Differentiation of Ia-reactive CD8+ murine T cells does not require Ia engagement. Implications for the role of CD4 and CD8 accessory molecules in T cell differentiation

The present study was undertaken to assess the Ia differentiation requirements of CD8+ class II-allospecific CTL, whose CD8+ phenotype is apparently "discordant" with their MHC class II reactivity. To do so, we compared the effect of in vivo anti-Ia blockade on the differentiation of Ia-reactive CD8+ CTL with its effect on the differentiation of CD4+ T cells. We found that anti-Ia blockade did not detectably interfere with the differentiation of CD8+ Ia-reactive CTL, even though it arrested the differentiation of CD4+ T cells. Thus, the differentiation of CD4+ T cells is strictly dependent upon Ia engagement, whereas the differentiation of CD8+ T cells, even those with reactivity against MHC class II alloantigens, does not require Ia engagement. These results support the concept that Ia-reactive CD8+ T cells are conventional CD8+ CTL, probably selected by self-class I MHC molecules during differentiation, whose receptors fortuitously crossreact on MHC class II alloantigens. Taken together, the present data indicate an intimate relationship between CD4/CD8 expression with MHC class specificity during T cell differentiation and selection. We suggest that an active triggering role for CD4 and CD8 accessory molecules in T cell differentiation is best able to explain these observations.     

Microchimerism maintains deletion of the donor cell-specific CD8+ T cell repertoire

Rare cases of stable allograft acceptance after discontinuation of immunosuppression are often accompanied by macrochimerism (> 1% donor cells in blood) or microchimerism (< 1% donor cells in blood). Here, we have investigated whether persistence of donor cells is the cause or the consequence of long-lasting CTL unresponsiveness. We found that engraftment of splenocytes bearing a single foreign MHC class I-restricted epitope resulted in lifelong donor cell microchimerism and specific CTL unresponsiveness. This status was reversed in a strictly time- and thymus-dependent fashion when the engrafted cells were experimentally removed. The results presented herein show that microchimerism actively maintains CTL unresponsiveness toward a minor histocompatibility antigen by deleting the specific repertoire and thus excluding dominant, T cell extrinsic mechanisms of CTL unresponsiveness independent of systemically persisting donor cell antigen.     

T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.     

多发性骨髓瘤患者T细胞亚群CD28表达及其临床意义

目的通过检测多发性骨髓瘤(MM)患者T细胞亚群CD28表达,探讨其T细胞免疫功能状态及临床意义。方法采用流式细胞术检测38例MM患者及20例健康对照外周血T细胞亚群CD28表达。结果①Ⅲ期MM患者CD4 T细胞较对照组及Ⅰ、Ⅱ期患者降低,为(29.1±7.7)%vs(37.2±5.9)%(、36.7±7.6)%、(36.4±6.4)%(P<0.05或<0.01);Ⅱ、Ⅲ期患者CD8 T细胞均高于对照组,为(38.0±6.4)%、(37.1±9.7)%vs(31.8±5.4)%(均P<0.05);Ⅲ期患者CD4 /CD8 比值较对照组及Ⅰ期患者降低,为0.8±0.3 vs 1.2±0.3、1.2±0.3(P<0.05或<0.01);CD4 CD28 、CD8 CD28 细胞在Ⅲ期患者均显著低于对照组及Ⅰ、Ⅱ期患者,分别为(25.0±8.2)%vs(35.3±5.6)%、(35.1±8.0)%(、33.8±5.7)%;(10.8±4.1)%vs(17.0±4.3)%、(18.3±4.8)%(、18.2±4.6)%(均P<0.01)。②进展期MM患者CD4 T细胞及CD4 /CD8 比值均低于对照组,分别为(32.0±8.3)%vs(37.2±5.9)%;0.9±0.3 vs 1.2±0.3(P<0.05或<0.01);CD8 T细胞比对照组增高,为(36.8±8.6)%vs(31.8±5.4)%(P<0.05),而其CD4 CD28 、CD8 CD28 细胞在进展期MM患者均低于对照组和稳定期,分别为(28.3±8.6)%vs(35.3±5.6)%(、35.6±7.4)%;(13.1±4.9)%vs(17.0±4.3)%(、20.5±5.9)%(P<0.05或<0.01)。③CD4 CD28 细胞百分率与β2微球蛋白水平呈负相关(r=-0.401,P<0.05)。结论MM患者存在T细胞亚群CD28表达缺陷,并与临床分期、病情进展及预后相关。     

尿毒症患者T细胞亚群共刺激分子CD28和CD152的表达分析

目的探讨尿毒症患者T细胞亚群表面共刺激分子CD28和CD152(CTLA4)的表达与细胞免疫功能紊乱的关系。方法采用流式细胞术测定尿毒症患者外周血T淋巴细胞上共刺激分子对CD28和CD152(CTLA4)的表达。结果尿毒症患者与健康对照组相比,CD4+T细胞相对计数和CD4/CD8比值均明显增加,共刺激分子CD28和CD152(CTLA4)在CD4+和CD8+T细胞上表达均明显增加,P值均<0.05。尿毒症患者CD4+T细胞表达的CD28明显高于CD8+T细胞,两组间T细胞亚群上共刺激分子CD28表达率差值间比较有显著性差异,P<0.05;患者的CD152分子在CD4+T细胞上的表达较CD8+T细胞上明显减少(P=0.027)。结论尿毒症患者的细胞免疫功能紊乱,T细胞亚群比例失衡,共刺激分子对CD28、CD152(CTLA4)在CD4+和CD8+T细胞上表达均增加,提示可能存在T细胞活化或抑制信号调节失衡。     

CD4+ CD7- CD28- T cells are expanded in rheumatoid arthritis and are characterized by autoreactivity.

Clonal expansion of CD4+ T cells is a characteristic finding in patients with RA and is only infrequently found in patients with psoriatic arthritis and healthy controls. Expanded CD4+ clonotypes are present in the blood, infiltrate into the joint, and persist over years. We have not addressed the question of whether the expanded clonotypes have unique functional and phenotypic properties which may explain the preferential in vivo expansion in RA. In contrast to most CD4+ T cells, expanded clonotypes lacked the expression of the CD28 and CD7 cell surface molecules. Accordingly, the subsets of CD4+ CD28- (9.7 vs 1.7, P = 0.00002) and CD4+ CD7- T cells (21.5 vs 12.26, P = 0.018) were increased in RA patients compared with age-matched normal individuals. Despite the lack of CD28 expression, clonally expanded CD4+ T cells were not anergic but proliferated in response to immobilized anti-CD3 and could be maintained in tissue culture. In vivo expanded CD4+ T cells were autoreactive to ubiquitously distributed autoantigens. They responded in an autologous mixed lymphocyte reaction, and T cell clones isolated from selected patients proliferated to autologous peripheral blood adherent cells. These data suggest that in RA patients selected CD4+ T cells which share the CD7- CD28- phenotype escape from peripheral tolerance.     

Cytotoxic T lymphocyte antigen 4 and CD28 modulate cell surface raft expression in their regulation of T cell function.

Coreceptors CD28 and cytotoxic T lymphocyte antigen (CTLA)-4 have opposing effects on TcR/CD3 activation of T cells. While CD28 enhances and CTLA-4 inhibits activation, the underlying molecular basis of these effects has yet to be established. In this context, ganglioside and cholesterol enriched membrane microdomains (rafts, GEMs) serve as centers of signaling in T cells. Although CD28 can promote TcR/raft colocalization, evidence is lacking on whether the surface expression of membrane rafts can be targeted by CTLA-4 in its modulation of T cell responses. In this study, we demonstrate that both CD28 and CTLA-4 profoundly alter the surface expression of membrane rafts during T cell activation. While CD28 increased expression and the number of peripheral T cells induced to express surface rafts in response to TcR ligation, CTLA-4 potently inhibited both TcR and TcR x CD28 induced raft expression on the surface of T cells. Consistent with this, CD28 increased the presence of the linker of activated T cells (LAT) in purified membrane rafts, while CTLA-4 coligation effectively blocked this increase. Further, the reversal of the CTLA-4 block with CD3/CD28 ligation was accompanied by an increase in surface raft expression and associated LAT. Our observations demonstrate for the first time that CTLA-4 targets the release of rafts to the surface of T cells, and provides a mechanism for the opposing effects of CD28 and CTLA-4 on costimulation.     

A role for CD9 molecules in T cell activation

Costimulation mediated by the CD28 molecule plays an important role in optimal activation of T cells. However, CD28-deficient mice can mount effective T cell-dependent immune responses, suggesting the existence of other costimulatory systems. In a search for other costimulatory molecules on T cells, we have developed a monoclonal antibody (mAb) that can costimulate T cells in the absence of antigen-presenting cells (APC). The molecule recognized by this mAb, 9D3, was found to be expressed on almost all mature T cells and to be a protein of approximately 24 kD molecular mass. By expression cloning, this molecule was identified as CD9, 9D3 (anti-CD9) synergized with suboptimal doses of anti-CD3 mAb in inducing proliferation by virgin T cells. Costimulation was induced by independent ligation of CD3 and CD9, suggesting that colocalization of these two molecules is not required for T cell activation. The costimulation by anti-CD9 was as potent as that by anti-CD28. Moreover, anti-CD9 costimulated in a CD28- independent way because anti-CD9 equally costimulated T cells from the CD28-deficient as well as wild-type mice. Thus, these results indicate that CD9 serves as a molecule on T cells that can deliver a potent CD28- independent costimulatory signal.     

CD8 and β2-microglobulin-free MHC class I molecules in T cell immunoregulationMHC class I molecules in T cell immunoregulation

Summary Intracellular assembly of MHC class I heavy chains with β₂-microglobulin occurs prior to the expression of the antigen-presenting complex on the cell surface. The association of β₂-microglobulin with newly synthesized class I heavy chains is thought to be a strict prerequisite for their transport to the cell surface. However, MHC class I molecules not associated with β₂-microglobulin (β₂-microglobulin-free class I heavy chains) have been detected on the surface of activated lymphoid cells. These molecules have different conformations. Therefore, their interactions with other membrane proteins and biological functions may be different from those assigned to β₂-microglobulin-associated MHC class I molecules. The two forms of MHC class I molecules on the surface of activated cells can self-associated and also form complexes with distinct proteins. Upon interaction with the appropriate ligands these molecular complexes transduce signals regulating cell activation. The ligand for β₂-microglobulin-free class I heavy chains appears to be soluble CD8. A model is presented describing a novel mechanism of immunoregulation mediated by both soluble and membrane-bound forms of CD8 and β₂-microglobulin-free class I heavy chains.     

抗B7分子抗体诱导T细胞免疫耐受研究

目的：联合应用抗B7分子的单克隆抗体（单抗）体外诱导T细胞免疫耐受模型，并探讨B7分子在T细胞免疫耐受中的作用及其机制。方法：体外联合应用抗B7单抗诱导T细胞免疫耐受，^3H-TdR掺入法检测T细胞增殖，逆转录－聚合酶链反应（RT－PCR）检测T细胞细胞因子mRNA合成。为了排除其它粘附分子的作用，应用联合转染DR7或（和）B7分子基因的CHO细胞系作为人工抗原递呈细胞（APC）介导混合淋巴细胞反应（MLR）。结果：T细胞增殖实验显示，B7－1（CD80）和B7－2（CD86）单抗单独应用可以部分阻MLR,人中CD86单抗的阻断作用更为明显。两种抗体联合应用时，可以明显阻断MLR。RT－PCR显示：应用抗B7单抗联合阻断后24h,IFN-γ及IL－2 mRNA合成明显减少，而IL－4 mRNA合成较前明显增加。人工APC介导的MLR显示，仅有第一信号（DR7）时也可以使T细胞增殖，但需要达到一定的信号强度。给予DR7信号同时给予共刺激信号CD80可以增强T细胞反应，增强效应可以被抗CD80单抗所阻断。结论：B7分子在T细胞免疫反应中具有重要的作用，可以非特异性增强T细胞免疫反应。阻断B7／CD28信号传导通路，可以诱导T细胞免疫耐受形成，其中CD86分子的作用可能更为重要。抗B7单抗诱导的T细胞无反应性后，向Th2方向分化，这可能是阻断B7分子共刺激后T细胞免疫耐受形成的机制之一。     

多发性硬化患者外周血CD4及共刺激分子CD28表达变化

目的 探讨CD4和CD28在多发性硬化患者中的表达变化.方法 采用流式细胞术检测29例多发性硬化患者外周血CD4＋、CD28＋、CD4＋ CD28＋的表达变化,采用EDSS量表对MS患者进行神经功能损伤评分.结果 复发期MS患者CD4＋表达明显高于缓解期MS和对照组,缓解期MS患者外周CD4＋细胞明显表达高于对照组;复发期MS患者CD28＋表达明显低于缓解期,缓解期明显低于对照组;CD4＋ CD28＋三组比较无统计学差异;重度MS患者外周血CD4＋表达高于中度,重度MS患者外周血CD28＋表达低于中度,而CD4＋ CD28＋表达二组比较无统计学差异.结论 多发性硬化患者CD4＋表达水平升高,CD28＋表达水平降低,CD28＋可能促进CD4＋T淋巴细胞活化.     

CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool

CD27, like CD28, acts in concert with the T cell receptor to support T cell expansion. Using CD27(-/-) mice, we have shown earlier that CD27 determines the magnitude of primary and memory T cell responses to influenza virus. Here, we have examined the relative contributions of CD27 and CD28 to generation of the virus-specific effector T cell pool and its establishment at the site of infection (the lung), using CD27(-/-), CD28(-/-), and CD27/CD28(-/-) mice. We find that primary and memory CD8+ T cell responses to influenza virus are dependent on the collective contribution of both receptors. In the primary response, CD27 and CD28 impact to a similar extent on expansion of virus-specific T cells in draining lymph nodes. CD27 is the principle determinant for accumulation of virus-specific T cells in the lung because it can sustain this response in CD28(-/-) mice. Unlike CD28, CD27 does not affect cell cycle activity, but promotes survival of activated T cells throughout successive rounds of division at the site of priming and may do so at the site of infection as well. CD27 was found to rescue CD28(-/-) T cells from death at the onset of division, explaining its capacity to support a T cell response in absence of CD28.     

共刺激分子CD28和B7在胃肠道肿瘤的表达

目的 检测胃肠道肿瘤患者外周血中CD28^＋T细胞数量及原代细胞表面B7表达水平,以探讨胃肠道肿瘤患者共刺激通路是否异常。方法 采用流式细胞术检测胃肠道肿瘤患者外周血中CD28^＋T细胞与胃肠道肿瘤患者原代细胞B7分子表达,并与胃肠道良性疾病进行比较。结果 胃肠道肿瘤患者CD28^＋T细胞数明显低于对照组（P〈O．01）;其原代细胞表面B7-1、B7-2表达水平低于对照组（P〈O．01）。结论胃肠道肿瘤患者外周血T细胞低表达CD28分子,胃肠道肿瘤细胞低表达B7分子,从而影响B7-CD28共刺激通路,使T细胞不能有效地清除肿瘤。     

Optimizing adoptive polyclonal T cell immunotherapy of lymphomas, using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains

We previously demonstrated the feasibility of generating therapeutic numbers of cytotoxic T lymphocyte (CTL) clones expressing a CD20-specific scFvFc:CD3zeta chimeric T cell receptor (cTCR), making them specifically cytotoxic for CD20+ B lymphoma cells. However, the process of generating and expanding he CTL clones was laborious, the CTL clones expressed the cTCR at low surface density, and they exhibited suboptimal proliferation and cytotoxicity. To improve the performance of the CTLs in vitro and in vivo, we engineered "second-generation' plasmid constructs containing a translational enhancer (SP163) and CD28 and CD137 costimulatory domains in cis with the CD3zeta intracellular signaling domain of the cTCR gene. Furthermore, we verified the superiority of generating genetically modified polyclonal T cells expressing the second-generation cTCR rather than T cell clones. Our results demonstrate that SP163 enhances the surface expression of the cTCR; that the second-generation cTCR improves CTL activation, proliferation, and cytotoxicity; and that polyclonal T cells proliferate rapidly in vitro and mediate potent CD20-specific cytotoxicity. This study provides the preclinical basis for a clinical trial of adoptive T cell immunotherapy for patients with relapsed CD20+ mantle cell lymphoma and indolent lymphomas.     

CD43 is a murine T cell costimulatory receptor that functions independently of CD28

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28- independent fashion since the mAb costimulates T cells from the CD28- deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.     

The in vivo cytotoxic activity of CD8+ T cell clones correlates with their levels of expression of adhesion molecules

CD8+ T cell clones specific for a defined epitope present in the circumsporozoite protein of Plasmodium yoelii display striking differences in their in vivo antiplasmodial activity. The adoptive transfer of certain clones (YA23 and YA26) into naive mice inhibits by 90% or more the development of liver stages of malaria parasites and protects against malaria infection. The adoptive transfer of two other T cell clones (YB8 and YA15) results, respectively, in partial or no inhibitory activity on parasite development. We found that "protective" and "nonprotective" cytotoxic T lymphocyte (CTL) clones do not differ in their fine epitope specificity and display similar levels of lysis and DNA degradation of target cells in vitro. Their pattern of production of lymphokines and granule-associated proteins also failed to correlate with their in vivo antiplasmodial activity. Histological studies combined with autoradiography showed that, upon adoptive transfer, only T cells from the protective CTL clones are capable of "associating" with a significant percentage of parasitized hepatocytes. Fluorescence-activated cell sorter analysis of surface molecules revealed pronounced differences in the levels of CD44 and VLA-4 expression by the different clones, correlating closely with their in vivo protective activity. The correlation between in vivo antiparasite activity and the expression of CD44 was further corroborated by the results of sorting, from the partially protective YB8 clone, two sub-populations expressing high and low levels of CD44. These were protective and nonprotective, respectively. The clones also differed in their adhesive properties. Cross-linking of CD44, using specific antibodies, induced LFA-1-mediated homotypic aggregation of protective clones, while nonprotective cells failed to aggregate.     

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.     

The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells.

Activation via the T lymphocyte cell surface molecule CD28 provides a potent amplification signal for interleukin 2 (IL-2) production in several in vitro systems. The B lymphocyte activation antigen, B7/BB1, is a natural ligand for CD28. Here we investigate the role of CD28 and B7/BB1 in primary activation of CD4+ T lymphocytes stimulated with allogeneic B lymphoblastoid cell lines. A subset of peripheral CD4+ T cells that is unresponsive to crosslinking of CD3/T cell receptor (TCR) with CD3 monoclonal antibody (mAb) does proliferate in response to allogeneic B lymphoblasts. TCR binding to allogeneic major histocompatibility complex antigens was an absolute requirement for activation of these cells because mAbs to either CD3 or human histocompatibility leukocyte antigen (HLA) class II completely inhibited activation. CD28 and B7/BB1 antibodies inhibited T cell proliferation 90% and 84%, respectively. Similar results were obtained with the total CD4+ T lymphocyte population. Crosslinking of HLA-DR antigens on small, resting B cells induced rapid expression of B7/BB1, which peaked at 6 h and returned to baseline levels within 18 h. These data demonstrate that CD28-B7/BB1 binding provides an important early second signal for alloactivation of CD4+ T lymphocyte by B lymphoblasts. The results also suggest that T cells interacting with allogeneic resting B cells may induce B7/BB1 expression in the alloantigen-presenting cell as a consequence of interaction between the TCR and class II molecules.