Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression

Recent data suggest that human effector CD8+ T cells express a distinct CD27-CD45RAhigh (CD57+CD28-CD11ahigh) phenotype. Here, we propose that CTL effector function correlates better with CD56 (neuronal cell adhesion molecule (NCAM)) surface expression. CD56 was absent on cord blood CD8+ T cells, but was expressed by 4-30% of freshly isolated circulating CD8+ T cells from 15 adults. Dramatic oligoclonal expansions in 3/3 individuals were confined to the CD56+ subset of CD8+ T cells. The CD56+ subset generally contained high amounts of intracellular perforin and granzyme B. Finally, direct cytolytic capacity was closely restricted to the CD56+(CD45RAhigh) cells, better than to CD27-CD45RAhigh cells in 5/5 individuals analyzed. Thus, the phenotype corresponding to the circulating effector CD8+ T cell pool may be simplified and more precisely defined by the use of just two surface markers: CD8 and CD56.     

Cutting edge: engagement of CD160 by its HLA-C physiological ligand triggers a unique cytokine profile secretion in the cytotoxic peripheral blood NK cell subset

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-gamma, TNF-alpha, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.     

LIGHT expression by mucosal T cells may regulate IFN-gamma expression in the intestine

The TNF superfamily of cytokines play an important role in T cell activation and inflammation. Sustained expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT) (TNFSF14) causes a pathological intestinal inflammation when constitutively expressed by mouse T cells. In this study, we characterized LIGHT expression on activated human T cell subsets in vitro and demonstrated a direct proinflammatory effect on regulation of IFN-gamma. LIGHT was induced in memory CD45RO CD4+ T cells and by IFN-gamma-producing CD4+ T cells. Kinetic analysis indicated rapid induction of LIGHT by human lamina propria T cells, reaching maximal levels by 2-6 h, whereas peripheral blood or lymph node-derived T cells required 24 h. Further analysis of intestinal specimens from a 41 patient cohort by flow cytometry indicated membrane LIGHT induction to higher peak levels in lamina propria T cells from the small bowel or rectum but not colon, when compared with lymph node or peripheral blood. Independent stimulation of the LIGHT receptor, herpesvirus entry mediator, induced IFN-gamma production in lamina propria T cells, while blocking LIGHT inhibited CD2-dependent induction of IFN-gamma synthesis, indicating a role for LIGHT in the regulation of IFN-gamma and as a putative mediator of proinflammatory T-T interactions in the intestinal mucosa. Taken together, these findings suggest LIGHT-herpesvirus entry mediator mediated signaling as an important immune regulatory mechanism in mucosal inflammatory responses.     

Spontaneous and lymphokine-induced cytotoxic activity of monkey intestinal mucosal lymphocytes

We determined the capacity of primate (macaque monkey) intestinal mucosal lymphocytes to mediate natural killer cytotoxicity, and characterized the nature of cells mediating this form of cytotoxicity in the intestine. Isolated macaque monkey intestinal mucosal lymphocytes were found to have intermediate levels of spontaneous cytotoxicity against K562 target cells compared to higher values found in lymphocytes of peripheral blood (PBL) and spleen and low values for mesenteric lymph node (MLN) lymphocytes. Intestinal lymphocytes were similar to PBL in having the same range of target cell specificites, in having augmentation of activity by interferon or interleukin 2, and in demonstrating specificity in cold target inhibition studies. Both intestinal and PBL spontaneous cytotoxic function in primates was mediated predominantly by cells bearing antigens cross-reactive with the anti-human monoclonal antibody Leu-11. The percentage of Leu-11+ lymphocytes was significantly lower in isolated intestinal, spleen, and MLN lymphocytes compared to peripheral blood. Furthermore, isolated intestinal lymphocytes differed from PBL in that intestinal Leu-11+ were predominantly Leu-15-, while Leu-11+ PBL were predominantly Leu-15+. These studies demonstrate that the lower spontaneous cytotoxic function of intestinal mucosal lymphocytes compared to PBL is associated with a lower number of effector cells and with effector cells which differ qualitatively in expression of the Leu-15 antigen.     

The human liver contains multiple populations of NK cells, T cells, and CD3+CD56+ natural T cells with distinct cytotoxic activities and Th1, Th2, and Th0 cytokine secretion patterns.

The human liver contains significant numbers of T cells, NK cells, and lymphocytes that coexpress T and NK cell receptors. To evaluate their functional activities, we have compared the cytotoxic activities and cytokines produced by normal adult hepatic CD3+CD56- (T) cells, CD3-CD56+ (NK) cells, and CD3+CD56+ (natural T (NT)) cells. In cytotoxicity assays using immunomagnetic bead-purified NK cell, T cell, and NT cell subpopulations as effectors, fresh hepatic NK cells lysed K562 targets, while NT cells could be induced to do so by culturing with IL-2. Both NT and T cells were capable of redirected cytolysis of P815 cells using Abs to CD3. Flow cytometric analysis of cytokine production by fresh hepatic lymphocyte subsets activated by CD3 cross-linking or PMA and ionomycin stimulation indicated that NT cells and T cells could produce IFN-gamma, TNF-alpha, IL-2, and/or IL-4, but little or no IL-5, while NK cells produced IFN-gamma and/or TNF-alpha only. The majority of NT cells produced inflammatory (Th1) cytokines only; however, approximately 6% of all hepatic T cells, which included 5% of Valpha24 TCR-bearing NT cells and 2% of gammadeltaTCR+ cells, simultaneously produced IFN-gamma and IL-4. The existence of such large numbers of cytotoxic lymphocytes with multiple effector functions suggests that the liver is an important site of innate immune responses, early regulation of adaptive immunity, and possibly peripheral deletion of autologous cells.     

Cutting edge: induction of follicular homing precedes effector Th cell development.

Transition from naive to Ag-experienced effector/memory CD4+ T cells is initiated during contact with APC in secondary lymphoid tissue. Here, we demonstrate that the CXCR5 is a marker for recently activated memory CD4+ T cells. CXCR5 is rapidly induced during contact with Ag-presenting dendritic cells, well before T cell expansion and effector cell development, and is irreversibly lost on terminally differentiated effector cells. Furthermore, immunization of human volunteers with a recall Ag results in rapid accumulation of Ag-responsive, CXCR5-expressing CD4+ T cells in peripheral blood. Early acquisition of a new migration program enables T zone CD4+ T cells to develop into follicular B helper T cells or, alternatively, into circulating memory CD4+ T cells. Together, CXCR5 unequivocally defines pre-effector memory CD4+ T cells generated during ongoing immune responses.     

CD4 positive Leu-8 negative helper-inducer T cells predominate in the human intestinal lamina propria

The regulatory function of peripheral blood CD4 T cells correlates with the presence or absence of the membrane glycoprotein recognized by anti-Leu-8 antibody; CD4,Leu8- T cells help Ig synthesis and CD4,Leu-8+ T cells suppress Ig synthesis. In contrast to CD4 T cells from the peripheral blood and organized gut-associated lymphoid tissues, intestinal lamina propria CD4 T cells were found to have diminished expression of the Leu-8 Ag. Therefore, studies were performed to determine whether the decreased expression of the Leu-8 Ag on lamina propria CD4 T cells correlates with a difference in the ability of peripheral blood and lamina propria CD4 T cells to regulate PWM-stimulated Ig synthesis. At high T cell to non-T cell ratios, the helper function of lamina propria CD4 T cells was significantly higher than that of peripheral blood CD4 T cells. When CD4 T cells were incubated with anti-Leu-8 antibody, the suppressor function of peripheral blood CD4 T cells was increased, but lamina propria CD4 T cells did not suppress Ig synthesis. No difference was found between the helper function of CD4,Leu-8- T cells and the suppressor function of CD4, Leu-8+ T cells isolated from either the peripheral blood or the lamina propria. Thus, the difference in the regulatory function of CD4 T cells from the peripheral blood and the lamina propria is due to the quantitative difference in CD4,Leu-8+ T cells in these sites. Consequently, the intestinal lamina propria is a site enriched in CD4,Leu-8- T cells which predominantly mediate help for Ig synthesis.     

基于流式细胞术快速检测人鼻黏膜组织淋巴细胞亚群的实验研究

目的：基于流式细胞术检测正常人的鼻黏膜组织中的淋巴细胞亚群(CD3+T、CD4+T、CD8+T、NK、CD19+B)的比例,初步探讨 鼻局部黏膜免疫功能的意义,为鼻局部黏膜免疫性疾病的研究提供更多的参考。方法：用鼻黏膜刮匙获取21 例正常人的鼻黏膜 组织,并抽取其外周血2 mL。采用流式细胞术分别检测其鼻黏膜和外周血中的淋巴细胞亚群(包括CD3+T、CD4+T、CD8+T、NK、 CD19+B)分别所占的比例。结果：鼻黏膜和外周血的淋巴细胞亚群存在很大差异,与外周血相比,CD3+T 比例增加（t=15.34,P<0. 0001),CD4+T 比例降低(t=5.952,P<0.0001)、CD8+T 比例增加(t=12.44,P<0.0001)、NK 比例降低(t=4.865,P<0.0001)、CD19+B 比例降 低(t=15.56,P<0.0001),CD4+T/CD8+T 降低。结论：流式细胞术可以用来检测鼻黏膜的淋巴细胞亚群,鼻黏膜的淋巴细胞亚群和外 周血的淋巴细胞亚群存在很大差异,这种差异体现鼻黏膜组织独特的局部黏膜免疫功能,本方法为变应性鼻炎的研究提供了新 的研究途径。     

Expression of CD161 (NKR-P1A) defines subsets of human CD4 and CD8 T cells with different functional activities

A subset of T cells in human peripheral blood expresses CD161 (NKR-P1A) receptors that are primarily associated with NK cells. In the current study we isolated blood T cell subsets according to the expression of CD161 and examined their contents of naive, central memory, and effector memory cells and their capacities for proliferation, cytokine secretion, and natural cytolysis. We found that CD4+CD161- and CD8+CD161- subsets contained predominantly naive T cells that secreted high levels of IL-2 after in vitro stimulation, and CD4+CD161int and CD8+CD161int subsets contained predominantly effector and central memory T cells that secreted high levels of IFN-gamma and TNF-alpha. All of these subsets showed vigorous proliferation after stimulation in vitro, but none had NK lytic activity. Unexpectedly, the CD8+CD161+ cells contained an anergic CD8alpha+CD8betalow/-CD161high T cell subset that failed to proliferate, secrete cytokines, or mediate NK lytic activity.     

Normal Human Pregnancy Results in Maternal Immune Activation in the Periphery and at the Uteroplacental Interface

Pregnancy poses a unique challenge to the human immune system: the semi-allogeneic fetus must be protected from maternal immune attack while immunity towards pathogens is maintained. Breakdown in maternal-fetal tolerance can lead to pregnancy-specific diseases with potentially high degrees of morbidity and mortality for both the mother and her fetus. Various immune cell-types could mediate these functions, but a comprehensive evaluation of the peripheral and local maternal T cell and regulatory T cell compartments in normal human pregnancy is lacking. In this case-control study, we apply the Human Immunology Project Consortium proposed gating strategies to samples from healthy 3^rd trimester human subjects compared with healthy non-pregnant controls. The proportions of HLA-DR+ and CD38+ effector- and effector memory CD8 T cells are significantly increased in the peripheral blood of pregnant women. Utilizing a novel technique that takes advantage of the standard protocol for intrauterine cleanup after cesarean section, we isolate lymphocytes resident at the uteroplacental interface (UPI). At the UPI, the CD4 and CD8 T cell compartments largely mirror the peripheral blood, except that the proportion of HLA-DR+ activated T regulatory cells is significantly increased in direct proportion to an observed increase in the number of activated CD8 T cells. We find that cryopreservation and delayed sample processing (>12 hours) decreases our ability to identify regulatory T cell subsets. Further, the Consortium proposed method for Treg identification underrepresents Resting and Cytokine Tregs compared with Activated Tregs, thus skewing the entire population. Better understanding of the changes in the immune system during pregnancy in the peripheral blood and at the uteroplacental interface are essential for progress in treatment of pregnancy diseases such as pre-eclampsia and recurrent miscarriage.     

Severe depletion of mucosal CD4+ T cells in AIDS-free simian immunodeficiency virus-infected sooty mangabeys

HIV-infected humans and SIV-infected rhesus macaques experience a rapid and dramatic loss of mucosal CD4+ T cells that is considered to be a key determinant of AIDS pathogenesis. In this study, we show that nonpathogenic SIV infection of sooty mangabeys (SMs), a natural host species for SIV, is also associated with an early, severe, and persistent depletion of memory CD4+ T cells from the intestinal and respiratory mucosa. Importantly, the kinetics of the loss of mucosal CD4+ T cells in SMs is similar to that of SIVmac239-infected rhesus macaques. Although the nonpathogenic SIV infection of SMs induces the same pattern of mucosal target cell depletion observed during pathogenic HIV/SIV infections, the depletion in SMs occurs in the context of limited local and systemic immune activation and can be reverted if virus replication is suppressed by antiretroviral treatment. These results indicate that a profound depletion of mucosal CD4+ T cells is not sufficient per se to induce loss of mucosal immunity and disease progression during a primate lentiviral infection. We propose that, in the disease-resistant SIV-infected SMs, evolutionary adaptation to both preserve immune function with fewer mucosal CD4+ T cells and attenuate the immune activation that follows acute viral infection protect these animals from progressing to AIDS.     

Cutting edge: size and diversity of CD4+CD25high Foxp3+ regulatory T cell repertoire in humans: evidence for similarities and partial overlapping with CD4+CD25- T cells

Both differentiation and function of CD4+CD25(high) naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the alphabetaTCR repertoire sizes of human peripheral blood Treg and CD4+CD25- T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted alphabeta TCR repertoire, the size and diversity of which are closely similar to those of CD4+CD25- T cells, with a mean estimated size of 3.5 x 10(6) distinct alphabeta TCR vs 4.7 x 10(6) distinct alphabetaTCR for CD4+CD25- T cells. In addition, a 24% overlap between the repertoires of these two CD4+ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.     

Altered CD4+ T cell phenotype and function determine the susceptibility to mucosal candidiasis in transgenic mice expressing HIV-1

The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans in these Tg mice.     

Interleukin-15 increases effector memory CD8+ t cells and NK Cells in simian immunodeficiency virus-infected macaques

Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 microg/kg of body weight, n = 3; high dose of IL-15, 100 microg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3- NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA-CD62L- and CD45RA+CD62L- effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-15 did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.     

Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression

A growing body of literature indicates that the Notch pathway can influence the activation and differentiation of peripheral murine T cells, though comparatively little is known about the effects of Notch signaling in human T cells. In the present report we demonstrate that Jagged-1-induced Notch signaling (using immobilized Jagged-1 fusion protein) during stimulation of purified human CD4+ and CD8+ T cells potently inhibits T cell proliferation and effector function, including both Th1- and Th2-associated cytokines. Inhibition of T cell activation is not due to apoptosis or disruption of proximal TCR signaling, but is associated with up-regulation of GRAIL (gene related to anergy in lymphocytes) in CD4+ T cells, with modest effects on other E3 ubiquitin ligases such as c-Cbl and Itch. When evaluated for its effects on CD4+ T cell differentiation, Jagged-1-mediated signaling inhibits T cell cytokine secretion with no significant effect on proliferative responses. Collectively, these data demonstrate that Notch signaling in human T cells induced by Jagged-1 promotes a novel form of T cell hyporesponsiveness that differs from anergy, whereby primary T cell proliferation and cytokine secretion are potently inhibited, and effector function but not proliferative capacity are ameliorated upon secondary stimulation.     

Programmed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis

Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.     

Activation through CD3 molecule leads to clonal expansion of all human peripheral blood T lymphocytes: functional analysis of clonally expanded cells

Monoclonal antibodies directed against CD3, a T cell-specific surface molecule essentially required for activation of these cells, are highly mitogenic for resting human peripheral blood T lymphocytes. A predetermined optimal concentration of anti-CD3 monoclonal antibody WT32 was employed to activate T cells cultured in limiting-dilution microcultures containing irradiated feeder cells and exogeneous interleukin 2. Frequencies of cells triggered into clonal expansion by WT32 under these culture conditions were 0.57 to 0.72 and 0.90 to 1.10 in peripheral blood mononuclear cells and E rosette-positive cells, respectively. It appeared that WT32 could induce virtually every human peripheral blood T lymphocyte to expand into a clonal progeny of 5 to 40 X 10(4) cells in 14 to 18 days of culture. This progeny was tested for cytolytic effector function with 51Cr-labeled murine P815 targets in the presence of PHA to detect all cytotoxic T lymphocytes (CTL) regardless of specificity, and was also assayed for natural killer like activity against K562 target cells. Frequencies of cells in the human peripheral blood T cell compartment giving rise to a clonal progeny expressing CTL function was 1/3, whereas 1/6 to 1/5 expanded into effector cell populations possessing NK activity. Frequency analysis of CD4-positive and CD8-positive populations, activated by WT32 in limiting dilution microcultures, demonstrated that 1 to 6% of the CD4-positive and 100% of the CD8-positive peripheral blood T lymphocytes expanded into CTL.     

Beyond help: direct effector functions of human immunodeficiency virus type 1-specific CD4(+) T cells

The immune correlates of protection in human immunodeficiency virus type 1 (HIV-1) infection remain poorly defined, particularly the contribution of CD4(+) T cells. Here we explore the effector functions of HIV-1-specific CD4(+) T cells. We demonstrate HIV-1 p24-specific CD4(+)-T-cell cytolytic activity in peripheral blood mononuclear cells directly ex vivo and after enrichment by antigen-specific stimulation. We further show that in a rare long-term nonprogressor, both an HIV-1-specific CD4(+)-T-cell clone and CD4(+) T cells directly ex vivo exert potent suppression of HIV-1 replication. Suppression of viral replication was dependent on cell-cell contact between the effector CD4(+) T cells and the target cells. While the antiviral effector activity of CD8(+) T cells has been well documented, these results strongly suggest that HIV-1-specific CD4(+) T cells are capable of directly contributing to antiviral immunity.