A QUANTITATIVE ANALYSIS OF CD45RLOW CD4 T CELLS IN THE SUBARACHNOID SPACE OF LEWIS RATS WITH AUTOIMMUNE ENCEPHALOMYELITIS

In order to quantify encephalitogenic effector T cells in the subarachnoid space (SAS) and spinal cord afflicted with experimental autoimmune encephalomyelitis (EAE), we immunized Lewis rats using myelin basic protein and complete Freund's adjuvants and analyzed the inflammatory cells by fluorescence-activated cell sorter (FACS) and immunohistochemistry. At the induction stage of EAE, the majority of observed inflammatory cells were determined by immunohistochemistry to be either CD4+ T cells or OX42+ macrophages. Among CD4+ T cells, both CD45R high (OX22+) and CD45R low (OX22-) T cells were found in the SAS, while in the neighboring subpial spinal cord parenchyma, CD45R low (OX22-negative)/CD4+ T cells predominated. FACS analysis showed that CD45RC low/CD4+ T cells was 83% of total CD4+ T cells in the SAS, while 94% of cells with the same phenotype were found in the parenchyma of rat spinal cords afflicted with EAE. This finding suggests that during the induction stage of EAE, effector T cells preferentially migrate into the subpial parenchyma from the SAS. Thereafter, suppressor T cells follow, which may lead to the spontaneous recovery from EAE paralysis.     

CD45R CD4 T cell subset-reconstituted nude rats: subset-dependent survival of recipients and bi-directional isoform switching.

High and low molecular weight variants (CD45R) of the leukocyte common antigen (CD45) divide CD4 T helper cells into subpopulations which display distinct characteristics. In vitro and in vivo evidence suggested that the presence of the high molecular weight splice variants CD45RA or CD45RB distinguished naive CD4 T cells from memory T cells which underwent an irreversible switch to the low molecular weight isoform as a consequence of antigen encounter. In the rat monoclonal antibody MRC OX22 identifies an epitope on the CD45RB molecule. We investigated this proposed differentiation pathway by reconstituting athymic nude rats with highly purified OX22+ or OX22- CD4 T lymphocyte subsets obtained from the thoracic duct (TDL) of euthymic, congenic, allotype-marked donors. Injection of CD4+CD45RB- (45R-) cells ensured long-term survival of nude recipients; recipients of CD4+CD45R+ (45R+) cells died within 2-3 months of injection. Early after transfer (3-4 weeks) the progeny of both 45R+ and 45R- TDL were uniformly 45R-. With time (by 2 months) progeny of both parental types expressed the high molecular weight CD45RB isoform. Nude recipients of 45R- TDL always generated progeny a proportion of which bore the 45R+ phenotype; 3 months to 2 years post-injection, 30%-50% of the donor-derived CD4 T cells were 45R+, 45R- progeny isolated from primary recipients of either 45R- or 45R+ cells transferred into secondary nude recipients induced skin allograft rejection with equal effectiveness and also generated 45R+ offspring. The results indicated that CD4 T cell subsets switched between CD45R isoforms and that the change between high and low molecular weight expression was bi-directional. The splice variants apparently are not lineage or maturation markers, but rather identify CD4 T cells that exist transiently in different functional states.     

Immune regulatory mechanisms influence early pathology in spinal cord injury and in spontaneous autoimmune encephalomyelitis

Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R- mice lack regulatory T cells. Our results show that 8 days after SCI, T/R- mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R- mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R- mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage.     

Identification of CD4- CD8- alpha beta T cells in the subarachnoid space of rats with experimental autoimmune encephalomyelitis. A possible route by which effector cells invade the lesions.

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats to elucidate the origin of effector T cells and the route by which they invade lesions. Since mouse studies have suggested that some autoimmune diseases are induced by extrathymic T cells in the liver, we focused our attention on the properties of mononuclear cells (MNC) isolated from the liver and other organs in rats with EAE. A small but significant proportion of LFA-1+ alpha beta T cells was identified in the liver as early as day 7 after immunization with myelin basic protein (MBP). Such LFA-1+ alpha beta T cells were also abundant among MNC attached to the spinal cord (i.e. subarachnoid space), and MNC infiltrated the spinal cord in rats with EAE (day 12). In electron microscopy, MNC attached to the spinal cord were found to be quite unique in terms of their large cell size with well-developed microvilli. More importantly, they were comprised of a considerably large proportion of double-negative CD4- CD8- T cells as well as single-positive CD4+ T cells. However, the cells which infiltrated the spinal cord were mainly CD4+. The present results raise the possibility that the subarachnoid space might be a major site for the expansion of extrathymic T cells in rats with EAE, and that only a limited population of CD4+ T cells invade the spinal cord directly through the outer layer and elicit EAE.     

Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ⁺ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4⁺ or CD8⁺. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4^?CD8^? T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4^?CD8^? T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.     

脐血CD4~+CD45~+调节性T细胞体外扩增及其抑制复发性流产患者免疫细胞功能研究

目的:建立有效的脐血CD4+CD45+调节性T(Treg)细胞体外培养体系,探讨其对复发性流产患者免疫细胞功能的抑制作用,为开发新的复发性流产免疫治疗方法奠定实验基础。方法:应用免疫磁珠分选法从脐血淋巴细胞中获得Treg细胞,体外进行长期培养、扩增,分别应用MTT法和51Cr释放法检测扩增后的Treg细胞对复发性流产患者效应T细胞和NK细胞功能的抑制作用;ELISA法检测Treg对复发性流产外周血Th1/Th2细胞因子的影响。结果:经过5、15、25天培养,Treg细胞的扩增倍数分别为(6.5±0.6)倍、(18.2±2.5)倍和(52.7±4.8)倍,Treg细胞的纯度分别为(93.2±3.4)%、(88.6±2.9)%和(81.9±3.3)%。培养25天后的脐血Treg细胞对复发性流产的CD4+CD25-效应T细胞和NK细胞杀伤活性具有明显抑制作用,能使外周血中IFNγ-水平下降,IL-10水平升高。结论:培养扩增后Treg细胞能在体外有效抑制复发性流产患者外周血免疫细胞功能,并能使外周Th1型细胞因子水平下降,Th2型细胞因子水平升高。     

OX40 (CD134) engagement drives differentiation of CD4+ T cells to effector cells

Naive, CD4+ T cells proliferate extensively but fail to differentiate when they are transferred into unirradiated recipients that express alloantigen or transgenic antigen on all MHC class II+ cells. Addition of an agonist antibody to OX40 (CD134), a costimulatory TNF receptor family member expressed on activated CD4+ T cells, enables the proliferating T cells to accumulate as differentiated effector cells and kill the host animals. The donor T cells from anti-OX40-treated animals express high levels of IL-2R alpha (CD25) and acquire the ability to secrete IFN-gamma when stimulated with IL-12 and IL-18. OX40 promotes differentiation by 48 h in T cell priming, before changes in Bcl-2 expression or cell recovery become apparent. We found that a Bcl-2 transgene or deficiency in Fas or TNFR1 failed to influence accumulation of differentiated donor cells, and found larger changes in expression of cytokine and cytokine receptor genes than in survival genes. Accumulation of differentiated CD4+ effector T cells is initiated directly through OX40, but some OX40-deficient donor cells can gain effector function as bystanders to OX40+/+ cells. Taken together, these data suggest that CD4+ T cell differentiation to effector function is an important effect of OX40 engagement under conditions of ubiquitous antigen presentation.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

CD62L is required for the priming of encephalitogenic T cells but does not play a major role in the effector phase of experimental autoimmune encephalomyelitis

CD62L (l ‐selectin, mel 14) regulates naïve T cell homing into lymph nodes and the migration of leucocytes to sites of inflammation. The requirement of CD62L in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has been demonstrated previously. However, it remains controversial as to whether CD62L is required for the induction or the effector phase of EAE. It is also unclear whether other non‐T effector cells need CD62L to enter the central nervous system (CNS) parenchyma and exert their damaging effects on myelin. We report that mice with a targeted mutation of CD62L are resistant to Myelin oligodendrocyte glycoprotein peptide‐induced EAE. CD62L‐deficient mice had no peptide‐specific T cell responses in the draining lymph nodes and had lower levels of peptide‐specific T cell responses in spleens at a later time point. Adoptive transfer studies showed that CD62L‐deficient mice were fully susceptible to adoptive transfer EAE induced by either wildtype or CD62L‐deficient T cells. Moreover, CD62L‐deficient, F4/80⁺ macrophages can be efficiently recruited into the CNS parenchyma. These data suggest that CD62L is required for the induction of encephalitogenic T cells during EAE development, but is not required by T and non‐T effector cells to attack the CNS parenchyma.     

DAB389IL-2 suppresses autoimmune inflammation in the CNS and inhibits T cell-mediated lysis of glial target cells

In multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), activated CD4 ⁺ T cells with upregulated IL-2R mediate inflammation and demyelination in the central nervous system (CNS). DAB₃₈₉IL-2, a chimeric fusion protein of IL-2 and diphtheria toxin, inhibits human and rodent IL-2 activated T cells that express the high affinity interleukin-2 receptor. In the present study, DAB₃₈₉IL-2 was used to treat rats with EAE. We wanted to investigate the possibility that DAB₃₈₉IL-2 could prevent tissue destruction within the CNS. We used a suboptimal dose of DAB₃₈₉IL-2 that allowed substantial transmigration of inflammatory cells across the blood–brain barrier. DAB₃₈₉IL-2 inhibited infiltration of CD4⁺, CD8⁺, CD25⁺ and TCR αβ⁺ associated mononuclear cells and inflammatory macrophages in the spinal cord on day 13 post-immunization, at the peak of disease. Gene expression study showed that DAB₃₈₉IL-2 treatment suppressed TNF-α and IFN-γ as well as IL-10 cytokine gene expression in the spinal cord of rats with EAE on day 13. DAB₃₈₉IL-2 in vitro treatment suppressed cytotoxicity of MBP-activated T cells from rats with EAE against oligodendrocytes in culture by 66%. Astrocytes were less targeted by MBP activated T cells in vitro. This study suggests that DAB₃₈₉IL-2 directly targets CD4⁺ and CD25⁺ (IL-2R) T cells and effector T cell function and also indirectly suppresses the activation of macrophage CD169⁺ (ED3⁺) and microglia CD11b/c (OX42⁺) populations in the CNS.     

Suppression of experimental autoimmune encephalomyelitis in Lewis rats by antibodies against CD2

The immunotherapeutic potential of three anti-rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti-CD2 mAb on the effector phase of disease. Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP-induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen-specific cellular response in MBP/CFA-immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen- or Ab-mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti-CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti-CD4 or anti-T cell receptor Ab.     

Hemorrhagic autoimmune encephalomyelitis induced by adoptive transfer of activated semiallogeneic spleen cells into irradiated rats.

Using lethal irradiation of recipients, adoptive transfer of experimental allergic encephalomyelitis (EAE) into Lewis (LEW) rats using (LEW x PVG/c)F1 (LPVGF1) spleen cells was successfully achieved. Recipient rats usually developed clinical signs of EAE 5 or 6 days after transfer. The EAE was characterized by the presence of a number of petechiae over the spinal cord. Immunohistochemical examination using OX27, a monoclonal antibody specific for RT1.Ac, revealed the localization of transferred F1 (RT1(1/c] cells in the LEW recipients (RT1(1]. Most of the inflammatory cells in the spinal cord lesions were stained positively for OX27, indicating that they were transferred cells. In mild EAE, more W3/25+ cells were found than OX8+ cells. OX8+ cells were predominant in severe EAE, however. Examination of the spleens of rats that developed EAE by OX27 staining revealed that transferred F1 cells gradually increased in number and reached a maximal level on days 5 and 6. In the spleens of rats that received irradiation and transfer but did not develop EAE, few transferred F1 cells were observed. In addition, bromodeoxyuridine (BrdU)-anti-BrdU immunohistochemistry was employed to demonstrate that cell proliferation really takes place in the spleen. It was revealed that the spleens of the recipients of lethal irradiation and F1 cells contained many BrdU+ cells. Because rats given lethal irradiation alone had extremely few BrdU-positive cells in their spleens, labeled cells in the recipients of radiation and transfer originated from transferred F1 cells. Collectively, these findings strongly suggest that transferred cells previously activated in vitro undergo further proliferation in the lymphoid organs of recipients to bring about the development of EAE.     

Distinct functions of autoreactive memory and effector CD4+ T cells in experimental autoimmune encephalomyelitis

The persistence of human autoimmune diseases is thought to be mediated predominantly by memory T cells. We investigated the phenotype and migration of memory versus effector T cells in vivo in experimental autoimmune encephalomyelitis (EAE). We found that memory CD4(+) T cells up-regulated the activation marker CD44 as well as CXCR3 and ICOS, proliferated more and produced more interferon-gamma and less interleukin-17 compared to effector T cells. Moreover, adoptive transfer of memory T cells into T cell receptor (TCR)alphabeta(-/-) recipients induced more severe disease than did effector CD4(+) T cells with marked central nervous system inflammation and axonal damage. The uniqueness of disease mediated by memory T cells was confirmed by the differential susceptibility to immunomodulatory therapies in vivo. CD28-B7 T cell costimulatory signal blockade by CTLA4Ig suppressed effector cell-mediated EAE but had minimal effects on disease induced by memory cells. In contrast, ICOS-B7h blockade exacerbated effector T cell-induced EAE but protected from disease induced by memory T cells. However, blockade of the OX40 (CD134) costimulatory pathway ameliorated disease mediated by both memory and effector T cells. Our data extend the understanding of the pathogenicity of autoreactive memory T cells and have important implications for the development of novel therapies for human autoimmune diseases.     

CD45 isoform expression is associated with different susceptibilities of human naive and effector CD4+ T cells to respond to IL-4

Interleukin-4 (IL-4) is the major factor promoting the development of T helper type 2 (Th2) cells from naive precursor T cells. Minute amounts of IL-4 produced by naive T cells seem to be sufficient; however, the molecular mechanisms explaining this efficient utilization of IL-4 are not yet known. Here, we show that human CD4+ CD45RA+ naive T cells, in contrast to CD4+ CD45R0+ effector T cells, show responsiveness to endogenous as well as exogenous IL-4 to proliferate and differentiate towards Th2 cells in vitro. Despite production levels of IL-4 below conventional detection limits, CD45RA+ T cell-derived IL-4 could clearly activate STAT6. Although the expression levels of IL-4R and STAT6 were not different between naive and effector T cells, only naive T cells responded to IL-4 in a STAT6-dependent reporter gene assay. Transfecting a trans-dominant negative form of STAT6 abrogated IL-4-induced proliferation in CD45RA+ cells. A significantly higher protein tyrosine phosphatase (PTPase) activity was detected in CD45R0+ T cells as compared to CD45RA+ T cells. Cross-linking CD45 potently reduced PTPase activity in CD45R0+ T cells and restored their ability to proliferate in response to IL-4. Thus, CD45 PTPase activity contributes to the susceptibility of naive and memory T cells to respond to IL-4.     

Allograft rejection in athymic nude rats by transferred T-cell subsets. I. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to complete allogeneic incompatibilities

PVG.rnu/rnu nude rats were pre-grafted with two allogeneic skin grafts, AO(RTlu) and BN(RTln), 6-14 days in advance of cell transfer. Cellular requirements for rejection were established by transferring graded numbers of B cell-depleted (Ig-) thoracic duct lymphocytes (TDL) or purified W3/25+ (CD4+) or OX8+ (CD8+) TDL subsets. Allografts were rejected by 10(5) to 5 x 10(6) Ig- TDL in a dose-dependent fashion. A similar dose-response relationship was found by transferring 5 x 10(5) to 5 x 10(6) Ig- OX8- TDL (purified by depletion of B cells and OX8+ cells). Larger numbers of Ig- OX8- TDL (10-30 x 10(6)) did not significantly accelerate rejection. W3/25+ TDL alone (10(5)), highly purified by fluorescence-activated cell sorting (FACS), were sufficient to induce allograft rejection in this athymic nude rat model. In contrast, 10 times more FACS purified OX8+ TDL (10(6)) were unable to initiate skin graft rejection despite the complete class I and class II MHC incompatibilities. Furthermore, the addition of 10(6) OX8+ cells did not accelerate or retard the rejection induced by 10(5) W3/25+ cells alone. Pre-grafted nude recipients, irradiated (500 R) 2 hr before W3/25+ TDL injection, in order to eliminate putative nude T cells, rejected allografts on the same day as unirradiated controls. We conclude that when confronted with complete MHC disparities, CD4+ T cells are necessary and sufficient to induce skin allograft rejection whereas CD8+ T cells do not appear to contribute.     

Two discreet subsets of CD8 T cells modulate PLP(91-110) induced experimental autoimmune encephalomyelitis in HLA-DR3 transgenic mice

Previously we showed that transgenic mice expressing human HLA-DR3 gene are susceptible to PLP(91-110) induced experimental autoimmune encephalomyelitis (EAE) and can serve as an animal model of multiple sclerosis (MS). HLA-DR3 mice with EAE showed increased number of CD8 T cells indicating their important role in disease pathogenesis. The role of CD8 T cells in MS, an inflammatory demyelinating disease of CNS, has been enigmatic as it has been assigned both regulatory and pathogenic roles. Therefore, to evaluate the role of CD8 T cells, we generated CD8 deficient HLA-DR3 transgenic mice (DR3.CD8(-/-)). Immunization with PLP(91-110) led to more severe EAE in DR3.CD8(-/-) mice compared to HLA-DR3 mice indicating a regulatory role for CD8 T cells. Interestingly, DR3.CD8(-/-) mice with EAE showed decreased CNS pathology compared to DR3 mice thus suggesting a pathogenic role for CD8 T cells. We show that these two subsets of CD8 T cells can be differentiated based on the surface expression of CD122 (IL-2 Rβ chain). CD8 T cells expressing CD122 (CD8+CD122+) play a regulatory role while CD8+CD122- T cells act as a pathogenic subset. CD122 expressing CD8 T cells are the regulatory subset of CD8 T cells and regulate the encephalitogenic CD4 T cells through direct modulation of antigen presenting cells and/or through the release of immunoregulatory cytokines such as IL-10, IFNγ and TGFβ. We also showed that adoptive transfer of CD8CD122- T cells caused increased spinal cord demyelination indicating that these are pathogenic subset of CD8 T cells. Our study suggests that CD8+ T cells play both regulatory as well as pathogenic role in disease pathogenesis of EAE. A better understanding of these subsets could aid in designing novel therapy for MS patients.     

The organotin-induced thymus atrophy, characterized by depletion of CD4+ CD8+ thymocytes, is preceded by a reduction of the immature CD4- CD8+ TcR alpha beta-/low CD2high thymoblast subset.

Thymic changes in the rat induced by the thymus atrophy-inducing organotin compound di-n-butyltin dichloride (DBTC) were examined using FACS analyses. The number of CD4+CD8+ thymocytes was reduced by DBTC treatment from Day 2 onwards and reached minimum level on Days 4 and 5 after dosing. On these days the CD4-CD8- and both the CD4-CD8+ and CD4+CD8- subsets were not affected. On Day 2 we observed a reduced proportion of transferrin receptor (CD71)-positive CD4-OX44- cells, representing the cycling immature CD4-CD8+ cells, and of CD71+OX44- cells, representing the cycling CD4+CD8+ cells, but not of CD71+CD4-CD8- cells. When compared to controls, the FSChigh cell population of DBTC-treated rats contained less CD4-OX44- and OX44- cells, which were further characterized as CD2high and T-cell receptor (TcR)alpha beta- low. Moreover, fewer TcR alpha beta high cells were detected in the OX44- thymoblast subset of DBTC-treated rats. The number of CD4-CD8- thymoblasts appeared marginally decreased while the numbers of CD4+OX44+ cells, representing mature CD4+ cells, were not affected. These data indicate that DBTC causes a preferential initial depletion of immature CD4-CD8+CD2high TcR alpha beta-low thymoblasts. This initial event may result in a decreased formation of CD4+CD8+ thymoblasts and of small CD4+CD8+ thymocytes. These characteristics of the initially depleted subset indicate a specific anti-proliferative effect of DBTC and may give clues for the mechanism involved in the induction of thymus atrophy.     

Distributional characteristics of CD25 and CD127 on CD4+ T cell subsets in chronic HCV infection

Attenuated CD4+ T-cell-mediated immune responses are involved in persistence of HCV infection, but the mechanism remains undefined. In this study, the proportions of CD4+ T cell subsets, naïve, central memory, effector memory and effector cells, along with CD25 (IL-2Rα) and CD127 (IL-7Rα) expression on different CD4+ T cell subsets, were measured by polychromatic flow cytometry in 24 chronic HCV-infected individuals and 21 healthy controls. A significant decrease in naïve CD4+ T cells and an increase of central memory and effector memory CD4+ T cells were found in HCV-infected patients compared with healthy controls. HCV-infected patients showed a lower level of CD127 expression in all CD4+ T cells subsets, especially in central memory and effector CD4+ T cells. In terms of total CD4+ T cells, an increase in CD25+ regular T cells (CD4+ CD25+ CD127lo) was found in HCV-infected patients. Interestingly, naïve CD4+ T cells showed increased CD25 expression, while effector memory and effector CD4+ T cells had lower CD25 expression. These data indicated that variations in different fractions of CD4+ T cells, including the phenotypic profile and expression level of CD25 and CD127, may be associated with low efficiency of immune response in chronic HCV infection. These results will strengthen the understanding of pathogenesis and dysfunction of CD4+ T cell immunity during long-term HCV persistence.     

Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division.

We recently showed that circulating human CD8(+) effector cells have a CD45RA+CD27(-) membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA+CD27(-) phenotype is dependent on antigenic stimulation and involves cellular division, TCR Vbeta usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8(+) subsets. FACS analysis showed that the TCR Vbeta repertoire of CD8(+)CD45RA+CD27(-) cells differed significantly from that of unprimed CD8(+)CD45RA+CD27(+) cells. Moreover, in two out of six individuals large expansions of particular Vbeta families were observed in the CD8(+)CD45RA+CD27(-) subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8(+)CD45RA+CD27(-) population most of the 22 tested Vbeta families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3+/-1.0 kb shorter in the CD8(+)CD45RA+CD27(-) subset compared with the unprimed CD8(+)CD45RA+CD27(+) population, but did not differ substantially from that of memory type, CD8(+)CD45RA-CD27(+) T cells. These findings indicate that the CD8(+)CD45RA+CD27(-) cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.     

Experimental allergic encephalomyelitis (EAE) in mice lacking CD4+ T cells

Like most experimental autoimmune disease experimental allergic encephalomyelitis (EAE) has been shown to be mediated by CD4⁺ helper T cells. In vivo antibody blocking studies with anti-CD4 and adoptive transfer of activated CD4⁺ T cells indicate the importance of CD4⁺ cells in disease induction. Fourth backcross generation mutant CD4—/— PL/J mice were immunized with myelin basic protein. Despite the lack CD4⁺ T cells some of these mice developed EAE, albeit, at a considerably reduced frequency and with variable severity. Furthermore, antigen-specific T cell proliferation can be demonstrated, indicating some residual helper activity that is major histocompatibility complex class II restricted. This demonstrates that, although the CD4⁺ T cell is the prime effector cell in EAE, in mice developmentally lacking in CD4, the expanded double-negative T cells may subserve helper and effector functions.