Mouse leukocyte-associated Ig-like receptor-1 (mLAIR-1) functions as an inhibitory collagen-binding receptor on immune cells

Leukocyte-associated Ig-like receptor-1 (LAIR-1) is a cell-surface molecule that functions as an inhibitory receptor on various immune cells. We developed mAbs to study the expression of mouse leukocyte-associated Ig-like receptor-1 (mLAIR-1) on primary immune cells and established that it is expressed on the majority of cells of the immune system, including T cells, NK cells, monocytes and dendritic cells. Furthermore, mLAIR-1 is inducibly expressed on blood granulocytes in vivo and is differentially expressed upon T cell activation in vitro. Unexpectedly, mLAIR-1 was not expressed on splenic and blood B220(+) B cells. Similar to its human homolog, mLAIR-1 interacted with high affinity with a wide range of collagen molecules. Furthermore, mLAIR-1 specifically interacted in a hydroxyproline-dependent manner with synthetic collagen Gly-Pro-Hyp peptides. We show, for the first time, that mLAIR-1 cross-linking with its ligands inhibits CD3-induced T cell stimulation in vitro. Given the similarities between the mouse and human receptors, mLAIR-1 may serve as a good model to assess the role of the LAIR-1 receptors in regulation of immune responses.     

The stromal cell antigen CD248 (endosialin) is expressed on naive CD8+ human T cells and regulates proliferation

CD248 (endosialin) is a transmembrane glycoprotein that is dynamically expressed on pericytes and fibroblasts during tissue development, tumour neovascularization and inflammation. Its role in tissue remodelling is associated with increased stromal cell proliferation and migration. We show that CD248 is also uniquely expressed by human, but not mouse (C57BL/6), CD8(+) naive T cells. CD248 is found only on CD8(+) CCR7(+) CD11a(low) naive T cells and on CD8 single-positive T cells in the thymus. Transfection of the CD248 negative T-cell line MOLT-4 with CD248 cDNA surprisingly reduced cell proliferation. Knock-down of CD248 on naive CD8 T cells increased cell proliferation. These data demonstrate opposing functions for CD248 on haematopoietic (CD8(+)) versus stromal cells and suggests that CD248 helps to maintain naive CD8(+) human T cells in a quiescent state.     

The degree of CD8 dependence of cytolytic T cell precursors is determined by the nature of the T cell receptor (TCR) and influences negative selection in TCR-transgenic mice

Although much has been learned about CD8 structure-function properties, it has so far not been tested whether the nature of the TCR is sufficient to transfer the property of CD8 dependence versus non-dependence to CD8⁺ cytotoxic T lymphocytes (CTL) and their precursors differentiating in T cell receptor (TCR)-transgenic (Tg) mice. In the present study, we compared the characteristics of dependence on CD8 for stimulation of CTL precursors and antigen-specific cytolysis by CD8⁺ T cells from two TCR-Tg mice expressing respectively the TCR (Tg) from a “CD8-dependent” and from a “CD8-independent” CTL clone, which were both reactive against the H-2K^b alloantigen and originated from H-2^k mice. The results indicate that the property of the Tg⁺CD8⁺ cells from H-2^k TCR-Tg mice corresponds to that of the CTL clone of origin, demonstrating that it is linked to the nature of the TCR. Consistent with this property, Tg⁺CD4⁺ cells could also differentiate into H-2K^b-specific CTL when originating from the “CD8-independent”, but not from the “CD8-dependent” Tg-TCR. The influence of the property of “CD8 dependence” on negative selection occurring in TCR-Tg H-2^klb mice was apparent at two levels: (i) in the thymus, the extent of deletion was much more pronounced for the “CD8-independent” TCR-Tg mice; (ii) in the periphery, Tg^+(hi) cells with low to negative CD8 expression were present for the “CD8-dependent” Tg-TCR, whereas only Tg⁺CD4^?CD8^? cells with low surface Tg-TCR and CD3 expression were found for the “CD8-independent” Tg-TCR, indicating that Tg⁺CD4^?CD8^? cells are susceptible to tolerance induction involving TCR/CD3 surface down-modulation. Furthermore, different in vitro conditions led to H-2K^b-induced stimulation of Tg⁺CD4^?CD8^? cells to differentiate into CTL detected in an anti-TCR clonotypic monoclonal antibody redirected cytolysis assay. Culture in interleukin-2 of H-2^klb Tg⁺CD4^?CD8^? cells was sufficient to induce CTL activity in the “CD8-independent” model, whereas stimulation with cells which overexpressed H-2K^b was required in addition to interleukin-2 to induce CTL differentiation in the “CD8-dependent” model. These data suggest that peripheral Tg⁺CD4^?CD8^? cells present in a situation of in vivo tolerance to H-2K^b can still be triggered by H-2K^b with a sensitivity correlated with the degree of CD8 dependence.     

抗原肽浓度对初始T细胞向Th1/Tc1、Th2/Tc2分化影响的研究

目的 研究不同浓度抗原肽(OVA)对初始CD4~+T细胞向Thl/Th2、初始CD8~+T细胞向Tc1/Tc2分化偏向性的影响.方法 采用系列浓度TCR特异性抗原肽与小鼠脾脏树突状细胞联合培养,然后刺激小鼠初始CD4~+或CD8~+T细胞.流式细胞术检测细胞内因子IFN-γ和IL-4的表达水平,同时用CFSE检测活化T细胞的增殖水平.结果 低浓度抗原肽刺激初始CD4~+T向Th2、初始CD8~+T向Tc2分化;而高浓度抗原肽刺激初始CD4~+T向Th1、初始CD8~+T向Tc1分化.Th细胞比Tc细胞受影响程度要明显.结论 抗原肽在很大浓度范围内均可以刺激初始T细胞的活化,但随着浓度的升高,分化方向从Th2、Tc2逐渐向Th1、Tc1倾斜.这一结论为动物实验中疫苗免疫剂量的控制提供了重要的参考价值.     

Regulated expression of the inhibitory receptor LAIR-1 on human peripheral T cells during T cell activation and differentiation

The leukocyte-associated Ig-like receptor-1 (LAIR-1) is capable of inhibiting immune cell function through interaction with collagens. LAIR is expressed on the majority of peripheral blood mononuclear cells. The abundant expression of both receptor and ligand calls for regulatory mechanisms to relieve the continuous interaction between collagens and LAIR-1. This regulation may occur at the expression level of the receptor. Here, we report that LAIR-1 is indeed differentially expressed during human T cell differentiation. Naive CD4(+) and CD8(+) T cells as well as CD8(+) T cells of the effector phenotype express higher levels of LAIR-1 compared to memory T cells. In vitro stimulation revealed a decrease in LAIR-1 expression upon activation, and the lower LAIR-1 expression on CD127(-) T cells suggests that activation-induced down-modulation of LAIR-1 may also occur in vivo. Furthermore, crosslinking of LAIR-1 on primary T cells results in an inhibition of T cell function. Our data suggest that regulated expression of LAIR-1 and the subsequent change in the threshold for activation may be a mechanism to modulate inhibition of the immune system.     

血小板T细胞活化抗原1分子在NK细胞上的作用研究

目的 研究血小板T细胞活化抗原1（PTA1）分子在NK细胞杀伤过程中的作用。方法 应用重民地向细胞毒实验,探讨了PTA1分子对混合淋巴细胞反应中活化的NK细胞杀伤作用的影响。结果 在重导向细胞毒实验中,PTA1 McAb能明显上调NK细胞及CTL的杀伤活性。结论 PTA1分子在NK细胞上具有刺激性受体的功能。     

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A^*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8×A2.1/K^b mouse-derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81–88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81–88), we found that the heterodimeric CD8αβ coreceptor, but not normally expressed homodimeric CD8αα, is required for tetramer binding and functional redirection of TCR-transduced human T cells. CD8⁺T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 antibody and required more peptide than wild-type (WT) MDM2 TCR⁺ T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) αβ domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expresion, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease. These two authors contributed equally to this work.     

The tetraspanin CD9 is preferentially expressed on the human CD4(+)CD45RA+ naive T cell population and is involved in T cell activation

Human CD4+ T cells can be divided into reciprocal memory and naive T cell subsets based on their expression of CD45 isoforms and CD29/integrin beta1 subunit. To identify unique cell surface molecules on human T cells, we developed a new monoclonal antibody termed anti5H9. Binding of anti5H9 triggers a co-stimulatory response in human peripheral blood T cells. Retrovirus-mediated expression cloning has revealed that the antigen recognized by anti5H9 is identical to the tetraspanin CD9. We now show that human CD9 is preferentially expressed on the CD4(+)CD45RA+ naive T cell subset, and that CD9(+)CD45RA+ T cells respond preferentially to the recombinant beta2-glycoprotein I, compared to CD9-CD45RA+ T cells. Furthermore, anti5H9 inhibits both the recombinant beta2-glycoprotein I- and the recall antigen tetanus toxoid-specific T cell proliferation. These results suggest that the tetraspanin CD9 plays an important role in T cell activation.     

A T cell receptor (TCR) antagonist competitively inhibits serial TCR triggering by low-affinity ligands,but does not affect triggering by high-affinity anti-CD3 antibodies

It has been demonstrated that modified peptides which fail to induce detectable T cell responses can act as T cell receptor (TCR) antagonists when presented together with agonist by the same antigen-presenting cell (APC). We report that a TCR antagonist competitively inhibits TCR triggering induced by low-affinity ligands such as agonistic peptides or bacterial superantigens. However, the same antagonist cannot inhibit TCR triggering and T cell activation induced by high-affinity anti-CD3 antibodies that engage most TCR at once. These results indicate that TCR antagonists inhibit T cell responses by interfering with the ongoing process of serial triggering, rather than by delivering an inhibitory signal to T cells.     

The minimal number of antigen-major histocompatibility complex class II complexes required for activation of naive and primed T cells

Although previous studies have shown that 50–200 antigen-major histocompatibility complex complexes (Ag-MHC) are sufficient to stimulate significant secretion of interleukin (IL)-2 from MHC class II-restricted T cell hybridomas, there have been no studies of this nature on more physiologically relevant T cell populations. In this study we have analyzed the ligand requirements for stimulation of responses from naive and previously primed T cells derived from T cell receptor (TCR)-transgenic animals whose TCR is specific for the pigeon cytochrome c (PCC) 88–104 peptide presented by I-E^k. Primed T cells were as sensitive as the previously reported T cell hybridomas, requiring about 100 Ag-MHC complexes to synthesize readily detectable quantities of IL-2, whereas naive T cells required 15 times more ligand to produce equivalent quantities of IL-2. Similarly, primed T cells required about 40 Ag-MHC complexes to produce a significant proliferative response, whereas naive T cells required about 400 complexes. In contrast to these results, naive and primed T cells showed similar ligand requirements when early events in the T cell activation pathway were analyzed; i.e. TCR down-modulation, CD69 and CD25 expression, and blast transformation. A further analysis of IL-2 and IL-2R expression indicated: 1) The first synthesis of IL-2 was detected at the same ligand concentration in both primed and naive T cells, but primed T cells made much more IL-2 as the ligand concentrations increased; 2) primed T cells expressed about fivefold more IL-2 receptor (R) than naive T cells, despite the fact that the antigen doseresponse curves with respect to the percentage of cells expressing IL-2R were identical. These results suggest that naive and primed T cells have the same threshold with respect to the number of Ag-MHC complexes required to initiate T cell activation, but that due to the inefficient expression of IL-2 and IL-2R, engagement of more complexes is needed to enable naive T cells to synthesize the necessary amounts of these two molecules to allow T cells to go through a complete cycle of replication.     

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0^brightRA^? or CD45R0^?-RA^bright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0^?RA^bright T cells, which converted to CD45R0^brightRA^? after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0^brightRA^?T cells compared to the CD45R0^?RA^bright subset. The poor responses of CD45R0^?RA^bright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0^?RA^bright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0⁺RA⁺). Alternatively, if primed CD45R0^?RA^bright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0^bright and CD45RA^bright expression identify memory and naive CD4⁺ T cells, respectively.     

The Qa-1 Dependent CD8^＋ T Cell Mediated Regulatory Pathway

The immune system has evolved a variety of regulatory mechanisms to ensure the peripheral self-tolerance as well as the optimal capacity to elicit effective anti-infection immunity. At present, there is no satisfactory conceptual framework to explain how the peripheral immunity is regulated at a biological system level, which enables the immune system to perform its essential functions to mount effective immunity to virtually any foreign antigens but avoid harmful immune responses to self. In this regard, during the past few years, an “affinity/avidity model of peripheral T cell regulation” has been proposed and tested, which opens up a new paradigm to understand how the peripheral immunity, to both self and foreign antigens, is regulated. The paradigm is based on the discovery of a subset CD8^＋ T cells with TCRs which specifically recognize a unique set of self-peptides presented by the MHC class Ib molecule Qa-I differentially expressed on T cells as a function of the affinity/avidity of T cell activation. These Qa-1 restricted CD8^＋ T cells represent an example of how the immune system utilizes a unified mechanism to regulate adaptive immunity to both self and foreign antigens. Thus, by selectively down-regulating T cells of intermediate affinity/avidity, to any antigens, the immune system controls the adaptive immunity without the necessity to distinguish self from non-self in the periphery at the level of T cell regulation. Cellular ＆ Molecular Immunology. 2005;2（3）：161-167.     

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is differentially expressed during human B cell differentiation and inhibits B cell receptor-mediated signaling.

Leukocyte-associated Ig-like receptor-1 (LAIR-1) belongs to the growing family of immunoreceptor tyrosine-based inhibitory motif-bearing receptors and is expressed on the majority of peripheral mononuclear cells, including NK cells, T cells, B cells, monocytes, and dendritic cells. In this study, we investigated the distribution and the capacity of LAIR-1 to function as an inhibitory receptor on human B cells. LAIR-1 is expressed from early on during B cell differentiation, but is absent on approximately half of the memory B cells, and all germinal center B cells, plasmablasts, and terminally differentiated plasma cells. In vitro stimulation of naive B cells via the B cell receptor (BCR) or CD40, triggering proliferation and differentiation into Ig-producing plasma cells, is accompanied by loss of LAIR-1 expression. We previously reported that LAIR-1 can function as an inhibitory receptor on NK cells and T cells. Here, we demonstrate that it can also function as a negative regulator of BCR-mediated signaling, since simultaneous cross-linking of LAIR-1 and the BCR reduces the increase of intracellular Ca(2+) evoked by BCR ligation. Taken together, this suggests that the inhibitory mechanism of LAIR-1 is functional in multiple components of the hematopoietic system.     

Lower numbers of circulating natural killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV‐1) associated neurological disease

Human T lymphotropic virus type 1 (HTLV‐1) infects 10–20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV‐1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4⁺ and fewer CD8⁺ cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4⁺ NK T subset are associated with HTLV‐1 disease progression.     

Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR alphabeta lymphocytes

A subset of CD8(+) T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8(+) T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7(+) memory cells and CCR7(-) effector cells. Since NKG2A can only be induced on naive CD8(+) T cells while CD94(-) memory cells are refractory, it is likely that commitment to the CD94:NKG2A(+) subset occurs during the first encounter with antigen. CCR7(+)CD94:NKG2A(+) T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-gamma. These cells occur as a separate CD94:NKG2A(+) T cell lineage with a distinct TCR repertoire that differs from that of the other CD8(+)CD94(-) T cells activated in situ.     

The role of TCR stimulation and TGF-beta in controlling the expression of CD94/NKG2A receptors on CD8 T cells

Following antigen recognition, murine CD8 T cells express CD94/NKG2A receptors. Our results show that this up-regulation occurs rapidly in vitro and is accompanied by an approximately 8-fold increase in CD94 and approximately 125-fold increase in NKG2A mRNA. In contrast, only a twofold increase in NKG2C mRNA is noted. The addition of TGF-beta, but not IL-10, IL-12 or IL-15, leads to a further increase in cell membrane expression of these receptors, as well as a approximately 6-fold increase in mRNA for both chains. TGF-beta also increases CD94/NKG2A expression on memory CD8 T cells that are re-exposed to antigen. The effect of TGF-beta on increasing CD94/NKG2A expression on both naive and memory CD8 T cells occurs only when there is a concurrent stimulation through the TCR. In contrast, TGF-beta does not increase expression of CD94/NKG2A on resting or activated NK cells. We also show by using purified CD8 T cells, that TGF-beta acts directly on these cells. These results implicate a role for both antigen and TGF-beta in increasing expression of inhibitory CD94/NKG2A receptors on CD8 T cells.