TCRγ4δ1-engineered αßT cells exhibit effective antitumor activity.

T cell engineering with T cell receptors (TCRs) specific for tumors plays an important role in adoptive T-cell transfer (ATC) therapy for cancer. Here, we present a novel strategy to redirect peripheral blood-derived αßT cells against tumors via TCRγ4δ1 gene transduction. The broad-spectrum anti-tumor activity of TCRδ1 cells in innate immunity is dependent on CDR3δ1. TCRγ4δ1-engineered αßT cells were prepared by lentiviral transduction and characterized by analyzing in vitro and in vivo cytotoxicity to tumors, ability of proliferation and cytokine production, and their potential role in autoimmunity. Results show TCRγ4δ1 genes were transduced to approximately 36% of polyclonal αßT cells. TCRγ4δ1-engineered αßT cells exhibited an effective in-vitro TCRγδ-dependent cytotoxicity against various tumor cells via the perforin-granzyme pathway. They also showed a strong proliferative capacity and robust cytokine production. TCRγ4δ1-engineered αßT cells neither expressed mixed TCR dimers nor bound/killed normal cells in vitro. More importantly, adoptive transfer of TCRγ4δ1-engineered αßT cells into nude mice bearing a human HepG2 cell line significantly suppressed tumor growth. Our results demonstrate a novel role for TCRγ4δ1 in gene therapy and ATC for cancer.

Human γδ T cells augment antigen presentation in Listeria Monocytogenes infection.

Circulating γδ T cells in healthy individuals rapidly respond to bacterial and viral pathogens. Many studies have demonstrated that γδ T cells are activated and expanded by Listeria monocytogenes (L.monocytogenes), a foodborne bacterial pathogen with high fatality rates. However, the roles of γδ T cells during L.monocytogenes infection are not clear. In the present study, we characterized the morphological characteristics of phagocytosis in γδ T cells after L.monocytogenes infection using transmission electron microscopy. Results show activation markers including HLA-DR and lymph node-homing receptor CCR7 on γδ T cells were upregulated after stimulation via L.monocytogenes. Significant proliferation and differentiation of primary αß T cells was also observed after co-culture of peripheral blood mononuclear cells with γδ T cells anteriorly stimulated by L.monocytogenes. L.monocytogenes infection decreased the percentage of γδ T cells in mouse IELs and increased MHC-II expression on the surface of γδ T cells in vivo. Our findings shed light on antigen presentation of γδ T cells during L.monocytogenes infection.

Vav1-phospholipase C-γ1 (Vav1-PLC-γ1) pathway initiated by T cell antigen receptor (TCRγδ) activation is required to overcome inhibition by ubiquitin ligase Cbl-b during γδT cell cytotoxicity.

T cell antigen receptor γδ (TCRγδ) and natural killer group 2, member D (NKG2D) are two crucial receptors for γδT cell cytotoxicity. Compelling evidences suggest that γδT cell cytotoxicity is TCRγδ-dependent and can be co-stimulated by NKG2D. However, the molecular mechanism of underlying TCRγδ-dependent activation of γδT cells remains unclear. In this study we demonstrated that TCRγδ but not NKG2D engagement induced lytic granule polarization and promoted γδT cell cytotoxicity. TCRγδ activation alone was sufficient to trigger Vav1-dependent phospholipase C-γ1 signaling, resulting in lytic granule polarization and effective killing, whereas NKG2D engagement alone failed to trigger cytotoxicity-related signaling to overcome the inhibitory effect of Cbl-b; therefore, NKG2D engagement alone could not induce effective killing. However, NKG2D ligation augmented the activation of γδT cell cytotoxicity through the Vav1-phospholipase C-γ1 pathway. Vav1 overexpression or Cbl-b knockdown not only enhanced TCRγδ activation-initiated killing but also enabled NKG2D activation alone to induce γδT cell cytotoxicity. Taken together, our results suggest that the activation of γδT cell cytotoxicity requires a strong activation signal to overcome the inhibitory effect of Cbl-b. Our finding provides new insights into the molecular mechanisms underlying the initiation of γδT cell cytotoxicity and likely implications for optimizing γδT cell-based cancer immunotherapy.

Ectopically expressed human tumor biomarker MutS homologue 2 is a novel endogenous ligand that is recognized by human γδ T cells to induce innate anti-tumor/virus immunity.

Human (h) MutS homologue 2, a nuclear protein, is a critical element of the DNA mismatch repair system. Our previous studies suggest that hMSH2 might be a protein ligand for TCRγδ. Here, we show that hMSH2 is ectopically expressed on a large panel of epithelial tumor cells. We found that hMSH2 interacts with both TCRγδ and NKG2D and contributes to Vδ2 T cell-mediated cytolysis of tumor cells. Moreover, recombinant human MSH2 protein stimulates the proliferation and IFN-γ secretion of Vδ2 T cells in vitro. Finally, hMSH2 expression is induced on the cell surface of Epstein-Barr virus-transformed lymphoblastoid cell lines, and the induction increases the sensitivity of these lymphoblastoid cell lines to γδ T cell-mediated cytolysis. Our data suggest that hMSH2 functions as a tumor-associated or virus infection-related antigen recognized by both Vδ2 TCR and NKG2D, and it plays a role in eliciting the immune responses of γδ T cells against tumor- and virus-infected cells. The recognition of ectopic surface-expressing endogenous antigen by TCRγδ and NKG2D may be an important mechanism of innate immune response to carcinogenesis and viral infection.

Ectopic expression of human MutS homologue 2 on renal carcinoma cells is induced by oxidative stress with interleukin-18 promotion via p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways.

Human MutS homologue 2 (hMSH2), a crucial element of the highly conserved DNA mismatch repair system, maintains genetic stability in the nucleus of normal cells. Our previous studies indicate that hMSH2 is ectopically expressed on the surface of epithelial tumor cells and recognized by both T cell receptor γδ (TCRγδ) and natural killer group 2 member D (NKG2D) on Vδ2 T cells. Ectopically expressed hMSH2 could trigger a γδ T cell-mediated cytolysis. In this study, we showed that oxidative stress induced ectopic expression of hMSH2 on human renal carcinoma cells. Under oxidative stress, both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways have been confirmed to mediate the ectopic expression of hMSH2 through the apoptosis-signaling kinase 1 (ASK1) upstream and activating transcription factor 3 (ATF3) downstream of both pathways. Moreover, renal carcinoma cell-derived interleukin (IL)-18 in oxidative stress was a prominent stimulator for ectopically induced expression of hMSH2, which was promoted by interferon (IFN)-γ as well. Finally, oxidative stress or pretreatment with IL-18 and IFN-γ enhanced γδ T cell-mediated cytolysis of renal carcinoma cells. Our results not only establish a mechanism of ectopic hMSH2 expression in tumor cells but also find a biological linkage between ectopic expression of hMSH2 and activation of γδ T cells in stressful conditions. Because γδ T cells play an important role in the early stage of innate anti-tumor response, γδ T cell activation triggered by ectopically expressed hMSH2 may be an important event in immunosurveillance for carcinogenesis.

Generation of human regulatory gammadelta T cells by TCRgammadelta stimulation in the presence of TGF-beta and their involvement in the pathogenesis of systemic lupus erythematosus.

As a component of the innate immune cell population, γδ T cells are involved in tumor immunosurveillance and host defense against viral invasion. In this study, we demonstrated a novel function of human γδ T cells as regulatory cells by detecting their suppressive effect on the proliferation of autologous naive CD4(+) T cells. These regulatory γδ T cells (γδ Tregs) could be generated in vitro by stimulating with anti-TCRγδ in the presence of TGF-ß and IL-2. Similar to CD4(+)Foxp3(+) Tregs, γδ Tregs also expressed Foxp3. Additionally, they primarily belonged to the Vδ1 subset with a CD27(+)CD25(high) phenotype. Furthermore, these γδ Tregs showed an immunoregulatory activity mainly through cell-to-cell contact. Importantly, this γδ regulatory population decreased in the peripheral blood of systemic lupus erythematosus patients, suggesting a potential mechanism in understanding the pathogenesis of systemic lupus erythematosus.

Flanking V and J sequences of complementary determining region 3 of T cell receptor (TCR) δ1 (CDR3δ1) determine the structure and function of TCRγ4δ1.

The γδ T cell receptor (TCR) differs from immunoglobulin and αß TCR in its overall binding mode. In human, genes δ1, δ2, and δ3 are used for TCRδ chains. Previously, we have studied antigen binding determinants of TCRδ2 derived from dominant γδ T cells residing in peripheral blood. In this study we have investigated the critical determinants for antigen recognition and TCR function in TCRδ1 originated from gastric tumor-infiltrating γδ T lymphocytes using three independent experimental strategies including complementary determining region 3 (CDR3) of TCRδ1 (CDR3δ1)-peptide mediated binding, CDR3δ1-grafted TCR fusion protein-mediated binding, and TCRγ4δ1- and mutant-expressing cell-mediated binding. All three approaches consistently showed that the conserved flanking V and J sequences but not the diverse D segment in CDR3δ1 determine the antigen binding. Most importantly, we found that mutations in the V and J regions of CDR3δ1 also abolish the assembly of TCR and TCR-CD3 complexes in TCRγ4δ1-transduced J.RT3-T3.5 cells. Together with our previous studies on CDR3δ2 binding, our finding suggests that both human TCRδ1 and TCRδ2 recognize antigen predominately via flanking V and J regions. These results indicate that TCRγδ recognizes antigens using conserved parts in their CDR3, which provides an explanation for a diverse repertoire of γδTCRs only recognizing a limited number of antigens.

The NKG2D ligand ULBP4 binds to TCRgamma9/delta2 and induces cytotoxicity to tumor cells through both TCRgammadelta and NKG2D.

UL16-binding proteins (ULBPs) belong to a family of ligands for NKG2D activating receptor of human natural killer (NK) cells. We previously reported that RAET1E2, a soluble isoform of the RAET1E (ULBP4), inhibits NKG2D-mediated NK cytotoxicity. In this study, we examined whether ULBP4 could be recognized by gammadeltaT cells via TCRgammadelta. Here we show that immobilized soluble ULBP4 (rULBP4) induces the proliferation of human ovarian epithelial carcinoma- or colonic carcinoma-derived Vdelta2(+) T cells in vitro. These Vdelta2(+) T cells secrete Th1 cytokines and display a strong cytolytic activity toward ULBP4-transfected targets. We also show that ULBP4 binds to a soluble chimeric protein containing TCRgamma9/delta2 and activates TCR(-) Jurkat T cells transfected with TCRgamma9/delta2. Moreover, both TCRgammadelta and NKG2D are involved in ULBP4-induced activation and cytotoxicity of gammadeltaT cells. We found that ULBP4 is expressed not only on human tumor cells, but also on Epstein-Barr virus (EBV)-infected peripheral blood cells. Taken together, our data suggest that ULBP4 functions as a ligand for both TCRgammadelta and NKG2D and may play a key role in immune surveillance of tumor development and clearance of viral infection.

The recognition of gammadelta TCR to protein antigen does not depend on the hydrophobic I97 residue of CDR3delta.

The crystal structure analysis demonstrated that the hydrophobic amino acid residue (isolecuine/leucine/valine) at conserved position 97 of Vdelta2 TCR plays an important role in recognizing the non-peptide antigen. But its importance to protein antigen remains unclear until now. In the present study, we focus on the role of hydrophobic amino acid residue at conserved position 97 of Vdelta2 TCR in complementarity determining region (CDR)3delta-mediated binding to protein antigen. We employed CDR3delta peptide and membrane-engineered gammadelta TCR as detecting molecules with mutated 97 hydrophobic amino acid residue in CDR3delta (nominated as OT10), a Vdelta2 CDR3 sequence derived from tumor infiltrating lymphocytes in ovarian epithelial carcinoma (OEC). Binding assays revealed that OT10 peptide and membrane-engineered gammadelta TCR (gammadelta TCR transfected cells with OT10 sequence) could bind specifically ovarian tumor cell line (SKOV3). The mutant analysis indicated that any amino acid substitution at position deltaI97 could abolish the response of the transfected cells to iso-butylamine, a known non-peptide antigen of gammadelta T cells. But amino acid substitution of isoleucine at position delta97 did not change the responsiveness of gammadelta TCR transfected cell to protein antigen. Our data suggested that a mechanism other than non-peptide antigen might mediate the recognition of Vdelta2gammadelta T cells for protein antigen. This finding may provide a possibility that gammadelta TCR recognize different ligands in diversity manners.

Identification of amino acids in highly pathogenic avian influenza H5N1 virus hemagglutinin that determine avian influenza species specificity.

To test the role of neutralizing antibodies (nAbs) and receptor adaptation in interspecies transmission of influenza virus, two H5N1 strains, isolated from human and avian hosts, with four amino acid differences in hemagglutinin (HA) and seven HA mutations were studied. We found that a mutation at amino acid position 90 in the H5N1 HA, outside the receptor-binding domain (RBD), could simultaneously induce changes in the RBD conformation to escape from nAb binding and alter the receptor preference through long-range regulation. This mutation was deemed a "key event" for interspecies transmission. It is likely a result of positive selection caused by antibodies, allowing the original invasion by new species-specific variants. A mutation at amino acid position 160 in the RBD only induced a change in receptor preference. This mutation was deemed a "maintaining adaptation", which ensured that influenza virus variants would be able to infect new organisms of a different species successfully. The mutation is the result of adaptation caused by the receptor. Our results suggest that continuing occurrence of these two types of mutations made the variants persist in the new host species.

[Selection of culture media for the mass production of gamma delta T cells used in adoptive immunotherapy].

OBJECTIVE: To select the optimal culture media for the mass production of gamma delta T cells used in adoptive immunotherapy. METHODS: Three different culture media (RPMI-1640, AIM-V, and OpTmizer, with or without autologous serum) were used to culture gamma delta T cells. The survival rate, purity, proliferation efficiency, and biological functions of the expanded gamma delta T cells were examined and compared. RESULTS: The survival rate of gamma delta T cells expanded in RPMI-1640 decreased over culture time. The purities of gamma delta T cells cultured in AIM-V or OpTmizer with or without serum were higher than those cultured in RPMI-1640. After two weeks of culture in the absence of serum, the purity and proliferation efficiency of gamma delta T cells cultured in OpTmizer were significantly higher than those cultured in RPMI-1640 (P < 0.05) and AIM-V (P < 0.05). gamma delta T cells in different culture media had similar CD107a expression and tumor necrosis factor-alpha production (P > 0.05). However, cells expanded in RPMI-1640 exhibited significantly weaker cytotoxicity against Daudi lymphoma cells than those expanded in OpTmizer (P < 0.05) and AIM-V (P < 0.05). CONCLUSION: Due to low serum-dependence, high proliferation efficiency, and favorable biology function of expanded cells, OpTmizer is the most suitable medium for the mass production of gamma delta T cells used in adoptive immunotherapy.

Antigen specificity of gammadelta T cells depends primarily on the flanking sequences of CDR3delta.

The structural basis that determines the specificity of gammadelta T cell receptor (TCR) recognition remains undefined. Our previous data show that the complementary determining region of human TCRdelta (CDR3delta) is critical to ligand binding. Here we used linear and configurational approaches to examine the roles of V, N-D-N, or J regions in CDR3delta-mediated antigen recognition. Surprisingly, we found that the binding activities of CDR3delta from different gammadelta TCRs to their target tissues and ligands depend on the conserved flanking sequences (V and J) but not as much on the D region of CDR3delta fragment. We further defined the key residues in the V and J regions of CDR3delta fragments, including the cysteine residue in the V fragment and the leucine residue in the J fragment that determine their ligand binding specificity. Our results demonstrate that TCRdelta primarily uses conserved flanking regions to bind ligands. This finding may provide an explanation for the limited number of gammadelta TCR ligands that have as yet been identified.

Four novel ULBP splice variants are ligands for human NKG2D.

UL16-binding proteins [ULBPs, also termed as retinoic acid early transcripts (RAET1) molecules] are frequently expressed by malignant transformed cells and stimulate anti-tumor immune responses mediated by NKG2D-positive NK cells, CD8(+) alphabeta T cells and gammadelta T cells in vitro and in vivo. In this study, we identified four novel functional splice variants of ULBPs including ULBP4-I, ULBP4-II, ULBP4-III and RAET1G3 in HepG2 liver carcinoma cells, WISH human amnion cells, Hep-2 larynx carcinoma cells and K562 leukemia cells, respectively, by reverse transcription-PCR and T vector cloning strategy. Analysis of alignments of amino acid sequences of the splice variants illustrated that there were important modifications between splice variants and their individual parental ULBP. All ULBP4 splice variants (ULBP4-I, ULBP4-II and ULBP4-III) were type 1 membrane-spanning molecules and had the ability to bind with human NKG2D receptor in vitro. Ectopic expressions of ULBP4 and ULBP4 splice variants resulted in the enhanced cytotoxic sensitivity of target cells against NK cells, which could be blocked by anti-NKG2D mAb. Moreover, co-culture-free soluble forms of ULBP4 splice variants (their alpha1 + alpha2 ectodomains) and RAET1G3 (soluble splice variant of RAET1G2) with NK cells down-regulated the cell surface expression of NKG2D. Finally, immobilized in a plate-bound form of RAET1G3 stimulated NK cells to secrete IFN-gamma. Taken together, all the identified functional splice variants will help to advance our knowledge regarding the overall functions of ULBP gene family.

miR-125b-5p and miR-99a-5p downregulate human γδ T-cell activation and cytotoxicity.

As an important component of innate immunity, human circulating γδ T cells function in rapid responses to infections and tumorigenesis. MicroRNAs (miRNAs) play a critical regulatory role in multiple biological processes and diseases. Therefore, how the functions of circulating human γδ T cells are regulated by miRNAs merits investigation. In this study, we profiled the miRNA expression patterns in human peripheral γδ T cells from 21 healthy donors and identified 14 miRNAs that were differentially expressed between peripheral αß T cells and γδ T cells. Of the 14 identified genes, 7 miRNAs were downregulated, including miR-150-5p, miR-450a-5p, miR-193b-3p, miR-365a-3p, miR-31-5p, miR-125b-5p and miR-99a-5p, whereas the other 7 miRNAs were upregulated, including miR-34a-5p, miR-16-5p, miR-15b-5p, miR-24-3p, miR-22-3p, miR-22-5p and miR-9-5p, in γδ T cells compared with αß T cells. In subsequent functional studies, we found that both miR-125b-5p and miR-99a-5p downregulated γδ T cell activation and cytotoxicity to tumor cells. Overexpression of miR-125b-5p or miR-99a-5p in γδ T cells inhibited γδ T cell activation and promoted γδ T cell apoptosis. Additionally, miR-125b-5p knockdown facilitated the cytotoxicity of γδ T cells toward tumor cells in vitro by increasing degranulation and secretion of IFN-γ and TNF-α. Our findings improve the understanding of the regulatory functions of miRNAs in γδ T cell activation and cytotoxicity, which has implications for interventional approaches to γδ T cell-mediated cancer therapy.

Evaluation of human major histocompatibility complex class I chain-related A as a potential target for tumor imaging.

To investigate the feasibility of using human major histocompatibility complex class I chain-related A (MICA) as a target for tumor imaging diagnosis, 10C6, a monoclonal antibody (mAb) that specifically recognizes MICA in vitro, was labeled with (99m)Tc (technetium) and administered into mice bearing MICA-positive human ovarian epithelial carcinoma line SKOV3. Measurement of organ-specific radioactivity showed that tumor accumulated radioactivity continuously, while the uptake in the other organs decreased over time. Scintigram showed that the tumor became clearly visible at 24h post-injection of radio-labeled 10C6 mAb. These results suggest that MICA is a promising target for tumor imaging and mAb 10C6 may be used clinically for early tumor diagnosis.

Rwdd1, a thymus aging related molecule, is a new member of the intrinsically unstructured protein family.

We had previously identified a novel protein termed Rwdd1 whose expression in thymus is decreased in aged or oxidatively stressed mice. In the present study, we found that Rwdd1 expressed in both prokaryotic and eukaryotic cells showed a slower migration rate on SDS-PAGE gel. In addition, Rwdd1 was more sensitive to proteinase proteolysis. Furthermore, being a highly acidic protein which contains an RWD domain, Rwdd1 shared a high level of sequence similarity with Gir2, a member of the intrinsically unstructured protein (IUP). These findings suggest that Rwdd1 is a novel member of the IUP family.

Identification and characterization of a novel thymus aging related protein Rwdd1.

By using DDRT-PCR and EST segment ligation, a novel mouse thymus involution related gene Rwdd1 was identified. The reading frame encoded a protein of 243 amino acid residues which contained an RWD domain at the N terminus. Rwdd1 expression in thymus was decreased in aged and oxidatively stressed mice. It was found to be expressed extensively in thymocytes and thymic epithelial cells. The expression level of Rwdd1 could affect the transactivation activity of androgen receptor (AR) in transiently transfected thymic epithelial cells. However, no direct interaction could be detected by fluorescence resonance energy transfer (FRET) analysis. In conclusion, Rwdd1 is a thymus involution related protein that may indirectly affect AR signaling pathway.

Gammadelta T cells recognize tumor cells via CDR3delta region.

The principles governing gammadelta T cell specificity and diversity remain unclear due to lack of detailed structural analysis. To elucidate key structural basis of the specificity of gammadelta TCR for tumors, we analyzed the binding activities of synthesized TCR Vdelta2 CDR3 peptides derived from tumor infiltrating lymphocyte (TIL) s in ovarian epithelial carcinoma (OEC) via biospecific interaction analysis approach, enzyme immunoassay and immunofluorescence assays. Besides, we used human CDR3delta grafted-Ig to repeat major tests. We found that synthesized OEC-derived CDR3delta peptides could bind specifically to tumor cell lines and tissues. CDR3delta-graft Ig showed a similar binding specificity with CDR3delta peptides, suggesting the determinant role of CDR3delta in antigen binding. Moreover, CDR3delta peptide-mediated binding specificity was blocked by pre-incubation with same peptide, which decreased the cytotoxicity of gammadelta T cells to OEC cells in vitro. Our finding indicates that CDR3delta peptide could mimic antigen-binding specificity of gammadelta TCR. Our strategy provides a novel, simple and convenient approach to investigate the binding activity and function of gammadelta TCR.

γδ T Cells Provide Protective Function in Highly Pathogenic Avian H5N1 Influenza A Virus Infection.

Given the high mortality rate (>50%) and potential danger of intrapersonal transmission, highly pathogenic avian influenza (HPAI) H5N1 epidemics still pose a significant threat to humans. γδ T cells, which participate on the front line of the host immune defense, demonstrate both innate, and adaptive characteristics in their immune response and have potent antiviral activity against various viruses. However, the roles of γδ T cells in HPAI H5N1 viral infection remain unclear. In this study, we found that γδ T cells provided a crucial protective function in the defense against HPAI H5N1 viral infection. HPAI H5N1 viruses could directly activate γδ T cells, leading to enhanced CD69 expression and IFN-γ secretion. Importantly, we found that the trimer but not the monomer of HPAI H5N1 virus hemagglutinin (HA) proteins could directly activate γδ T cells. HA-induced γδ T cell activation was dependent on both sialic acid receptors and HA glycosylation, and this activation could be inhibited by the phosphatase calcineurin inhibitor cyclosporin A but not by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002. Our findings provide a further understanding the mechanism underlying γδ T cell-mediated innate and adoptive immune responses against HPAI H5N1 viral infection, which helps to develop novel therapeutic strategies for the treatment of H5N1 infection in the future.

MicroRNA-146a Overexpression Impairs the Positive Selection during T Cell Development.

MicroRNAs play crucial roles in modulating immune system. miR-146a, a potent feedback suppressor of NF-κB signaling, was shown to limit the innate immune response and myelopoiesis in a knockout mouse model. Here, we observed high lymphopoiesis demonstrated as mild splenomegaly and severe lymphadenopathy in a miR-146a transgenic mouse model. Overexpression of miR-146a resulted in enhanced proliferation and reduced apoptosis of T cells. More activated CD4+ T cells or effector memory T cells were observed in transgenic mice even under physiological conditions. Importantly, as one of the key steps to generate central tolerance, the positive selection of thymocytes is impaired in transgenic mice, resulting in more CD4+CD8+ double-positive thymocytes but fewer CD4+CD8- and CD4-CD8+ single-positive thymocytes. The maturation of selected CD4-CD8+ thymocytes was also impaired, leading to more severe loss of CD4-CD8+ than CD4+CD8- thymocytes in thymus of transgenic mice. Gene expression profiling analysis identified nine positive selection-associated genes, which were downregulated in transgenic mice, including genes encoding major histocompatibility complex class I/II molecules, IL-7 receptor α chain, and Gimap4, whose downregulation may contribute to the impairment of positive selection. Gimap4 was verified as a novel target of miR-146a. These findings further extend our understanding of the function of miR-146a in T cell biology and identify a novel regulatory mechanism underlying the positive selection during T cell development.