Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro

Using a novel system that supports positive selection in vitro, we have investigated the cellular requirements for this process by testing the ability of individual thymic and nonthymic stromal cell types to support the maturation of CD4+CD8+ thymocytes into CD4+ or CD8+ T cells. We show that thymic cortical epithelial cells are unique in their ability to mediate this maturation, and suggest that in addition to TCR ligation, these cells supply specific signals for positive selection. Moreover, by demonstrating positive selection on ECDI (1- ethyl-3-[3'dimethyl-aminopropyl]-carbodiimide)-fixed epithelial cells in this system, we provide direct evidence that the provision of these signals involves interactions with epithelial cell surface molecules rather than the release of soluble factors.     

CD8 is required during positive selection of CD4-/CD8+ T cells

Interactions between self-MHC molecules and T cells are necessary for the proper development of mature T cells, in part due to an absolute requirement for self-MHC-TCR interactions. Recently, we showed that CD4-mediated interactions also participate in shaping the T cell repertoire during thymic maturation. We now examine the possible role of the CD8 molecule during in vivo T cell development. Our results demonstrate that perinatal thymi treated with intact anti-CD8 mAb fail to generate CD8 single-positive T cells, while the generation of the other main phenotypes remains unchanged. Most importantly, the use of F(ab')2 anti-CD8 mAb fragments gave identical results, i.e., lack of generation of CD4-/CD8+ cells, with no effect on the generation of CD4+/CD8+. Furthermore, selective blocking of one CD8 allele with F(ab')2 mAbs in F1 mice expressing both CD8 alleles did not interfere with the development of CD4-/CD8+ cells, demonstrating that the absence of CD8+ T cells in homozygous mice is not due to depletion, but rather is caused by a lack of positive selection. This is most likely attributable to a deficient CD8-MHC class I interaction. Our findings strongly advocate that CD8 molecules are vital to the selection process that leads to the development of mature single-positive CD8 T cells.     

The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+3(2+) thymocytes as post- selection intermediates leading to mature T cells

Cortical thymocytes from adult mice, separated on the basis of coexpression of CD4 and CD8 or of binding of high levels of peanut agglutinin (PNA), were subdivided according to the level of expression of the T cell receptor (TCR)-CD3 complex. The incidence of dividing cells in the resultant subpopulations was determined by DNA staining. Precursor-product relationships and the timing of TCR-CD3 acquisition were studied using continuous in vivo [3H]TdR labeling and radioautography. The extent of intrathymic selection for TCR specificity in the subpopulations was determined from the incidence of cells bearing V beta 6 or V beta 17a in different mouse strains. The majority of dividing CD4+8+ blast cells expressed extremely low levels of TCR-CD3, indicating that TCR expression and specificity selection generally occurred after division ceased. The [3H]TdR-labeling studies indicated that postdivision TCR expression was rapid, and that those nondividing cortical thymocytes which had not expressed significant levels of TCR by day 1, remained extremely low or negative for their entire 3.6-d lifespan. Small cortical thymocytes which expressed moderate levels of TCR-CD3, were predominantly an unselected population with a lifespan of 3.8 d. A small subgroup of CD4+8+ PNA+ cortical thymocytes expressing high levels of TCR-CD3 was identified as a nondividing intermediate between the small cortical thymocytes expressing moderate levels of TCR and mature medullary thymocytes. These intermediates showed a 1-d lag in [3H]TdR labeling, then a 3.4-d transit time. The cell flux through this intermediate subpopulation was approximately 10(6) cells/d, similar to the rate of turnover of mature thymocytes; thus, although only 3-4% of thymocytes progressed to this intermediate state, once reaching it most then progressed to full maturity. In accordance with this, the incidence of the V beta selection markers within the intermediate subpopulation indicated that both positive and negative selection had already occurred. Selection for TCR specificity in the systems studied appeared to take place among CD4+8+ thymocytes expressing intermediate levels of TCR.     

脐血CD34^＋细胞体外诱导分化为成熟巨核细胞及产出血小板

背景:据作者查新检索,国外尚无通过体外诱导干细胞生成具有功能的血细胞并形成产品的报道.目的:体外诱导脐血CD34+细胞向成熟巨核细胞分化,并观察血小板产出情况.设计、时间及地点:细胞学体外观察,于200412006在湘雅医院及湘雅三医院中心实验室完成.材料:脐带来源于足月妊娠健康产妇,由湘雅医院提供.方法:免疫磁珠法分选脐血CD34+细胞,按5×107L-1密度接种于24孔培养板,加入含L-谷氨酰胺、铁饱和的人转铁蛋白、CaCl2、胰岛素、去离子牛血清白蛋白及重组人血小板牛成素的StemPro-34无血清培养基,置于37℃、体积分数为0.05的CO2饱和湿度条件F向巨核细胞诱导培养14～21d.吸取细胞培养液,离心取上清,再次离心弃上清,余下物质即为细胞培养液中比重较小的血小板样颗粒.同法分离正常富血小板血浆中的血小板.主要观察指标:培养细胞与上清液中血小板样颗粒的形态变化、免疫组织化学染色结果、显微及超微结构观察,血小板聚集情况及CD41的表达.结果:培养第10天,巨核细胞诱导培养体系中出现丝状物质,片有血小板样颗粒产生,第16天达高峰:培养细胞强阳性表达血小板特异件抗原GP Ⅱb Ⅲa:光镜观察培养细胞呈成熟巨核细胞形态,但也可见幼稚巨核细胞样,巨核细胞旁可见血小板样颗粒:电镜观察培养细胞大多呈成熟巨核细胞形态,少量呈凋亡状态,上清液中血小板样颗粒与富血小板血浆中的血小板大小、超微结构基本一致,有的血小板表面光滑,有的则呈现不规则表面.上清液中血小板样颗粒与正常富血小板血浆中的血小板均能对凝血酶产生聚集反应,流式细胞仪检测两者具有同样的CD41高表达率.结论:脐血CD34+细胞能在体外诱导生成高纯度且成熟的巨核细胞,并产出血小板.     

IL-12 is required for differentiation of pathogenic CD8+ T cell effectors that cause myocarditis

Cardiac antigen-specific CD8(+) T cells are involved in the autoimmune component of human myocarditis. Here, we studied the differentiation and migration of pathogenic CD8(+) T cell effector cells in a new mouse model of autoimmune myocarditis. A transgenic mouse line was derived that expresses cardiac myocyte restricted membrane-bound ovalbumin (CMy-mOva). The endogenous adaptive immune system of CMy-mOva mice displays tolerance to ovalbumin. Adoptive transfer of naive CD8(+) T cells from the ovalbumin-specific T cell receptor-transgenic (TCR-transgenic) OT-I strain induces myocarditis in CMy-mOva mice only after subsequent inoculation with ovalbumin-expressing vesicular stomatitis virus (VSV-Ova). OT-I effector T cells derived in vitro in the presence or absence of IL-12 were adoptively transferred into CMy-mOva mice, and the consequences were compared. Although IL-12 was not required for the generation of cytolytic and IFN-gamma-producing effector T cells, only effectors primed in the presence of IL-12 infiltrated CMy-mOva hearts in significant numbers, causing lethal myocarditis. Furthermore, analysis of OT-I effectors collected from a mediastinal draining lymph node indicated that only effectors primed in vitro in the presence of IL-12 proliferated in vivo. These data demonstrate the importance of IL-12 in the differentiation of pathogenic CD8(+) T cells that can cause myocarditis.     

Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection

Few hepatitis C virus (HCV) infections resolve spontaneously but those that do appear to afford protective immunity. Second infections are usually shorter in duration and are less likely to persist but mechanisms of virus control in immune individuals have not been identified. In this study we investigated whether memory helper and/or cytotoxic T lymphocytes provide protection in chimpanzees serially reinfected with the virus. Clearance of the first infection took 3-4 mo and coincided with the delayed onset of CD4+ and CD8+ T cell responses. High frequencies of memory T cells targeting multiple HCV proteins were stable over 7 yr of follow-up. Animals were infected for a second time to assess the protective role of memory T cells. In contrast to the prolonged course of the first infection, viremia was terminated within 14 d. Control of this second infection was kinetically linked to rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in blood. The importance of memory CD8+ T cells in control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset before a third infection. Virus replication was prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections and was not terminated until HCV-specific CD8+ T cells recovered in the liver. These experiments demonstrate an essential role for memory CD8+ T cells in long-term protection from chronic hepatitis C.     

Presentation of antigen by endothelial cells and chemoattraction are required for homing of insulin-specific CD8+ T cells

Activated insulin-specific CD8(+) T cells (IS-CD8(+) cells) home to the pancreas, destroy beta cells, and cause rapid diabetes upon transfer into diabetes-prone NOD mice. Surprisingly, they also cause diabetes in mouse strains that are free of preexistent inflammation. Thus, we hypothesized that islet-specific homing may be in part dependent on IS-CD8(+) cells' recognition of the cognate major histocompatibility complex (MHC)/peptide complexes presented by pancreatic endothelial cells, which acquire the antigen (insulin) from beta cells. In fact, islet-specific homing was abrogated in mice that lack MHC class I expression, or presentation of the specific peptide, or have impaired insulin secretion. Moreover, we found that IS-CD8(+) cells directly recognized pancreatic endothelial cells in islet organ cultures. Triggering of IS-CD8(+) cells' T cell receptor (TCR) led to activation of integrins expressed by these cells. In addition, chemokines, particularly SLC (CCL21), were also required for IS-CD8(+) cells' adhesion to endothelial monolayers and for successful homing in vivo. Thus, signaling through TCR and chemokine receptors work in concert to assure firm adhesion of T cells to the pancreatic endothelium. The antigen cross-presentation ability of endothelia may therefore contribute to the specificity of homing of activated T lymphocytes to the tissues where antigens are generated by other cell types.     

Response of mature unprimed CD8+ T cells to Mlsa determinants

Contrary to existing dogma, evidence is presented that proliferative responses of mature unprimed T cells to Mlsa antigens involve CD8+ cells as well as CD4+ cells. The response of CD8+ cells to Mlsa antigens proved to be heavily dependent on help from CD4+ cells, and responses were stronger in three I-E+ strain combinations than in an I-E- combination. In I-E+ combinations, CD8+ blast cells accounted for 20-25% of the blasts generated from unseparated T cells responding to Mlsa-bearing stimulator cells in vitro; similar findings applied to blast cells generated in vivo. The observation that the majority (greater than or equal to 50%) of Mlsa-stimulated CD8+ cells (and CD4+ cells) were V beta 6+ indicated that CD8+ cells respond to Mlsa antigens, per se, rather than to nonspecific stimuli. Whether CD4+ and CD8+ cells use the same or different H-2-restricting elements to respond to Mlsa antigens has yet to be resolved.     

T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2- dependent differentiation pathway of CD8 alpha+/beta- T cells

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hunig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.     

Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta chain-independent pathway

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.     

Sublethal gamma-radiation induces differentiation of CD4-/CD8- into CD4+/CD8+ thymocytes without T cell receptor beta rearrangement in recombinase activation gene 2-/- mice

DNA recombination of the immunoglobulin (Ig) or T cell receptor (TCR) gene loci is an essential step in the production of lymphocytes bearing antigen-specific receptors. Mice that lack the ability to rearrange their Ig and TCR gene loci are devoid of mature B and T cells. Complete rearrangement and expression of the TCR-beta chain has been suggested to allow immature thymocytes to switch from the CD4-/CD8- to the CD4+/CD8+ stage of thymic development. Thus, thymocytes from severe combined immune deficient (SCID) mice or mice deficient in recombinase activation genes (RAG), which do not undergo proper DNA rearrangement, are arrested at the early CD4-/CD8- stage of development. B cell precursors in SCID or RAG mice do not progress from the B220+/sIgM- /heat stable antigen (HSA)+/CD43+ to the B220+/sIgM-/HSA+/CD43- stage. In an attempt to reconstitute RAG-2-/- mice with bone marrow- or fetal liver-derived progenitor cells, we subjected these mice to sublethal doses of gamma-radiation. It is surprising that in the absence of donor cells, irradiated RAG-2-/- mice revealed a dramatic change in their lymphoid phenotype. 14 d after irradiation, the majority of thymocytes had advanced to the CD4+/CD8+ stage of T cell development and a small number of bone marrow precursors had progressed to the CD43-, HSAhi stage of B cell development. Analysis of the resulting CD4+/CD8+ thymocytes revealed no surface expression of the TCR/CD3 complex and no V-D-J rearrangement of the TCR-beta gene locus. Our findings provide evidence for a novel pathway that allows the transition of thymocytes from the CD4-/CD8- to the CD4+/CD8+ stage and that does not appear to require TCR-beta chain rearrangement.     

In vitro proliferation and differentiation of human CD34+ cells from peripheral blood into mature red blood cells with two different cell culture systems

BACKGROUND: An in vitro erythropoiesis assay is a powerful tool for investigating red blood cell (RBC) development and diseases of the erythroid lineage. Most assays, however, failed in either proliferation or terminal differentiation. Here two liquid cultures (LCs) for in vitro generation of RBCs from peripheral blood CD34+ cells were compared. STUDY DESIGN AND METHODS: Granulocyte-colony-stimulating factor-mobilized CD34+ cells were cultured for 16 days in a two-phase LC (2P-LC; Days 1-8, stem cell factor [SCF], erythropoietin [EPO], insulinlike growth factor [IGF]-1, and steroids; Days 9-16, EPO and insulin) and for 21 days in a three-phase LC (3P-LC; Days 1-7, SCF, thrombopoetin, and Flt3-ligand; Days 8-14, SCF, EPO, and IGF-1; Days 15-21, EPO and IGF-1). Maturation was analyzed by flow cytometry (CD36, CD71, glycophorin A [GPA]) and microscopy. RESULTS: In the 2P-LC, cell numbers increased from 0.5 x 10(6) to 25.7 x 10(6) +/- 15.1 x 10(6) cells per mL. More than 95 percent were GPA+ and showed morphologic characteristics of normoblasts (52 +/- 15%) and enucleated reticulocytes (43 +/- 18%). In the 3P-LC, a higher overall proliferation to 55.7 x 10(6) +/- 37.7 x 10(6) cells per mL was achieved (p < 0.05). This was also accompanied by a high degree of normoblasts (36 +/- 16%) and reticulocytes (48 +/- 24%). The amount of GPA+ cells was slightly lower (88.4 +/- 16.4%), associated with a significantly higher contamination by nonerythroid cells (15.8 +/- 19.3% vs. 3.9 +/- 2.9%, p < 0.05). CONCLUSION: Both LCs were able to generate fully matured RBCs and represent powerful tools for fundamental research in erythroid development and diseases targeting the erythroid lineage. A slightly higher proliferation was achieved in the 3P-LC. This was associated with a limited homogeneity due to more nonerythroid cells, however. Therefore the 2P-LC is favored, also saving additional culture days and growth factors.     

Unraveling a revealing paradox: Why major histocompatibility complex I-signaled thymocytes "paradoxically" appear as CD4+8lo transitional cells during positive selection of CD8+ T cells

The mechanism by which T cell receptor specificity determines the outcome of the CD4/CD8 lineage decision in the thymus is not known. An important clue is the fact that major histocompatibility complex (MHC)-I-signaled thymocytes paradoxically appear as CD4+8lo transitional cells during their differentiation into CD8+ T cells. Lineage commitment is generally thought to occur at the CD4+8+ (double positive) stage of differentiation and to result in silencing of the opposite coreceptor gene. From this perspective, the appearance of MHC-I-signaled thymocytes as CD4+8lo cells would be due to effects on CD8 surface protein expression, not CD8 gene expression. But contrary to this perspective, this study demonstrates that MHC-I-signaled thymocytes appear as CD4+8lo cells because of transient down-regulation of CD8 gene expression, not because of changes in CD8 surface protein expression or distribution. This study also demonstrates that initial cessation of CD8 gene expression in MHC-I-signaled thymocytes is not necessarily indicative of commitment to the CD4+ T cell lineage, as such thymocytes retain the potential to differentiate into CD8+ T cells. These results challenge classical concepts of lineage commitment but fulfill predictions of the kinetic signaling model.     

Stimulation of mature unprimed CD8+ T cells by semiprofessional antigen-presenting cells in vivo.

To test whether unprimed CD8+ cells can recognize class I alloantigens presented selectively on non-bone marrow (BM)-derived cells, unprimed parental strain CD8+ cells were transferred to long-term parent-->F1 BM chimeras prepared with supralethal irradiation. Host class I expression in the chimeras was undetectable on BM-derived cells and, in spleen, was limited to low-level staining of vascular endothelium and moderate staining of follicular dendritic cells (a population of nonhemopoietic cells in germinal centers). Despite this restricted expression of antigen, acute blood-to-lymph recirculation of parental strain T cells through the chimeras led to selective trapping of 95% of CD8+ cells reactive to normal F1 spleen antigen presenting cells (APC) in vitro. Subsequently, a small proportion of the trapped cells entered cell division and gave rise to effector cells expressing strong host-specific CTL activity. The activation of host-specific CD8+ cells was also prominent in double-irradiated chimeras, and cell separation studies showed that the effector cells were generated from resting precursor cells rather than from memory-phenotype cells. It is suggested that the non-BM-derived cells in the chimeras acted as semiprofessional APC. These cells were nonimmunogenic for most host-reactive CD8+ cells but were capable of stimulating a small subset of high-affinity T cells. The possible relevance of the data to the prolonged immunogenicity of vascularized allografts in humans is discussed.     

CD8 T cells are required for the formation of ectopic germinal centers in rheumatoid synovitis

The assembly of inflammatory lesions in rheumatoid arthritis is highly regulated and typically leads to the formation of lymphoid follicles with germinal center (GC) reactions. We used microdissection of such extranodal follicles to analyze the colonizing T cells. Although the repertoire of follicular T cells was diverse, a subset of T cell receptor (TCR) sequences was detected in multiple independent follicles and not in interfollicular zones, suggesting recognition of a common antigen. Unexpectedly, the majority of shared TCR sequences were from CD8 T cells that were highly enriched in the synovium and present in low numbers in the periphery. To examine their role in extranodal GC reactions, CD8 T cells were depleted in human synovium-SCID mouse chimeras. Depletion of synovial CD8 T cells caused disintegration of the GC-containing follicles. In the absence of CD8 T cells, follicular dendritic cells disappeared, production of lymphotoxin-alpha1beta2 markedly decreased, and immunoglobulin (Ig) secretion ceased. Immunohistochemical studies demonstrated that these CD8 T cells accumulated at the edge of the mantle zone. Besides their unique localization, they were characterized by the production of interferon (IFN)-gamma, lack of the pore-forming enzyme perforin, and expression of CD40 ligand. Perifollicular IFN-gamma+ CD8 T cells were rare in secondary lymphoid tissues but accounted for the majority of IFN-gamma+ cells in synovial infiltrates. We propose that CD8+ T cells regulate the structural integrity and functional activity of GCs in ectopic lymphoid follicles.     

CD4+ but not CD8+ cells are essential for allorejection

The generation of knockout mice with targeted gene disruption has provided a valuable tool for studying the immune response. Here we describe the use of CD4 and CD8 knockout mice to examine the role of CD4+ and CD8+ cells in initiating allotransplantation rejection. Pretreatment with a brief course of depletive anti-CD4 monoclonal antibody therapy allowed permanent survival of heart, but not skin, allografts transplanted across a major histocompatibility barrier. However, skin as well as heart grafts were permanently accepted in the CD4 knockout mice. Transfer of CD4+ cells into CD4 knockout recipient mice 1 d before skin engraftment reconstituted rejection, demonstrating that CD4+ cells are necessary for initiating rejection of allogeneic transplants. Major histocompatibility complex disparate heart and skin allografts transplanted into CD8 knockout recipients were rejected within 10 d. This study demonstrates that CD4+ but not CD8+ T cells are absolutely required to initiate allograft rejection.     

H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes

Studies using major histocompatibility complex (MHC)-Ia-deficient mice have shown that MHC-Ib-restricted CD8+ T cells can clear infections caused by intracellular pathogens such as Listeria monocytogenes. M3-restricted CD8+ T cells, which recognize short hydrophobic N-formylated peptides, appear to comprise a substantial portion of the MHC-Ib-restricted T cell response in the mouse model of L. monocytogenes infection. In this study, we isolated formyltransferase (fmt) mutant strains of L. monocytogenes that lacked the ability to add formyl groups to nascent polypeptides. These fmt mutant Listeria strains did not produce antigens that could be recognized by M3-restricted T cells. We showed that immunization of MHC-Ia-deficient mice with fmt mutant Listeria resulted in stimulation of a protective memory response that cleared subsequent challenge with wild-type L. monocytogenes, despite the fact that M3-restricted CD8+ T cells did not proliferate in these mice. These data suggest that M3-restricted T cells are not required for protection against L. monocytogenes and underscore the importance of searching for new antigen-presenting molecules among the large MHC-Ib family of proteins.     

Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells

The TCR/CD3 complex plays a central role in antigen recognition and activation of mature T cells, and, therefore, abnormalities in the expression of the complex should induce unresponsiveness of T cells to antigen stimulus. Using flow cytometry, we detected and enumerated variant cells with loss or alteration of the surface TCR/CD3 expression among human mature CD4+ T cells. The presence of variant CD4+ T cells was demonstrated by isolating and cloning them from peripheral blood, and their abnormalities can be accounted for by alterations in TCR expression such as defects of protein expression and partial protein deletion. The variant frequency in peripheral blood increased with aging in normal donors and was highly elevated in patients with ataxia telangiectasia, an autosomal recessive inherited disease with defective DNA repair and variable T cell immunodeficiency. These findings suggest that such alterations in TCR expression are induced by somatic mutagenesis of TCR genes and can be important factors related to age-dependent and genetic disease-associated T cell dysfunction.