotoxicity of CD8+ BZ-4 T cells. CD4+ T cells isolated from healthy donor PBMCs were transfected via Sleeping Beauty electroporation with the control MART-1 reactive TCR (DMF5), BZ-4, or BZ-4 alongside the CD8 co-receptor (BZ-4-8). CD8+ T cells were similarly transfected with DMF5 or BZ-4. Following magnetic bead enrichment, 66% to 84% (mean 77%) of transfected T cells stained positive for Vβ7.1. TCR engineered T cells were subsequently tested for their ability to lyse a) luciferase transduced HLA A-11 expressing RCC tumors and b) a T2 cell line modified to express both HLA A-11 and luciferase that was peptide-pulsed with decreasing concentrations (5 × 10−1, 5 × 10−2, 5 × 10−3 & 5 × 10−4 ug/mL) of CT-RCC-1. CD4+ T cells expressing BZ-4 or BZ-4-8 as well as CD8+ T cells expressing BZ-4 or DMF5 were used either alone or in combination in cytotoxicity assays. For combined T cell groups, the total effector/target ratio was maintained at the same ratio as individual T cell groups. For T-2 targets, CD4+ BZ-4 T cells exhibited no cytolytic activity in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which were highly cytolytic to target cells. For RCC tumor targets, CD4+ BZ-4 T cells exhibited no cytolytic activity at 48, 72 and 96 hours in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which exhibited high levels of tumor killing at these timepoints. Remarkably, CD4+ BZ-4-8 T cells alone or in combination with CD8+ BZ-4 T cells demonstrated tumor cytotoxicity that was comparable to that observed with CD8+ BZ-4 T cells alone. In conclusion, these data show that CD4+ BZ-4 T cells transfected with the CD8 co-receptor acquire the ability to lyse RCC tumors at levels comparable to that observed in CD8+ BZ-4 T cells. Additional experiments are planned to evaluate whether mixtures of CD8+ BZ-4 T cells with CD4+ BZ-4-8 T cells will augment the ability of TCR modified T cells to proliferate in vivo and kill human RCC tumors compared to CD8+ BZ-4 T cells alone in tumor bearing NSG mice. Citation Format: Muna Igboko, Long Chen, Stefan Barisic, Elena Cherkasova, David Allan, Mala Chakraborty, Joseph Clara, Angie Parrizzi, Kyra Carney, Rosa Nadal Rios, Robert Reger, Richard Childs. Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3601.

Abstract 1329: Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors

Abstract T cell receptor (TCR)-T based therapies, such as MAGE-A4 TCR-T cells, have shown compelling data demonstrating effective infiltration into and targeting of solid tumors with clinical responses across various solid cancers. However, heterogeneity and down-regulation of target antigen and HLA expression limit the durability and curative potential of these types of treatments. On the other hand, owing to their clinical potency, favorable safety profile and applicability as “off the shelf” therapy, natural killer (NK) cells have emerged as promising modalities in recent years. Unguided NK cells have shown limited potential against solid cancers, due to lacking infiltration into tumors. With our proprietary TCR-NK platform, we combine the solid tumor targeting capabilities of TCRs with the pan-cancer sensing and cytotoxic potential of highly potent killer cells, redirecting and arming NK cells with a fully functional TCR-CD3 complex. Here we present our lead product, an optimized TCR-NK cell product, ZI-MA4-1a, expressing an affinity enhanced TCR (KVL-a) directed to the HLA class I-restricted clinically validated cancer-testis antigen MAGE-A4, that is broadly expressed across solid tumors. The KVL-a TCR was successfully engineered from a naturally occurring wild-type TCR, increasing the affinity to MAGE-A4-peptide-HLA complex and resulting in enhanced recognition of tumor cells expressing the target antigen. ZI-MA4-1a is produced by transducing healthy peripheral blood derived NK cells with the full CD3 complex, the KVL-a TCR and a CD8 coreceptor. The process has been optimized to enrich TCR-NK cells, increasing process robustness, diminishing the impact of donor variability, and producing a higher performing TCR-NK cell product. We show the dual functionality of ZI-MA4-1a: TCR driven cytotoxicity as well as innate NK sensing of antigen knockout or HLA negative cancers. We demonstrate killing of heterogenous tumor cell models in vitro, as well as the multifunctionality of ZI-MA4-1a with killing, degranulation and multicytokine secretion. Interestingly, we observe enhanced proliferation of ZI-MA4-1a cells after activation by MAGE-A4 expressing tumor cells, indicating that TCR-NK cells acquire further proliferative capacity when the TCR is triggered. Finally, we demonstrate higher activity of ZI-MA4-1a compared to KVL-a and benchmark TCR transduced T cells against antigen positive targets, and activity against antigen negative tumor cells escaping TCR-T cell recognition. Citation Format: Sarah Vollmers, Artur Cieslar-Pobuda, Justyna Kmiecik, Giulia Malachin, Michelle L. Sætersmoen, Frida Høsøien Haugen, Margherita Boieri, Maja Sandve, Dennis Clement, Ines Cardoso, Pimthanya Wanichawan, Martha E. Haugstøyl, Anja Oldenburg, Liliane Christ, Pranav Oberoi, Luz Mora-Velandia, Anders Holm, Namir J. Hassan, Emilie Gauthy, Julia Ino, Sylvie Pollmann, Luise U. Weigand. Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1329.

Abstract 1859: Diverse CD28-binding IgG and heavy chain-only antibodies for T-cell engager development

Abstract Engagement of the CD28 co-receptor by T-cell engager (TCE) molecules can enhance activation, proliferation, and anti-tumor activities, particularly in immunosuppressive tumor microenvironments. In this study, we present data on a diverse panel of CD28-binding IgG and heavy chain-only (HCAb) antibodies for T-cell co-stimulation. Our diverse panel of CD28-binding antibodies add co-stimulatory building blocks to our TCE repertoire. Using single B cell-screening, we identified fully human IgG and HCAb binders to human and non-human primate CD28 with a high degree of sequence diversity. We selected a subset of antibodies for expression and assessed binding to both human and cynomolgus CD28-expressing cells. These antibodies displayed a wide range of binding avidities in the sub-nanomolar to micromolar range, and epitope binning analysis identified seven epitope communities. Co-stimulatory activity for a subset of molecules was assessed in vitro. Antibodies displayed a wide range of crosslinked T-cell activation with the majority showing no activation independent of crosslinking. Data presented here describe a diverse set of CD28-binding antibodies ready for engineering into co-stimulatory TCEs. Combining these molecules with our TCE platform or other T-cell activating strategies will enable development of novel TCE modalities with co-stimulatory function for diverse tumor targets. Citation Format: Katherine Lam, Shirley Zhi, Lucas Kraft, Elena Vigano, Wei, Cristina Faralla, Esther Odekunle, Kelly Bullock, Erin Marshall, Melissa Cid, Grace Leung, Lindsay DeVorkin, Sherie Duncan. Diverse CD28-binding IgG and heavy chain-only antibodies for T-cell engager development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1859.

Kidney double positive T cells have distinct characteristics in normal and diseased kidneys

Multiple types of T cells have been described and assigned pathophysiologic functions in the kidneys. However, the existence and functions of TCR+CD4+CD8+ (double positive; DP) T cells are understudied in normal and diseased murine and human kidneys. We studied kidney DPT cells in mice at baseline and after ischemia reperfusion (IR) and cisplatin injury. Additionally, effects of viral infection and gut microbiota were studied. Human kidneys from patients with renal cell carcinoma were evaluated. Our results demonstrate that DPT cells expressing CD4 and CD8 co-receptors constitute a minor T cell population in mouse kidneys. DPT cells had significant Ki67 and PD1 expression, effector/central memory phenotype, proinflammatory cytokine (IFNγ, TNFα and IL-17) and metabolic marker (GLUT1, HKII, CPT1a and pS6) expression at baseline. IR, cisplatin and viral infection elevated DPT cell proportions, and induced distinct functional and metabolic changes. scRNA-seq analysis showed increased expression of Klf2 and Ccr7 and enrichment of TNFα and oxidative phosphorylation related genes in DPT cells. DPT cells constituted a minor population in both normal and cancer portion of human kidneys. In conclusion, DPT cells constitute a small population of mouse and human kidney T cells with distinct inflammatory and metabolic profile at baseline and following kidney injury.

A combined biochemical and cellular approach reveals Zn2+-dependent hetero- and homodimeric CD4 and Lck assemblies in T cells

The CD4 or CD8 co-receptors' interaction with the protein-tyrosine kinase Lck initiates the tyrosine phosphorylation cascade leading to Tcell activation. A critical question is: to what extent are co-receptors and Lck coupled? Our contribution concerns Zn2+, indispensable for CD4- and CD8-Lck formation. We combined biochemical and cellular approaches to show that dynamic fluctuations of free Zn2+ in physiological ranges influence Zn(CD4)2 and Zn(CD4)(Lck) species formation and their ratio, although the same Zn(Cys)2(Cys)2 cores. Moreover, we demonstrated that the affinity of Zn2+ to CD4 and CD4-Lck species differs significantly. Increased intracellular free Zn2+ concentration in Tcells causes higher CD4 partitioning in the plasma membrane. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly.

A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature

Lymphocyte-specific protein tyrosine kinase (LCK) is an SRC-family kinase critical for initiation and propagation of T-cell antigen receptor (TCR) signaling through phosphorylation of TCR-associated CD3 chains and recruited downstream molecules. Until now, only one case of profound T-cell immune deficiency with complete LCK deficiency [1] caused by a biallelic missense mutation (c.1022T>C, p.L341P) and three cases of incomplete LCK deficiency [2] caused by a biallelic splice site mutation (c.188-2A>G) have been described. Additionally, deregulated LCK expression has been associated with genetically undefined immune deficiencies and hematological malignancies. Here, we describe the second case of complete LCK deficiency in a 6-month-old girl born to consanguineous parents presenting with profound T-cell immune deficiency. Whole exome sequencing (WES) revealed a novel pathogenic biallelic missense mutation in LCK (c.1393T>C, p.C465R), which led to the absence of LCK protein expression and phosphorylation, and a consecutive decrease in proximal TCR signaling. Loss of conventional CD4+ and CD8+ αβT-cells and homeostatic T-cell expansion was accompanied by increased γδT-cell and Treg percentages. Surface CD4 and CD8 co-receptor expression was reduced in the patient T-cells, while the heterozygous mother had impaired CD4 and CD8 surface expression to a lesser extent. We conclude that complete LCK deficiency is characterized by profound T-cell immune deficiency, reduced CD4 and CD8 surface expression, and a characteristic TCR signaling disorder. CD4 and CD8 surface expression may be of value for early detection of mono- and/or biallelic LCK deficiency.

CD3 downregulation identifies high-avidity human CD8 T cells.

CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.

Legend or Truth: Mature CD4+CD8+ Double-Positive T Cells in the Periphery in Health and Disease.

The expression of CD4 and CD8 co-receptors defines two distinct T cell populations with specialized functions. While CD4+ T cells support and modulate immune responses through different T-helper (Th) and regulatory subtypes, CD8+ T cells eliminate cells that might threaten the organism, for example, virus-infected or tumor cells. However, a paradoxical population of CD4+CD8+ double-positive (DP) T cells challenging this paradigm has been found in the peripheral blood. This subset has been observed in healthy as well as pathological conditions, suggesting unique and well-defined functions. Furthermore, DP T cells express activation markers and exhibit memory-like features, displaying an effector memory (EM) and central memory (CM) phenotype. A subset expressing high CD4 (CD4bright+) and intermediate CD8 (CD8dim+) levels and a population of CD8bright+CD4dim+ T cells have been identified within DP T cells, suggesting that this small subpopulation may be heterogeneous. This review summarizes the current literature on DP T cells in humans in health and diseases. In addition, we point out that strategies to better characterize this minor T cell subset's role in regulating immune responses are necessary.

αβ-T cell receptor transduction gives superior mitochondrial function to γδ-T cells with promising persistence

Adoptive cell therapy using allogeneic γδ-T cells is a promising option for off-the-shelf Tcell products with a low risk of graft-versus-host disease (GVHD). Long-term persistence may boost the clinical development of γδ-T cell products. In this study, we found that genetically modified Vγ9+Vδ2+ Tcells expressing a tumor antigen-specific αβ-TCR and CD8 coreceptor (GMC) showed target-specific killing and excellent persistence. To determine the mechanisms underlying these promising effects, we investigated metabolic characteristics. Cytokine secretion by γδ-TCR-stimulated nongene-modified γδ-T cells (NGMCs) and αβ-TCR-stimulated GMCs was equally suppressed by a glycolysis inhibitor, although the cytokine secretion of αβ-TCR-stimulated GMCs was more strongly inhibited by ATP synthase inhibitors than that of γδ-TCR-stimulated NGMCs. Metabolomic and transcriptomic analyses, flow cytometry analysis using mitochondria-labeling dyes and extracellular flux analysis consistently suggest that αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function. In conclusion, αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function with promising persistence.

High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

A dynamic biomimetic model of the membrane-bound CD4-CD3-TCR complex during pMHC disengagement

The coordinated (dis)engagement of the membrane-bound T cell receptor (TCR)-CD3-CD4 complex from the peptide-major histocompatibility complex (pMHC) is fundamental to TCR signal transduction and T cell effector function. As such, an atomic-scale understanding would not only enhance our basic understanding of the adaptive immune response but would also accelerate the rational design of TCRs for immunotherapy. In this study, we explore the impact of the CD4 coreceptor on the TCR-pMHC (dis)engagement by constructing a molecular-level biomimetic model of the CD3-TCR-pMHC and CD4-CD3-TCR-pMHC complexes within a lipid bilayer. After allowing the system complexes to equilibrate (engage), we use steered molecular dynamics to dissociate (disengage) the pMHC. We find that 1) the CD4 confines the pMHC closer to the T cell by 1.8 nm at equilibrium; 2) CD4 confinement shifts the TCR along the MHC binding groove engaging a different set of amino acids and enhancing the TCR-pMHC bond lifetime; 3) the CD4 translocates under load increasing the interaction strength between the CD4-pMHC, CD4-TCR, and CD4-CD3; and 4) upon dissociation, the CD3-TCR complex undergoes structural oscillation and increased energetic fluctuation between the CD3-TCR and CD3-lipids. These atomic-level simulations provide mechanistic insight on how the CD4 coreceptor impacts TCR-pMHC (dis)engagement. More specifically, our results provide further support (enhanced bond lifetime) for a force-dependent kinetic proofreading model and identify an alternate set of amino acids in the TCR that dominate the TCR-pMHC interaction and could thus impact the design of TCRs for immunotherapy.