Anti-TCR antibody treatment activates a novel population of nonintestinal CD8 alpha alpha+ TCR alpha beta+ regulatory T cells and prevents experimental autoimmune encephalomyelitis

CD8alphaalpha+CD4-TCRalphabeta+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8alphaalpha+CD4-TCRalphabeta+ regulatory T cells (CD8alphaalpha Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vbeta8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vbeta8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8alphaalpha regulatory T cells (Tregs) and found that they predominantly use the TCR Vbeta6 gene segment. In vivo injection of anti-TCR Vbeta6 mAb results in activation of the CD8alphaalpha Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8alphaalpha Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.     

Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder.

There are experimental data which suggest that the primary immune effector cell responsible for maintaining immune surveillance against the outgrowth of EBV-transformed B cells in humans is the CTL, but in vivo proof of this is lacking. In this study we perform a series of cellular and molecular assays to characterize an autologous, endogenous immune response against a transplantation-associated, monoclonal, EBV+ posttransplant lymphoproliferative disorder (PTLD). Following allogeneic bone marrow transplantation, a patient developed a monoclonal PTLD of donor B cell origin. With a decrease in immune suppression, we document the emergence of endogenous, donor-derived CD3+CD8+ CTLs, followed by regression of the PTLD. The TCR Vbeta repertoire went from a polyclonal pattern prior to the development of PTLD to a restricted TCR Vbeta pattern during the outgrowth and regression of PTLD. Donor-derived CD3+CD8+ T lymphocytes displayed MHC class I-restricted cytolytic activity against the autologous EBV+ B cells ex vivo without additional in vitro sensitization. The striking temporal relationship between the endogenous expansion of a TCR Vbeta-restricted, CD3+CD8+ population of MHC class I-restricted CTL, and the regression of an autologous monoclonal PTLD, provides direct evidence in humans that endogenous CD3+CD8+ CTLs can be responsible for effective immune surveillance against malignant transformation of EBV+ B cells.     

Molecular analysis of TCR and peptide/MHC interaction using P18-I10-derived peptides with a single D-amino acid substitution

For the structural analysis of T-cell receptor (TCR) and peptide/MHC interaction, a series of peptides with a single amino acid substitution by a corresponding D-amino acid, having the same weight, size, and charge, within P18-I10 (aa318-327: RGPGRAFVTI), an immunodominant epitope of HIV-1 IIIB envelope glycoprotein, restricted by the H-2Dd class I MHC molecule, has been synthesized. Using those peptides, we have observed that the replacement at positions 324F, 325V, 326T, and 327I with each corresponding D-amino acid induced marked reduction of the potency to sensitize targets for P18-I10-specific murine CD8+ cytotoxic T lymphocytes (CTLs), LINE-IIIB, recognition. To analyze further the role of amino acid at position 325, the most critical site for determining epitope specificity, we have developed a CTL line [LINE-IIIB(325D)] and its offspring clones specific for the epitope I-10(325v) having a D-valine (v) at position 325. Taking advantage of two distinct sets of CD8+ CTLs restricted by the same Dd, three-dimensional structural analysis on TCR and peptide/MHC complexes by molecular modeling was performed, which indicates that the critical amino acids within the TCRs for interacting with 325V or 325v appear to belong to the complementarity-determining region 1 but not to the complementarity-determining region 3 of Vbeta chain.     

Regulation of immunity by a novel population of Qa-1-restricted CD8alphaalpha+TCRalphabeta+ T cells

Regulatory mechanisms involving CD8+ T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRalphabeta+ Tregs that control activated Vbeta8.2+ CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8alphaalpha homodimer and recognize a peptide from a conserved region of the TCR Vbeta8.2 chain in the context of the Qa-1a (CD8alphaalpha Tregs). They secrete type 1 cytokines but not IL-2. CD8alphaalpha Tregs kill activated Vbeta8.2+ but not Vbeta8.2- or naive T cells. The CD8alphaalpha Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.     

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

Defective CD8+ T cell activation and cytolytic function in the absence of LFA-1 cannot be restored by increased TCR signaling.

Signaling through the TCR as well as engagement of costimulatory molecules are required for efficient T cell activation and progression into differentiated effector cells. The beta2 integrin LFA-1 (CD11a/CD18) has been implicated in TCR costimulation as well as in cell-cell adhesion function, but its exact role is still ambiguous. The present study focuses on the requirement for LFA-1 in CD8+ T cell activation and effector function using LFA-1-deficient cells expressing the 2C transgenic TCR as a model system. The lack of LFA-1 expression in 2C T cells resulted in severely diminished proliferative response toward allogeneic BALB/c splenocytes. Increase in TCR signaling alone by pulsing stimulators with high affinity peptides, p2Ca or QL9, had minimal effects in restoring proliferation. Addition of exogenous IL-2, however, enhanced the effect of peptide pulsing on proliferation of LFA-1-deficient 2C T cells. LFA-1-deficient 2C CTLs generated from alloantigen stimulation exhibited a defective cytotoxic activity when tested on a variety of target cells. Cytolysis could be improved, but not fully rectified by peptide pulsing of target cells. Thus, in the 2C TCR model, LFA-1 has a requisite role for optimal CD8+ T cell activation and effector function, which cannot be overcome by increasing peptide/MHC density on either the APCs or target cells, respectively.     

TCR rearrangement in lymphocytes infiltrating melanoma metastases after administration of autologous dinitrophenyl-modified vaccine

Administration of a vaccine consisting of autologous melanoma cells modified with a hapten, dinitrophenyl (DNP), induces T cell infiltration of metastatic sites. We have reported an analysis of these infiltrating T cells, indicating that certain TCR-Vbeta gene segments are greatly overexpressed. In this study, we investigate the rearrangement of the TCR-Vbeta as well as the junctional diversity in T cells infiltrating melanoma metastases following treatment with DNP vaccine. In 19 of 26 control specimens, V-D-J length analysis showed the expected polyclonal patterns. In contrast, postvaccine tumors from 9 of 10 patients showed dominant peaks of V-D-J junction size in one or more Vbeta families. Dominant peaks were seen most frequently in six Vbeta families (Vbeta7, 12, 13, 14, 16, and 23) and were never seen in seven others. Further analysis of the oligoclonal Vbeta products showed dominant peaks in the J region as well. Of particular interest was the finding that Vbeta and Jbeta peaks were similar in inflamed metastases obtained at different times or from different sites from the same patient. Although 6 of 10 patients expressed HLA-A1, there was no common pattern of TCR rearrangements among them. Finally, the amplified PCR products from seven of these specimens were cloned and sequenced and the amino acid sequence of the complementarity-determining region 3 was deduced. In six of seven specimens, the same complementarity-determining region 3 sequence was repeated in at least two clones and in five of seven in at least three clones. Our study indicates that DNP vaccine induces the expansion of particular T cell clones that may be agents of its antitumor effects.     

Activated human γδ T cells induce peptide-specific CD8+ T-cell responses to tumor-associated self-antigens

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

Reconstitution of CD8+ T cells by retroviral transfer of the TCR alpha beta-chain genes isolated from a clonally expanded P815-infiltrating lymphocyte

Gene transfer of TCR alphabeta-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vbeta subfamily usage of CD8(+) TIL in the tumor sites demonstrated that T cells using Vbeta10 efficiently infiltrated and expanded clonally. The TCR alpha- and beta-chain sequences derived from a tumor-infiltrating CD8(+)/Vbeta10(+) single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR alphabeta-chain genes were retrovirally introduced into CD8(+) T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2L(d) mAb. Furthermore, T cells containing both TCR alpha- and beta-chains, but not TCR beta-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8(+) T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.     

Differential requirement for tapasin in the presentation of leader- and insulin-derived peptide antigens to Qa-1b-restricted CTLs

The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.     

Broad TCR usage in functional HIV-1-specific CD8+ T cell expansions driven by vaccination during highly active antiretroviral therapy

During chronic HIV-1 infection, continuing viral replication is associated with impaired proliferative capacity of virus-specific CD8+ T cells and with the expansion and persistence of oligoclonal T cell populations. TCR usage may significantly influence CD8+ T cell-mediated control of AIDS viruses; however, the potential to modulate the repertoire of functional virus-specific T cells by immunotherapy has not been explored. To investigate this, we analyzed the TCR Vbeta usage of CD8+ T cells populations which were expanded following vaccination with modified vaccinia virus Ankara expressing a HIV-1 gag/multiepitope immunogen (MVA.HIVA) in HIV-1-infected patients receiving highly active antiretroviral therapy. Vaccinations induced the re-expansion of HIV-1-specific CD8+ T cells and these showed broad TCR Vbeta usage which was maintained for at least 1 year in some individuals. By contrast, virus-specific CD8+ T cell populations in the same donors which failed to expand after vaccination and in unvaccinated controls were oligoclonal. Simultaneously, we observed that CD8+ T cells recognizing vaccine-derived HIV-1 epitopes displayed enhanced capacity to proliferate and to inhibit HIV-1 replication in vitro, following MVA.HIVA immunizations. Taken together, these data indicate that an attenuated viral-vectored vaccine can modulate adaptive CD8+ T cell responses to HIV-1 and improve their antiviral functional capacity. The potential therapeutic benefit of this vaccination approach warrants further investigation.     

An MHC class Ib-restricted TCR that cross-reacts with an MHC class Ia molecule

TCR transgenic 6C5 T cells recognize an insulin B chain epitope presented by the nonclassical class I MHC molecule, Qa-1(b). Positive selection of these T cells was shown previously to require Qa-1(b). Despite dedicated specificity for Qa-1(b), evidence presented in the current study indicates that 6C5 T cells can cross-recognize a classical class I molecule. Clonal deletion was observed unexpectedly in 6C5.H-2(bxq) mice, which do not express I-E MHC class II molecules and thus should not be subject to superantigen-mediated negative selection. 6C5 T cells were observed to respond in vivo and in vitro to spleen cells from allogeneic H-2(q) mice, and specificity was mapped to D(q). Evidence was obtained for direct recognition of D(q), rather than indirect presentation of a D(q)-derived peptide presented by Qa-1(b). Polyclonal CD8(+) T cells from class Ia-deficient K(b)D(b-/-) mice reacted in vitro to allogeneic spleen cells with an apparent frequency comparable to conventional class Ia-restricted T cells. Our results provide a clear example of a Qa-1-specific TCR that can cross-react with a class Ia molecule and evidence supporting the idea that this may be a common property of T cells selected by class Ib molecules.     

Role of the complementarity-determining region 3 (CDR3) of the TCR-beta chains associated with the V alpha 14 semi-invariant TCR alpha-chain in the selection of CD4+ NK T Cells

The NK1.1(+)TCRalphabeta(int) CD4(+), or double negative T cells (NK T cells) consist of a mixture of CD1d-restricted and CD1d-unrestricted cells. The relationships between CD4(+)NK1.1(+) T cells and conventional T cells are not understood. To compare their respective TCR repertoires, NK1.1(+)TCRalphabeta(int), CD4(+) T cells have been sorted out of the thymus, liver, spleen, and bone marrow of C57BL/6 mice. Molecular analysis showed that thymus and liver used predominantly the Valpha14-Jalpha281 and Vbeta 2, 7, and 8 segments. These cells are CD1d restricted and obey the original definition of NK T cells. The complementarity-determining region 3 (CDR3) sequences of the TCR Vbeta8.2-Jbeta2.5 chain of liver and thymus CD4(+) NK T cells were determined and compared with those of the same rearrangements of conventional CD4(+) T cells. No amino acid sequence or usage characteristic of NK T cells could be evidenced: the Vbeta8.2-Jbeta2.5 diversity regions being primarily the same in NK T and in T cells. No clonal expansion of the beta-chains was observed in thymus and liver CD1d-restricted CD4(+)NK T cells, suggesting the absence of acute or chronic Ag-driven stimulation. Molecular analysis of the TCR used by Valpha14-Jalpha281 transgenic mice on a Calpha(-/-) background showed that the alpha-chain can associate with beta-chains using any Vbeta segment, except in NK T cells in which it paired predominately with Vbeta 2, 7, and 8(+) beta-chains. The structure of the TCR of NK T cells thus reflects the affinity for the CD1d molecule rather than a structural constraint leading to the association of the invariant alpha-chain with a distinctive subset of Vbeta segment.     

Chronic modulation of the TCR repertoire in the lymphoid periphery

Using TCR V beta 5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4+V beta 5+ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8+V beta 5+ T cells transit through a CD8lowV beta 5low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4-CD8-V beta 5+ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR V alpha repertoire of each of these cellular compartments in both V beta 5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of V alpha families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.     

Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity.However,cholesterol metabolism as a key component of this regulation remains largely unexplored.Herein,we found that the low-density lipoprotein receptor (LDLR),which has been previously identified as a transporter for cholesterol,plays a pivotal role in regulating CD8+ T cell antitumor activity.Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion,we also found a non-canonical function of LDLR in CD8+ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling,thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs).Furthermore,we found that the tumor microenvironment (TME) downregulates CD8+ T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs.Moreover,genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the suppressive effect on CD8+ T cells and consequently inhibit tumor progression.While previously established as a hypercholesterolemia target,this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.