Dissecting the complexity of the memory T cell response

Memory immune responses are classically attributed to the reactivation of long-lived, antigen-specific T lymphocytes that persist in a quiescent state. Determining mechanisms for the generation of memory T cells and dissecting the functional nature of the memory T cell pool has been encumbered by an inability to distinguish recently activated effector T cells from memory T cells. We have established new activation and biochemical criteria that distinguish effector and memory T cells and have applied these criteria to follow memory generation from activated cells in vivo. We found that the resultant memory T cell pool is heterogeneous and consists of effector-like and resting memory-like subsets that differ in expression of the homing receptor, CD62L. We discuss these findings in the context of memory T cell heterogeneity identified in human and mouse systems. These results suggest that more than one type of previously activated T cell can mediate recall or memory immune responses and that elucidating the fundamental phenotypic and functional features of memory T cell subsets is therefore critical to deciphering the complex nature of the memory immune response.     

An increased frequency of autoantibody-inducing CD4+ T cells in pre-diseased lupus-prone mice

Pathogenic autoantibody production in murine models of lupus is dependent on autoreactive CD4+ helper T cells. However, the mechanisms which permit the selection and maintenance of this autoantibody-inducing CD4+ T-cell repertoire are currently unknown. We hypothesized that the peripheral CD4+ T-cell repertoire of lupus-prone mice was enriched with autoantibody-inducing specificities. To test this, we utilized the splenic focus assay to determine if pre-diseased lupus-prone (NZB x NZW)F(1) mice have an elevated frequency of autoreactive CD4+ T lymphocytes capable of supporting autoantibody production. The splenic focus limiting dilution assay permits anti-nuclear antibodies to be generated from contact-dependent T-B interactions in vitro. We show that young, pre-diseased lupus-prone mice have an elevated frequency of autoantibody-inducing CD4+ T cells. Interestingly, these autoantibody-inducing CD4+ T-cell responses are also present in the thymus. Therefore, an elevated frequency of autoantibody-inducing CD4+ T cells predisposes lupus-prone mice to the development of autoantibodies.     

Effects of 17 beta-oestradiol on function of lymphocytes from normal donors and patients with common variable immunodeficiency (CVID).

Effects of oestradiol (E2) have been studied on the in vitro T cell-dependent differentiation of B cells from peripheral blood and spleen using normal donors and patients with the antibody deficiency disease CVID. We also studied whether it modifies T cell DNA synthesis. The effect of E2 was examined on cultures of B cells with T cells for IL-2-driven immunoglobulin secretion or of T cells for phytohaemagglutinin (PHA)-driven DNA synthesis. Interestingly, in control experiments without E2, the normal sex difference in immunoglobulin production is reversed in CVID. The data show that for normal individuals there is no major difference between male and female donors in the in vitro actions of E2 on blood B and T lymphocytes. With normal blood B cells, E2 failed to affect IgM production, but it did inhibit IgG. In normal splenic cells, E2 increased both IgM and IgG secretion in a similar way to the tonsillar cell data previously reported. E2 on normal blood T cell DNA synthesis was stimulatory. With blood cells from CVID patients an interesting contrast was seen. As with normal B cells, E2 had no effect on IgM secretion by those CVID blood B cells able to secrete IgM. However, a difference between patients and normals was that E2 did not inhibit the IL-2-driven IgG production by those CVID B cells able to secrete IgG. For T cell function, the stimulatory effect of E2 on CVID T cell DNA was as in normal T cells. However, E2 failed to restore CVID B and T cell function to normal levels. These data suggest that there may be subtle defects in the pathway of action of E2 in CVID lymphocytes.     

A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies.

Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 x 10(-13) to 5.7 x 10(-11) M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10(-8) M (immunotoxin containing gelonin) and 5 x 10(-9) M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S less than or equal to 10(-11) M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes.     

Death of peripheral CD8+ T cells in the absence of MHC class I is Fas-dependent and not blocked by Bcl-xL

Productive immune responses require an appropriate environment to support peripheral CD8(+) T cell survival. Although host MHC class I molecules appear to be required for this process, the cellular and molecular requirements have not been comprehensively studied. Using adoptive transfer of 2C/recombinase-activating gene-2 (RAG-2)(-/-) TCR-transgenic T cells, we found that the survival of both naive and effector CD8(+) T cells was dependent upon host expression of the same MHC class I alleles that supported thymic selection. Expression of appropriate MHC class Iby either bone marrow- or non-bone-marrow-derived cells was sufficient, suggesting that professional antigen-presenting cells were not mandatory. In contrast to MHC class I, neither T cell expression of CD28 nor host expression of ICAM-1 was required for peripheral T cell survival. Finally, T cell death in the absence of appropriate host MHC class I was overcome by elimination of Fas signaling but not by overexpression of Bcl-x(L) by CD8(+) T cells. These results suggest that, in the absence of a survival signal provided by engagement of host MHC/self peptide complexes, CD8(+) T cells die via a Fas-dependent, mitochondria-independent pathway.     

Clinical,hematologic, and immunologic effects of interleukin-10 in humans

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25g/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25g/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10g/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon- (IFN) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFN.     

Superantigen responses and co-stimulation: CD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo

Co-stimulation via the CD28/CTLA-4 system appears critical forT cell proliferation to peptide antigens presented in associationwith MHC. In this study, we examine the roles of CD28 and CTLA-4in the response of murine T cells to the superantigen staphylococcalenterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2or Fab fragments of anti-CD28 antibodies significantly inhibitthe response of splenocytes to SEB. Conversely, Fab fragmentsof anti-CTLA-4 antibodies augment the proliferative response.Further, addition of blocking antibodies directed against B7-1/B7-2augment proliferation co-stimulated by intact anti-CD28 antibodies.These data support the hypothesis that CD28 and CTLA-4 exertopposing effects upon early T cell activation. In vivo, Intactanti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28appear to have similar inhibitory effects upon the expansionof V_ß8⁺ T cells. In contrast, both intact and Fabfragments of anti-CTLA-4 appear to amplify this expansion. Weconclude that the SEB response is significantly augmented byCD28-derived signaling and this in turn may be attenuated bysignals through CTLA-4.     

卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。     

Beneficial effect of amrinone on murine cardiac allograft survival.

Amrinone is a non-glycoside positive inotropic agent with an inhibitory effect on a cyclic adenosine monophosphate (AMP) phosphodiesterase isoenzyme. In the present study, we examined the immunosuppressive action of amrinone, since several other cyclic AMP-elevating agents have been shown to suppress T lymphocyte activation. First, the in vivo effects of amrinone were investigated. Oral amrinone treatment, at 40 mg/kg per day, significantly prolonged median cardiac allograft survival compared with non-treated controls (22.0 days versus 10.5 days, P < 0.01) when DBA/2 mouse hearts (H-2d) were heterotopically transplanted into C57B1/6 mice (H-2b). Histopathological examination showed that there was less prominent cellular infiltration in the amrinone-treated than in the non-treated allografts. Plasma amrinone concentrations of mice after a single oral dose of 40 mg/kg were within the range of clinical relevance. To clarify the mechanism of action, in vitro studies were done. The generation of specific cytotoxic T lymphocytes after mixed lymphocyte culture was significantly suppressed by addition of amrinone to the culture medium at 5 micrograms/ml. The production of IL-2 and the interferon-gamma during mixed lymphocyte culture was also suppressed by amrinone at 5 micrograms/ml. However, the level of intracellular cyclic AMP in mouse splenic lymphocytes was not affected significantly by the same dose of amrinone. In conclusion, amrinone has immunosuppressive actions at the therapeutic doses, and it may be a beneficial agent for therapy against acute cardiac allograft rejection.     

Analysis of susceptibility of mature human T lymphocytes to dexamethasone-induced apoptosis

We present evidence that dexamethasone (Dex), a synthetic glucocorticosteroid, causes apoptosis in mature human T cells, similarly to what has been reported for murine T lymphocytes. Human T cell clones and short-term activated T lymphocytes treated with Dex show the characteristic pattern of apoptotic cells, such as hypodiploid nuclei, chromatin condensation and DNA fragmentation into oligonucleosomal fragments. However, Dex susceptibility of T cells to apoptosis is cell cycle-dependent. The progression in the proliferative cell cycle (G1 versus S) rescues Dex-treated T cells from apoptosis. Moreover, occupancy of the T cell receptor reverses Dex-induced apoptotic phenomena. These observations suggest that glucocorticoids contribute to the regulation of the proliferative or the suicidal response of antigen-activated human T cells.