Cassette vectors directing expression of T cell receptor genes in transgenic mice

We describe a pair of cassette vectors that can be used to express rearranged T cell receptor genes in transgenic mice. Short DNA fragments containing rearranged V and Vβ segments are readily amplified from T cells and introduced between artificial cloning sites. Transgene-derived mRNAs are transcribed under the control of the natural TCR and -β promoter/enhancer elements. Using this vector, we have obtained transgenic mouse lines which display transgene-encoded TCR and β chains on a majority of T cells.     

T cells with dual antigen specificity in T cell receptor transgenic mice rejecting allografts

Allelic exclusion of T cell receptor (TCR) genes is incomplete: a significant percentage (10–30%) of normal human and mouse peripheral T cells express two surface TCR α chains, and a small percentage of peripheral human T cells have been reported to express two surface TCR β chains. A proportion of thymocytes in TCR transgenic mice rearrange endogenous T cell receptor genes, and peripheral T cells with two TCR α chains, transgenic and endogenous, have been reported. T cell clones with more than a single TCR heterodimer on their surface might be expected to show specificity for more than one cognate antigen: we report here a T cell clone with dual antigen specificity, isolated from an F5 TCR influenza nucleoprotein (NP 366–374/D^b)-specific transgenic female mouse which had rejected an H-2-matched male skin graft. It was selected in vitro by stimulation with male H-2^b spleen cells in the absence of the NP366–374 peptide but has specificity for both H-Y/D^b and NP366–374. This contrasted with the single NP366–374/D^b specificity shown by a control clone isolated from a Rag1–/– F5 mouse. The dual antigen specificity was associated with the rearrangement of endogenous TCR genes and cell surface expression of these as well as the TCR transgene.     

P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed-type hypersensitivity

The role of P-selectin in T lymphocyte accumulation and injury was studied in delayed-type hypersensitivity (DTH) responses in the skin and glomeruli of rats. Sprague Dawley rats were sensitized to sheep globulin and challenged 5 days later in the skin by subcutaneous injection and simultaneously in glomeruli by intravenous injection of a subnephritogenic dose of sheep anti-rat glomerular basement membrane globulin. This resulted in cutaneous and glomerular T lymphocyte-dependent macrophage influx and injury characteristic of DTH. Up-regulation of P-selectin expression on endothelial cells was observed in both inflammatory lesions. Treatment of rats with anti-CD5 antibody immediately prior to antigen challenge prevented the development of injury as assessed by measurement of proteinuria and skin swelling, as well as local T cell and macrophage accumulation in the glomerulus and in the skin, but did not block up-regulation endothelial cell P-selectin. Treatment with anti-CD4 antibody produced similar results. Blocking P-selectin in vivo with a functionally inhibitory antibody prevented development of proteinuria and skin swelling following antigen challenge. Local accumulation of T cells and macrophages was markedly attenuated in glomeruli and the skin and up-regulation of endothelial cell P-selectin was prevented. These data demonstrate that P-selectin is locally up-regulated on endothelial cells in T cell-dependent glomerular and cutaneous inflammation and suggests a pivotal functional role for P-selectin in local T cell recruitment and subsequent injury in DTH.     

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4⁺ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4⁺ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-A^b or from λ represser with specificity to I-A^d as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I-A^b/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non-self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.     

Impaired survival of T cell receptor Vγ3+ cells in interleukin-4 transgenic mice

The mouse epidermis contains a network of Thy-1⁺ dendritic T cells. Most of these cells express a homogeneous T cell receptor (TCR) configuration (Vγ3/ Vδ1) with only negligible junctional diversity. Because fetal thymocytes are precursors of these dendritic epidermal T cells (DETC) and the addition of interleukin (IL)-4 to fetal thymic organ cultures causes an early arrest in thymopoiesis, we examined DETC development in transgenic (tg) mice expressing IL-4 under the control of major histocompatibility complex class I regulatory sequences. Immunohistologic examination of epidermal sheets and polymerase chain reaction analysis of total skin RNA from IL-4 tg mice failed to reveal TCR Vγ3⁺ DETC and Vγ3 mRNA, respectively. In contrast, the sizes of TCR γδ subpopulations in lymphoid organs were unchanged in these mice. Although the numbers and staining intensities of TCR Vγ3⁺ thymocytes in early fetal (days 14–17) IL-4 tg mice were similar to those of littermate controls, we observed a preferential death of these cells in thymic organ cultures from IL-4 tg mice. We observed further that epidermal sheets prepared from 9-day-old mice whose mothers had been treated with an IL-4-neutralizing antibody from day 12 to day 18 of pregnancy contained DETC numbers similar to those of controls. However, upon termination of the anti-IL-4 treatment, DETC ceased to expand. We conclude that IL-4 impairs the survival of TCR Vγ3⁺ cells.     

Dominance of the BV17 element in nickel-specific human T cell receptors relates to severity of contact sensitivity

Hypersensitivity to nickel (Ni) represents the most common manifestation of contact allergy in humans. The role of metal-specific T cells in this disease is well established, but the molecular interactions involved in their activation are poorly understood. We examined the T cell receptor (TCR) repertoire in T cells activated with either NiSO₄ or NiSO₄-treated human serum albumin from six allergic patients. For the three most hyperreactive donors, we found a strong over-represention of the TCR BV17 element. TCR sequencing for one of these donors revealed an additional skewing for AV1 as well as a selection for an N region encoded argine at position 95 of the BV17 complementarity determining region (CDR)3. Since Arg is not known to participate in Ni complexing, we suppose that this selection is driven by contacts with peptide rather than nickel. However, the CDR1 of BV17 contains a unique combination of amino acids (HDA) that bears similarities to known motifs in Ni-binding proteins or peptides. We therefore propose that the severe hypersensitivity reactions found in BV17 over-expressors may be the result of Ni²⁺ ions bridging the germ-line-encoded BV17 CDR1 loop to corresponding sites in the major histocompatibility complex/peptide complex and thereby creating a superantigen-like enhancement of weak TCR-peptide contacts.     

尿酸钠对BALB/c小鼠抗体应答及树突状细胞和迟发型超敏反应的影响

目的:探讨尿酸钠作为佐剂对BALB/c小鼠体液和细胞免疫应答的影响。方法:利用尿酸钠悬浮液为佐剂、天花粉蛋白(TCS)为免疫原对BALB/c小鼠进行免疫,以酶联免疫测定法检测特异性抗体IgG的效价。体外诱导小鼠树突状细胞(DC),流式细胞术分析DC表型,评价尿酸钠体外对DC成熟的效应。以二硝基氟苯建立迟发型超敏反应(DTH)模型,分析尿酸钠在体内对细胞免疫应答的影响。结果:传统弗氏佐剂可极大地增强小鼠对TCS的抗体应答,尿酸钠佐剂对抗体应答不但没有促进,与单独使用免疫原相比,抗体应答反而明显降低。流式细胞术分析显示,尿酸钠对DC表达CD11c和CD83没有影响,但可明显提高MHCII的表达水平。DTH模型中,尿酸钠增强致敏原引发的耳廓肿胀程度,并促进DTH小鼠淋巴细胞的体外增殖能力。结论:尿酸钠悬浮液作为佐剂,对细胞免疫有显著增强作用,而对体液免疫应答却有一定的抑制作用,提示该佐剂在疫苗研究中有潜在应用前景。     

Regulation of T cell production in T cell receptor transgenic mice

The thymus produces many more cells than it releases into the periphery. According to generally accepted models of T cell development most of this loss occurs in the thymic cortex, among CD4⁺8⁺ thymocytes. An interesting situation arises in the case of T cell receptor (TcR) transgenic mice in which all cells can potentially be positively selected, leading to a theoretical increase of about 30-fold in the survival rate of CD4⁺8⁺ cells and in their transition to mature CD4⁺8^?or CD4^?8⁺ thymocytes. This in turn should lead to a 30-fold increase in the size of the thymic medulla, in the emigration rate and in the size of the peripheral Tcell pool. Increases in medullary or peripheral pool sizes of this magnitude are not seen in TcR transgenic mice. The question was therefore asked whether some form of homeostatic process regulated the size of the mature T cell pool and at what level it might operate. In this report we demonstrate that the increased rate of double-positive to single-positive transition in the TcR transgenic mice is directly reflected in an increased emigration rate, and that the medulla seems to be relatively efficient regardless of the number of cells passing through it. However, the potential increases in emigrant numbers in TcR transgenic mice are offset by the reduced size of the CD4⁺8⁺ thymocyte pool. It would appear then that regulation of T cell production, if it occurs, probably does so through regulation of the size of the CD4⁺8⁺ thymocyte pool. Mechanisms for regulation of this kind are not yet known.     

Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell-mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8⁺ T cells producing interferon (IFN)-γ which mediate the response and CD4⁺ T cells producing interleukin (IL)-4 and IL-10 which negatively regulate the magnitude and duration of the response. Since CD4⁺ T cell development to either IFN-γ- (Th1) or IL-4/IL-10- (Th2)-producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4⁺ T cell induction in a Th1-promoting environment would not only alter the CD4⁺ T cell cytokine-producing phenotype but also the course of the CHS response. Administration of the Th1-promoting cytokine IL-12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten-sensitized mice depleted of CD8⁺ T cells, treatment with IL-12 induced effector CD4⁺ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed-type hypersensitivity than CHS. Hapten-primed CD4⁺ T cells from IL-12 treated, sensitized mice produced IFN-γ, but not IL-4 in response to T cell receptor-mediated stimulation. Use of neutralizing anti-IFN-γ antibody indicated that IL-12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN-γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4⁺ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.     

Macrophages regulate induction of delayed-type hypersensitivity and experimental allergic encephalomyelitis in SJL mice

The relationship between the delayed-type hypersensitivity (DTH) response and susceptibility to experimental allergic encephalomyelitis (EAE) was examined using a unique age-dependent defect in the DTH response in an EAE-susceptible mouse strain. Young adult male SJL mice (< 10 weeks of age) are defective in DTH responses following immunization with a variety of soluble antigens. By contrast, they respond to antigens applied to the skin, demonstrating a normal contact sensitivity response. In this report, we show that the nonresponder male SJL are also unable to mount a DTH response to soluble neuroantigens or neuroantigens emulsified in complete adjuvant, and are additionally resistant to actively induced EAE. This contrasts with the DTH response in older males (> 10 weeks of age) and young adult females (6 weeks of age), which are both DTH responders and susceptible to EAE. By contrast, all three groups are susceptible to EAE mediated by the transfer of activated effector T cells, suggesting that the defect in young adult males is in the induction of effectors. Furthermore, transfer of a macrophage population from female responders to young male non-responders mediates the induction of both DTH responsiveness and EAE susceptibility. The phenotype of this antigen-presenting cell is (I-A⁺, Mac-1⁺, Mac-2^?, Mac-3⁺), identical to the phenotype of the macrophage regulating DTH responsiveness in this strain of mice. These data are consistent with the hypothesis that a defect in this cell inhibits induction of both CD4⁺ T_h1 DTH and EAE effector T cells.     

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.     

Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells

CD4⁺ helper T lymphocytes and CD8⁺ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4⁺ CD8⁺ thymocytes into mature ‘single-positive’ T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4⁺ CD8⁺ thymocytes determines their fate to become either CD4⁺ or CD8⁺ T cells, the molecular signals that direct precursor thymocytes to become CD4⁺ and CD8⁺ T cells are unclear. By using ZAP-70^? mutant thymus organ cultures in which T cell development is arrested at the CD4⁺ CD8⁺ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4⁺ and CD8⁺ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4⁺CD8^? TCR^high cells consistent with previous results. On the other hand, we find that the generation of CD4^? CD8⁺ TCR^high cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4⁺ and CD8⁺ T cells, and that biochemical treatment can manipulate the development and choice of CD4⁺ and CD8⁺ T cells.     

Tolerance is overcome in beef insulin-transgenic mice by activation of low-affinity autoreactive T cells

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self- reactive BI-specific T cells in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1–14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.     

Naive T-cell receptor transgenic T cells help memory B cells produce antibody

Injection of the same antigen following primary immunization induces a classic secondary response characterized by a large quantity of high-affinity antibody of an immunoglobulin G class produced more rapidly than in the initial response - the products of memory B cells are qualitatively distinct from that of the original naive B lymphocytes. Very little is known of the help provided by the CD4 T cells that stimulate memory B cells. Using antigen-specific T-cell receptor transgenic CD4 T cells (DO11.10) as a source of help, we found that naive transgenic T cells stimulated memory B cells almost as well (in terms of quantity and speed) as transgenic T cells that had been recently primed. There was a direct correlation between serum antibody levels and the number of naive transgenic T cells transferred. Using T cells from transgenic interleukin-2-deficient mice we showed that interleukin-2 was not required for a secondary response, although it was necessary for a primary response. The results suggested that the signals delivered by CD4 T cells and required by memory B cells for their activation were common to both antigen-primed and naive CD4 T cells.     

卵清白蛋白特异性T细胞免疫诱导BALB/c小鼠的调节性免疫应答

目的：研究T细胞免疫后正常小鼠的调节性免疫应答，方法：应用体外扩增的卵清白蛋白（OVA）特异的T细胞克隆免疫BALB／c小鼠，3H－TdR掺入法分析细胞增殖，3H－TdR标记靶细胞检测杀伤T细胞的杀伤效应，间接免疫荧光法分析血清中抗T细胞抗体水平。结果：T细胞免疫后能诱导BALB/c小鼠产生调节性T细胞的增殖反应，对靶细胞的杀伤效应以及针对于活化的T细胞的体液免疫应答，并进一步降低机体对OVA抗原的应答，结论：T细胞免疫能诱导正常机体的调节性免疫应答。     

CD69 upregulation on T cells as an in vitro marker for delayed-type drug hypersensitivity

Background:  T cells play a key role in delayed-type drug hypersensitivity reactions. Their reactivity can be assessed by their proliferation in response to the drug in the lymphocyte transformation test (LTT). However, the LTT imposes limitations in terms of practicability, and an alternative method that is easier to implement than the LTT would be desirable.
Methods:  Four months to 12 years after acute drug hypersensitivity reactions, CD69 upregulation on T cells of 15 patients and five healthy controls was analyzed by flow cytometry.
Results:  All 15 LTT-positive patients showed a significant increase of CD69 expression on T cells after 48 h of drug-stimulation exclusively with the drugs incriminated in drug-hypersensitivities. A stimulation index of 2 as cut-off value allowed discrimination between nonreactive and reactive T cells in LTT and CD69 upregulation. T cells (0.5–3%) showed CD69 up-regulation. The reactive cell population consisted of a minority of truly drug reactive T cells secreting cytokines and a higher number of bystander T cells activated by IL-2 and possibly other cytokines.
Conclusions:  CD69 upregulation was observed after 2 days in all patients with a positive LTT after 6 days, thus appearing to be a promising tool to identify drug-reactive T cells in the peripheral blood of patients with drug-hypersensitivity reactions.     

Peripheral tolerance in T cell receptor-transgenic mice: Evidence for T cell anergy

T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I-E^k. Antigen-specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene-bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen-specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen-specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.     

由人免疫球蛋白转基因小鼠制备人抗乙酰胆碱受体单克隆抗体

目的：从人免疫球蛋白（Ig）转基因小鼠制备人源性抗乙酰胆碱受体（AChR）单克隆抗体。方法：以电鳐（Torpedo）AChR（tAChR）作为基础免疫原，分别以tAChR或人AChR（hAChR）α亚单位1～210位氨基酸（Hα1-210）与thioredoxin（Trx）融合制备的融合蛋白Trx-Hα1-210作为加强免疫原，注射人Ig转基因小鼠。应用ELISA检查由该小鼠制备的杂交瘤细胞培养上清液中人源性抗tAChR或抗Trz-Hα1-210单克隆抗体的分泌，应用放射免疫测定法（RIA）测定抗tAChR或抗Trx-Hα1-210单克隆抗体与hAChR的交叉反应性。结果：从tAChR作为加强免疫原组小鼠得到433株分泌人源性抗tAChR单克隆抗体的细胞株，从抗Trx-Hα1-210作为加强免疫原组小鼠得到20株分泌人源性抗Trx-Hα1-210单克隆抗体的细胞株。但这些人源性抗体均不能与hAChR起交叉反应。结论：由人Ig转基因小鼠制备人源性抗AChR单克隆抗体是可行的。