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.     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

Cyclosporine A prevents the generation of single positive mature T cells in newborn mice

The effect of Cyclosporin A (CsA) during T cell development was investigated in newborn mice. CsA treatment completely blocked the generation of peripheral single positive (SP) mature T cells: the lymphatic tissues were hypoplastic. However, double negative (DN) T3 expressing lymphocytes were still detectable. Thymuses from CsA-treated mice lacked the SP L3T4(CD4)+ subset, DN and double positive (DP) thymocytes were still present. We further defined a SP Lyt2(CD8)+ thymic subpopulation which lacked CD3 expression and displayed no functional activity in vitro. Thus, CsA critically interferes with the maturation of SP T lymphocytes; we found no evidence for 'leaky' autoreactive peripheral SP T cells in CsA-treated newborn mice.     

Mouse models of autoimmune disease suggest that self-tolerance is maintained by unresponsive autoreactive T cells.

Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.     

Depletion of CD52‐positive cells inhibits the development of central nervous system autoimmune disease,but deletes an immune‐tolerance promoting CD8 T‐cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis

The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction by way of intravenous antigen administration such that it could be used to either improve efficiency of multiple sclerosis (MS) inhibition or inhibit secondary autoimmunities that may occur following alemtuzumab use in MS. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses were assessed clinically. A dose–response of CD4 monoclonal antibody depletion indicated that the 60–70% functional CD4 T‐cell depletion achieved in perceived failed trials in MS was perhaps too low to even stop disease in animals. However, more marked (~75–90%) physical depletion of CD4 T cells by CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly, in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness through a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T‐cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rationale for the rapid B‐cell hyper‐repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T‐cell regulatory mechanisms that promote immune tolerance may account for the secondary B‐cell autoimmunities, which occur following alemtuzumab treatment of MS.     

Importance of CD80/CD86-CD28 interactions in the recognition of target cells by CD8+CD122+ regulatory T cells

CD8+CD122+ regulatory T cells are a newly identified, naturally occurring type of regulatory T cell that produce interleukin-10 (IL-10) and effectively suppress interferon-gamma (IFN-gamma) production from both CD8+ and CD4+ target cells. Molecular mechanisms responsible for the recognition of target cells by CD8+CD122+ regulatory T cells were investigated in this study by using an in vitro culture system that reconstitutes the regulatory action of these cells. CD8+CD122( regulatory T cells did not produce IL-10 and did not suppress the IFN-gamma production of allogeneic target T cells when they were stimulated by immobilized anti-CD3 antibody alone, but they clearly produced IL-10 and suppressed the IFN-gamma production of target cells when stimulated by anti-CD3 plus anti-CD28-coated beads. IFN-gamma production by major histocompatibility complex-class I-deficient T cells was also suppressed by CD8+CD122+ regulatory T cells stimulated with anti-CD3 plus anti-CD28 antibody but was not suppressed by cells stimulated by anti-CD3 alone. Experiments examining the blockade of cell surface molecules expressed on either the regulatory cells or the target cells by adding specific neutralizing antibodies in the culture indicated that CD80, CD86, and CD28 molecules were involved in the regulatory action, but cytotoxic T lymphocyte antigen-4, inducible costimulatory molecule (ICOS) and programmed death-1 (PD-1) molecules were not. Finally, CD8+CD122+ cells isolated from CD28-knockout (CD28-/-) mice showed no regulatory activity. These results indicate that CD8+CD122(+) regulatory T cells recognize target T cells via the interaction of CD80/CD86-CD28 molecules to become active regulatory cells that produce suppressive factors such as IL-10.     

Estrogen-induced protection against experimental autoimmune encephalomyelitis is abrogated in the absence of B cells

Increased remissions in multiple sclerosis (MS) during pregnancy suggest that elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2=17β-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Studies demonstrate that depletion of B cells prior to induction of EAE exacerbates disease severity, implicating regulatory B cells. We thus evaluated pathogenic and E2-induced protective mechanisms in B-cell-deficient (μMT(-/-)) mice. EAE-protective effects of E2 were abrogated in μMT(-/-) mice, with no reduction in disease severity, cellular infiltration or pro-inflammatory factors in the central nervous system compared to untreated controls. E2 treatment of WT mice selectively upregulated expression of PD-L1 on B cells and increased the percentage of IL-10-producing CD1d(high) CD5(+) regulatory B cells. Upregulation of PD-L1 was critical for E2-mediated protection since E2 did not inhibit EAE in PD-L1(-/-) mice. Direct treatment of B cells with E2 significantly reduced proliferation of MOG(35-55)-specific T cells that required estrogen receptor-α (ERα). These results demonstrate, for the first time, a requirement for B cells in E2-mediated protection against EAE involving direct E2 effects on regulatory B cells mediated through ERα and the PD-1/PD-L1 negative co-stimulatory pathway. E2-primed B cells may represent an important regulatory mechanism in MS and have strong implications for women receiving current MS therapies that cause B-cell depletion.     

T cell- and perforin-dependent depletion of B cells in vivo by staphylococcal enterotoxin A.

Bacterial superantigens bind to major histocompatibility complex (MHC) class II and subsequently activate both CD4+ and CD8+ T lymphocytes expressing certain T-cell receptor (TCR)-Vbeta chains. In response to superantigen exposure these subsets proliferate, produce large amounts of proinflammatory cytokines and in addition CD8+ cytotoxic T lymphocytes (CTL) are induced. Previous studies in vitro have shown that these CTL effectively lyse MHC class II-expressing cells presenting the proper superantigen. However, it is unknown whether superantigens induce a similar response towards MHC class II+ antigen-presenting cells in vivo. In this study we demonstrate that administration of repeated injections of the superantigen staphylococcal enterotoxin A (SEA) to TCR-Vbeta3 transgenic mice results in a loss of MHC class II-expressing cells in the spleen. Analysis of different MHC class II+ subsets revealed a selective depletion of CD19+ B cells, while F4/80+ macrophages increased in number. Depletion of T cells with anti-CD4 or anti-CD8 monoclonal antibody indicated that CD8+ T cells were crucial for SEA-induced cytotoxicity in vivo. Repeated injections of SEA to perforin-deficient mice resulted in significantly less B-cell depletion compared with control mice. This suggests that superantigen-activated CD8+ T cells lyse MHC class II+ antigen-presenting cells in a perforin-dependent manner in vivo. It is suggested that this represents a novel bacterial immune escape mechanism, which may particularly impair local humoral immune responses.     

Maturation of CD4-8- alpha/beta TCR+ T cells induced by CD2-stimulation in vivo and in vitro.

Newly generated ('virgin') rat thymocytes of the immature CD4+8+ double positive (DP) subset were treated in suspension culture for 2 days with the stimulatory pair of anti-CD2 monoclonal antibodies OX-54 and OX-55. Approximately 50% of the recovered cells had downregulated CD4 and CD8 and upregulated the T cell antigen receptor (TCR). CD2-stimulated, but not control thymocytes proliferated in response to TCR plus IL-2 stimulation. In vivo, postnatal injection of OX-54/55 led to a dramatic and selective increase in functionally mature CD4-CD8- double negative (DN) alpha/beta--TCR(high) thymocytes and peripheral T cells. These findings show that CD2 stimulation can promote T cell differentiation and suggest that DN TCR(high) thymocytes can be generated from DP thymocytes via alternative pathways of T cell maturation.     

IL-15-secreting γδT cells induce memory T cells in experimental allergic encephalomyelitis (EAE) mice

With the most recent data suggesting γδT cells as primary producers of the pro-inflammatory autoimmune-associated cytokine, the relationship between γδT cells and Th17 in experimental allergic encephalitis (EAE) mice requires more extensive investigation. By flow cytometry and qPCR, we identified a new subset of IL-15-secreting γδT (γδT15) cells that increased in EAE mice. The capacity of IL-15-secreting γδT cells inducing memory T cells and memory T cells inducing IL-17⁺Th17 was examined by transferring into EAE mice and 7-week-old female nude mice, respectively. We found that γδT15 induced CD44^hi memory T cells by secreting IL-15. γδT15-induced memory T cells induced EAE by transforming into pathogenic Th17 cells. The data suggest that a new subset of IL-15-secreting γδT cells mediated the production of memory T cells which transformed into pathogenic Th17 cells in EAE mice.     

Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells

Naturally arising CD4(+)CD25(+) regulatory T (T(R)) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4(+)CD25(-) T cells also possesses regulatory activity. Programmed death-1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4(+)CD25(-)PD-1(+) T cells in the spleen of naive mice that constitutively expressed CTLA-4 and FoxP3 and was hypoproliferative in response to anti-CD3 antibody stimulation in vitro. However, the CD4(+)CD25(-)PD-1(+) T cells uniquely produced large amounts of IL-4 and IL-10 in response to anti-CD3 and anti-CD28 mAb stimulation, unlike the CD4(+)CD25(+) T(R) cells. The CD4(+)CD25(-)PD-1(+) T cells exhibited a suppressor activity against the proliferation of anti-CD3 antibody-stimulated CD4(+)CD25(-)PD-1(-) T cells in vitro, which was partially abrogated by anti-CTLA-4 mAb, but not by anti-IL-10 or anti-PD-1 mAb. Remarkably, the CD4(+)CD25(-)PD-1(+) T cells inhibited the development of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into C.B17-scid/scid mice, albeit to a lesser extent than CD4(+)CD25(+) T(R) cells, in a CTLA-4-dependent manner. These results indicate that the CD4(+)CD25(-)PD-1(+) T cells contain substantial amounts of T(R) cells that are involved in the maintenance of peripheral tolerance.     

Regulation of experimental autoimmune encephalomyelitis by CD4+, CD25+ and CD8+ T cells: analysis using depleting antibodies

Experimental Autoimmune Encephalomyelitis (EAE) can be induced in mice of the C57BL/6 strain by subcutaneous immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide p35-55 in CFA, administered twice at an interval of one week and supplemented with Bordetella pertussis toxin given IV. Here, we studied the effect on the induction of EAE of depleting antibodies to CD4, CD8, or CD25 administered before either the first or the second dose of MOG p35-55. We found that anti-CD4 abolished EAE when given before the first immunization; anti-CD4 did not affect the disease when it was given before the second immunization. Anti-CD8 enhanced EAE induction when given before either of the two immunizations. Anti-CD25 enhanced EAE to the same degree as anti-CD8 when given before the first immunization, but anti-CD25 was even more effective in enhancing EAE when given before the second immunization. The anti-CD25 treatment led to significantly enhanced IFNgamma production by T cells responding to MOG p35-55 and persisting anti-MOG antibodies detectable 56 days after the first immunization. Administration of anti-CD8 or anti-CD25 abolished the need for pertussis toxin to induce EAE. These findings are compatible with the idea that CD4 T cells are required for the initial induction of EAE and that the disease is down-regulated by T cells expressing CD8 or CD25. These regulatory T cells exist prior to MOG immunization, but the CD25+ regulators appear to be further amplified by immunization.     

Modulation of CD4 co-receptor limits spontaneous autoimmunity when high-affinity transgenic TCR specific for self-antigen is expressed on a genetically resistant background

Myelin proteolipid protein (PLP) 139-151 is an immunodominant peptide that induces experimental autoimmune encephalomyelitis (EAE) in H-2(s) SJL/J mice. While PLP 139-151-specific TCR transgenic (tg) 4E3 mice develop fulminant spontaneous disease on the susceptible SJL/J background, spontaneous EAE is dramatically reduced on the H-2(s) congenic B10.S background. On this resistant background, we observed a high frequency of positively selected tg CD4-CD8- (DN) thymocytes and peripheral DN tg T cells. Splenic DN tg T cells responded to anti-CD3 stimulation similarly to CD4+ cells, but proliferative and cytokine responses to PLP 139-151 were blunted, implying that CD4 co-receptor down-regulation modulated T cell responses to the self-antigen in vitro. Adoptive transfer of tg DN CD3hi cells into RAG-deficient wild-type (WT) recipients induced EAE less efficiently than transfer of CD4+ T tg cells indicating the blunted responses of DN tg T cells to self-antigen in vivo. The frequency of tg DN T cells was irrespective of thymic expression of the autoantigen. These data implicate that down-regulation of CD4 co-receptor in the thymus, which is independent from the expression of thymic autoantigen, results in a blunted response to the autoantigen in the periphery and limits the incidence of spontaneous autoimmunity in genetically resistant mice bearing a large autoreactive tg T cell repertoire.     

CD4+CD25+ naturally occurring regulatory T cells and not lymphopenia play a role in the pathogenesis of iodide-induced autoimmune thyroiditis in NOD-H2h4 mice

NOD-H2(h4) mice, which express I-A(k) on the NOD background, spontaneously develop autoimmune thyroiditis, a model of Hashimoto thyroiditis in humans, by adding iodide in the drinking water. Parental NOD mice have previously been shown to have intrinsic numerical abnormalities in peripheral lymphocytes and lymphocyte subpopulations such as CD4(+)CD25(+) naturally occurring regulatory T cells (Treg). Therefore we first investigated whether the similar abnormalities exist in NOD-H2(h4) mice. We observed that, compared with other non-autoimmune disease prone BALB/c and C57BL/6 mice, NOD-H2(h4) mice have lower numbers of splenocytes, CD3(+)T, CD4(+)T and CD8(+)T cells but the ratios of Treg to CD4(+)T cells were comparable. Increasing the numbers of peripheral lymphocytes by Complete Freund's Adjuvant immunization or splenocyte transfer did not affect development of thyroiditis, indicating that lymphopenia does not play a critical role in the pathogenesis of thyroiditis. We next examined the significance of Treg by depleting this lymphocyte subset with anti-CD25 antibody. Treg depletion, performed 4days before the administration of NaI water for 8 weeks, significantly exacerbated thyroiditis (p<0.01). Anti-thyroglobulin antibody titers also increased by Treg depletion (p<0.01) without changing the IgG2b to IgG1 ratios. In addition, expression levels of mRNA for IFN-gamma and IL-4 were enhanced in parallel. However, T(4) levels were similar between antibody-treated and untreated groups. Additional anti-CD25 administration at 3 weekly intervals did not influence these results. These data, together with previous studies on other mouse models of inducible thyroiditis and Graves' disease, indicate the role played by Treg in keeping anti-thyroid autoimmune reaction in check in experimental autoimmune thyroid diseases.     

CD75s is a marker of murine CD8(+) suppressor T cells

We have previously described a monoclonal antibody, 984, which specifically recognizes murine CD8(+) suppressor T cells (Ts) but not CD8(+) cytolytic T lymphocytes (CTLs). Removal of 984(+) cells abrogates the suppressive effect of CD8(+) Ts generated either in vivo or in vitro while having no effect upon CTL. In this report, the molecules recognized by 984 are identified as 2-6 sialylated neolacto series gangliosides, which are members of the newly defined CD75s cluster. We proceed to demonstrate that like 984, a separate anti-CD75s antibody (CRIS-4), recognizes primary CD8(+) Ts cells. In addition, the 2,6 sialyltransferase responsible for the synthesis of the 984 epitope is identified, allowing the manipulation and study of the regulation of this epitope. This is the first report of CD75s on murine cells and the first report that delineates lymphocyte function based upon CD75s expression. In addition to contributing to the growing body of evidence that lineage dependent gangliosides are expressed by T lymphocytes, these findings suggest that CD8(+) CD75s(+) T lymphocytes represent a functionally distinct subset of CD8(+) T cells with negative regulatory function.     

F(ab')2 anti-CD4 and intact anti-CD4 monoclonal antibodies inhibit the accumulation of CD4+ T cells, CD8+ T cells, and B cells in the kidneys of lupus-prone NZB/NZW mice

Murine lupus in NZB/NZW (B/W) mice is characterized by immune-complex glomerulonephritis and lymphocytic infiltration of several organs, including the kidney. We recently showed that treatment of B/W mice with F(ab')2 anti-CD4 monoclonal antibody retards autoimmunity by inhibiting the function of CD4+ cells and not by depleting them. To determine if treatment with F(ab')2 anti-CD4 prevented lymphocytic infiltration of kidneys or simply inhibited the function of the infiltrating lymphocytes, long-term survivors of treatment with F(ab')2 anti-CD4 and intact anti-CD4 were sacrificed for immunohistochemical analysis of their kidneys. Untreated B/W mice had large lymphocytic aggregates under the surface epithelium of the renal calyces. Most of these lymphocytes were CD4+ T cells, but CD8+ T cells and B cells were also present. In contrast, treatment with either intact anti-CD4 or F(ab')2 anti-CD4 substantially reduced, and in many cases prevented, the development of renal infiltrates. Treatment with either form of anti-CD4 not only inhibited renal infiltration by CD4+ T cells, but also prevented the accumulation of CD8+ T cells and B cells. These observations suggest a role for the CD4+ T cell in the accumulation of lymphocytes in target organs.     

Beta interferon restricts the inflammatory potential of CD4+ cells through the boost of the Th2 phenotype, the inhibition of Th17 response and the prevalence of naturally occurring T regulatory cells

Beta-interferon (IFN-beta) is a valuable therapy for multiple sclerosis (MS) which is also effective in the animal model of experimental autoimmune encephalomyelitis (EAE). However, the accurate mechanisms to explain its anti-inflammatory activity in the disease are not fully revealed. Available data support that T lymphocytes are among the main cell targets of IFN-beta. We have found that in vitro anti-CD3 stimulation of uncommitted murine na?ve T cells under IFN-beta treatment results in skewing the T cell differentiation process towards the T2 phenotype, in a prevention from apoptosis of naturally occurring CD4+ T regulatory cells (nTreg) in correlation with an increase in Bcl-XL expression, and in a decrease of IL-17 expression. Elimination of nTreg from the primary culture of na?ve CD4+ cells abolished the down-regulation of IL-17 driven by IFN-beta, what suggests the interaction between Th17 and nTreg subsets. Experiments in EAE induced in SJL mice, showed in vivo evidence for the accumulation of spleen CD4+CD25+GITR+Foxp3+ cells after IFN-beta treatment. On the other hand, treated animals showed a striking decrease of IL-17 expression by peripheral CD4+ cells (Th17) and MBP-specific spinal cord cells. Both the in vivo and in vitro results point out new targets through which IFN-beta could exert its therapeutic action.     

Peripheral T lymphocyte depletion by apoptosis after CD4 ligation in vivo: selective loss of CD44- and 'activating' memory T cells.

We have demonstrated that a single intravenous bolus of rat anti-CD4 MoAb caused a small but prolonged increase in apoptosis in murine lymph nodes. We have quantified this process using the novel Highly Optimized Microscope Environment (HOME) interactive images analysis system and shown that the increase in apoptosis was sufficient to account for the observed depletion of the peripheral CD4+ T cell subset. This occurred in the absence of any other exogenous signal. Furthermore, there was no evidence of an inflammatory or necrotic response in the tissues, indicating that this was unlikely to be Fc or complement-mediated antibody killing. The anti-CD4-induced depletion selectively removed CD44- T cells. Using mice previously immunized with yeast-derived HIV-1 p24 recombinant protein there was sparing of memory T cell function after in vivo anti-CD4 treatment, except during a window of less than 24 h duration, when simultaneous exposure to antigen and anti-CD4 antibody resulted in the depletion of specific memory T lymphocyte function. This indicated that a very minor alteration in the frequency of apoptosis had a marked effect on cell number over time, and suggested that opportunistic infection associated with CD4+ T cell depletion may be explained by loss of memory cells when there is antigenic stimulation at the same time as CD4 ligation. These results have implications for the pathology of HIV-associated disease which is associated with ligation of CD4 molecules in vivo.     

不同临床类型小鼠肝炎模型T细胞亚群的初步研究

目的 探讨3型鼠肝炎病毒(MHV-3)感染不同品系小鼠所致不同临床疾病类型小鼠肝炎模型后体内T细胞亚群的动态变化.方法 取Balb/cJ和A/J小鼠各8只,腹腔注射100 pfu 3型鼠肝炎病毒(MHV-3),采用流式细胞仪检测感染后0、24、48、72 h外周血、脾细胞悬液以及肝脏组织中T淋巴细胞亚群包括CD3 CD4 CD8-、CD3 CD4-CD8 及CD3 CD4-CD8-T(DN T) 细胞的细胞数及各自在T淋巴细胞中所占比率.结果 Balb/cJ小鼠于感染后24 h外周血、脾脏以及肝脏T淋巴细胞中DN T细胞比率明显升高直至小鼠3 d后全部死亡,CD3 CD4 CD8-和CD3 CD4-CD8 T淋巴细胞比率相应下降;A/J小鼠感染后24～96 h外周血和脾脏T细胞各亚群变化不明显.结论 两种不同品系小鼠感染MHV-3 后的不同转归及DN T 细胞比率的不同改变提示DN T 细胞可能在病毒性肝炎发生、发展中起着一定的作用.     

Differential effect of CD69 targeting on bystander and antigen-specific T cell proliferation

In spite of an initially proposed role as a costimulatory molecule for CD69, in vivo studies showed it as a regulator of immune responses and lymphocyte egress. We found constitutive CD69 expression by T cell subsets and pDC. We examined a possible effect of CD69 on T cell proliferation using transfer models and in vitro assays. In mice locally expressing or receiving antigen, anti-CD692.2 treatment did not affect the proliferation of antigen-specific transgenic T cells in ADLN, although we observed the presence of proliferated T cells in non-ADLN and spleen. This was not affected by FTY720 treatment and thus, not contributed by increased egress of proliferated lymphocytes from ADLN. In the absence of antigen, anti-CD69 2.2 treatment induced bystander proliferation of transferred memory phenotype T cells. This proliferation was mediated by IL-2, as it was inhibited by anti-IL-2 or anti-CD25 antibodies in vitro and by anti-CD25 antibodies in vivo. It was also dependent on CD69 expression by donor T cells and recipient cells. CD69 targeting on T cells enhanced IL-2-mediated proliferation and CD25 expression. However, it did not lead to increased early IL-2 production by T cells. No T cell subset was found to be specifically required in the recipient. Instead, CD69 targeting on pDC induced their expression of IL-2 and CD25, and pDC depletion showed that this subset was involved in the proliferation induction. These results indicate that CD69 targeting induces bystander T cell proliferation through pDC IL-2 production and T cell sensitization to IL-2 without affecting antigen-driven T cell proliferation.