Bidirectional NK/DC interactions promote CD4 expression on NK cells, DC maturation, and HIV infection

Interactions between natural killer (NK) and dendritic cells (DCs) are integral to immune response development, potentially leading to bidirectional NK/DC activation. We demonstrate that autologous NK/DC interactions induce CD4 expression on NK cells, influencing degranulation. Cell contact is required, with high NK:DC ratios and mature DCs most effectively inducing CD4 expression. CD4(+) NK cells, in turn, mediate DC maturation via contact-dependent and independent pathways, more effectively maturing DCs than CD4(-) NK cells. Bidirectional NK/DC interactions also impact HIV infection, as NK-matured DCs effectively deliver infectious HIV to T cells, via trans-infection. DC-induced CD4 expression also renders NK cells susceptible to HIV infection. Focusing on NK/DC interactions, DCs can transfer infectious virus and enhance HIV infection of CD4(+) NK cells, strongly suggesting that these interactions influence HIV pathogenesis. Findings provide new insight regarding NK/DC interactions, defining a mechanism by which cellular interactions in the absence of pathogens promote DC-mediated amplification of HIV infection.     

NK4基因的克隆及其在Raji细胞中的表达

目的： 克隆NK4基因,构建其重组真核表达载体,并观察其在Raji细胞中的表达。方法：从人肝组织中提取总RNA,行RT-PCR获得NK4基因cDNA,与载体pVITRO2-mcs构建重组真核表达载体,转染Raji细胞。潮霉素B筛选后,采用实时荧光定量PCR、ELISA、细胞免疫组化和半固体培养等方法检测NK4基因在Raji细胞的表达,筛选稳定表达的细胞株。结果：NK4基因获成功克隆并构建了重组真核表达载体pVITRO2-mcs-NK4;转染NK4基因后的Raji细胞经RT-PCR发现存在特异性DNA片段。NK4基因在Raji细胞可稳定表达NK4 mRNA和蛋白,且可抑制Raji细胞的增殖、迁徙和侵袭。结论：NK4基因成功克隆并构建了重组真核表达载体,转染Raji细胞后表达稳定。     

Potential confusion of contaminating CD16+ myeloid DCs with anergic CD16+ NK cells in chimpanzees

Precise identification of NK-cell populations in humans and nonhuman primates has been confounded by imprecise phenotypic definitions. A common definition used in nonhuman primates, including chimpanzees, is CD3(-) CD8α(+) CD16(+) , and this is the dominant NK-cell phenotype in peripheral blood. However, recent data suggest that in chimpanzees a rare CD8α(-) CD16(+) population also exists. Herein, we present evidence validating the existence of this rare subset in chimpanzee peripheral blood, but also demonstrating that gating on CD3(-) CD8α(-) CD16(+) cells can inadvertently include a large number of CD16(+) myeloid DCs (mDCs). We confirmed the inclusion of mDCs in CD3(-) CD8α(-) CD16(+) gated cells by demonstrating high expression of CD11c, BDCA-1 and HLA-DR, and by the lack of expression of NKp46 and intracellular perforin. We also functionally validated the CD8α(-) NK-cell and mDC populations by mutually exclusive responsiveness to a classical NK-cell stimulus, MHC class I-deficient cells, and a prototypic mDC stimulus, poly I:C, respectively. Overall, these data demonstrate common problems with gating of NK cells that can lead to erroneous conclusions and highlight a critical need for consensus protocols for NK-cell phenotyping.     

CD3+CD56+ NK T cells are significantly decreased in the peripheral blood of patients with psoriasis

Psoriasis is a chronic, inflammatory, hyperproliferative skin disease, in which autoimmunity plays a great role. Natural killer T cells (NK T cells), are suggested to be involved in the pathogenesis of different autoimmune diseases. To examine the involvement of CD3+CD56+ NK T cells in the pathogenesis of psoriasis, we investigated the lymphocyte subpopulations obtained from blood samples of psoriatic patients before and after treatment, and of healthy controls, using two-colour flow cytometry. We found no significant differences between total T cells, total B cells, T helper cells, T cytotoxic cells and NK cells in patients with psoriasis before and after treatment and in controls. Increased percentage of memory T cells and decreased percentage of naive T cells was detected in psoriatic patients compared to controls, but these changes were not statistically significant. The CD3+CD56+ cells of psoriatic patients were significantly decreased relative to controls. The percentage of CD3+CD56+ cells increased after different antipsoriatic therapies, but remained significantly lower than those found in controls. CD3+CD56+ cells of healthy controls were capable of rapid activation, while in psoriatic patients activated NK T cells were almost absent. The decrease in the number of CD3+CD56+ cells may represent an intrinsic characteristic feature of patients with psoriasis, which is supported by the fact that after treatment NK T cells do not reach the values found in controls. In conclusion our results suggest that CD3+CD56+ NK T cells could be actively involved in the development of Th1 mediated autoimmune diseases.     

CD73 expression on extracellular vesicles derived from CD4+CD25+Foxp3+ T cells contributes to their regulatory function

CD4⁺CD25⁺Foxp3⁺ Treg cells maintain immunological tolerance. In this study, the possibility that Treg cells control immune responses via the production of secreted membrane vesicles, such as exosomes, was investigated. Exosomes are released by many cell types, including T cells, and have regulatory functions. Indeed, TCR activation of both freshly isolated Treg cells and an antigen‐specific Treg‐cell line resulted in the production of exosomes as defined morphologically by EM and by the presence of tetraspanin molecules LAMP‐1/CD63 and CD81. Expression of the ecto‐5‐nucleotide enzyme CD73 by Treg cells has been shown to contribute to their suppressive function by converting extracellular adenosine‐5‐monophosphate to adenosine, which, following interaction with adenosine receptors expressed on target cells, leads to immune modulation. CD73 was evident on Treg cell derived exosomes, accordingly when these exosomes were incubated in the presence of adenosine‐5‐monophosphate production of adenosine was observed. Most importantly, CD73 present on Treg cell derived exosomes was essential for their suppressive function hitherto exosomes derived from a CD73‐negative CD4⁺ T‐cell line did not have such capabilities. Overall our findings demonstrate that CD73‐expressing exosomes produced by Treg cells following activation contribute to their suppressive activity through the production of adenosine.     

CD4+CD25+ T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence

We hypothesized that advanced age and medical conditions had an impact on the accumulation of CD4+CD25+ T regulatory cells (Treg), which in turn could deteriorate cytotoxic activity of CD8+ T and NK cells. Volunteers were divided according to the Senieur Protocol into healthy young and elderly and non-healthy young and elderly subjects. The numbers of Treg cells in peripheral blood, their influence on CD8+ T and NK cells and production of IL2 as well as apoptosis intensity of Treg cells were measured. The number of Treg cells was higher in both elderly groups than in respective young ones. Compared to healthy subjects, those with medical conditions were revealed to have higher numbers of Treg cells. In addition, the highest accumulation of Treg cells in non-healthy elderly could be a result of their resistance to undergo apoptosis. The frequency of Treg cells correlated inversely with the activity of autologous cytotoxic cells in PBMC and production of IL2 by autologous CD4+CD25- Th cells. Thus, these parameters were the most highly decreased in non-healthy subjects, notably in the elderly. However, these parameters improved in the cultures of pure sorted cells. The only subset capable of decreasing them to the levels noted in PBMC when added back was Treg cells, which proved the link between the number of Treg cells, cytotoxic activity and production of IL2. Concluding, we found that Treg accumulated as a result of ageing and/or medical conditions were capable of decreasing cytotoxic activity of CD8+ T and NK cells and production of IL2.     

CD4/CD8 ratio,comorbidities, and aging in treated HIV infected individuals on viral suppression

The progression of the human immunodeficiency virus (HIV) infection to acquired immunodeficiency syndrome (AIDS) can be efficiently interrupted by antiretroviral therapy (ART). However, even successfully treated HIV-infected individuals are prone to develop non-AIDS-related diseases that affect the metabolism and several organs and systems. Biomarkers that predict the occurrence of comorbidities may help develop preventive measures. Current research shows that CD4⁺ T cell counts and viral load do not predict the development of non-AIDS-related diseases. The CD4/CD8 ratio has been indicated as a suitable marker of persistent immune dysfunction and the occurrence of non-AIDS-related events in treated HIV-positive patients. In this study, we explored the relationship between CD4/CD8 ratios, comorbidities, and aging in ART-treated HIV patients on viral suppression. We collected and evaluated data from 352 HIV-positive adults who were virologically suppressed (<40 copies/mL) on ART and with CD4 counts above 350 cells/mm³. The median age for participants was 46 years, 218 individuals had at least one comorbidity, and 239 had inverted CD4/CD8 ratios (<1). Current CD4/CD8 ratios were predicted by baseline CD4/CD8 ratios and nadir CD4 counts. Despite the high rates of inverted CD4/CD8 ratios and prevalence of comorbidities, no association between them was observed. The prevalence of comorbidities was significantly higher in older individuals, though aging alone did not explain the rate of all individual comorbidities. Low CD4/CD8 ratios were linked to neurocognitive disorders, suggesting that persistent T cell dysfunction contributes to neurocognitive decline.     

Differential usage of VLA-4 and CXCR4 by CD3+CD56+ NKT cells and CD56+CD16+ NK cells regulates their interaction with endothelial cells

The mechanism that regulates the preferential accumulation of NKT cells in the BM is unknown. The BM endothelium constitutively expresses selectins, the integrin ligands VCAM-1 and ICAM-1, and the chemokine CXCL12. Both NK and NKT subsets of cells exhibited similar tethering and rolling interactions on both P-selectin and E-selectin and expressed similar levels of the integrins, VLA-4 and LFA-1. Although NKT cells express higher levels of CXCR4 than NK cells, CXCL12 (the ligand for CXCR4) rapidly stimulates similar levels of adhesion of NK and NKT cells to VCAM-1 and ICAM-1. In both subsets, the arrest on VCAM-1 was dependent on high affinity VLA-4 and the homing of these cells to the BM of NOD/SCID was VLA-4-dependent. However, as opposed to the situation for NK cells, CXCL12 preferentially triggers, under shear flow, the rolling on VCAM-1 and transendothelial migration of NKT cells. Moreover, over-expression of high levels of CXCR4 on the YT NK cell line enables them to migrate in response to CXCL12. This study therefore suggests an important role for CXCR4 levels of expression and for VLA-4 in regulating the accumulation of NKT cells in the BM.     

HIV persistence in subsets of CD4+ T cells: 50 shades of reservoirs

Antiretroviral therapy controls HIV replication but does not eliminate the virus from the infected host. The persistence of a small pool of cells harboring integrated and replication-competent HIV genomes impedes viral eradication efforts. The HIV reservoir was originally described as a relatively homogeneous pool of resting memory CD4+ T cells. Over the past 20 years, the identification of multiple cellular subsets of CD4+ T cells endowed with distinct biological properties shed new lights on the heterogeneity of HIV reservoirs. It is now clear that HIV persists in a large variety of CD4+ T cells, which contribute to HIV persistence through different mechanisms. In this review, we summarize recent findings indicating that specific biological features of well-characterized subsets of CD4+ T cells individually contribute to the persistence of HIV. These include an increased sensitivity to HIV infection, specific tissue locations, enhanced survival and heightened capacity to proliferate. We also discuss the relative abilities of these cellular reservoirs to contribute to viral rebound upon ART interruption. Together, these findings reveal that the HIV reservoir is not homogeneous and should be viewed as a mosaic of multiple cell types that all contribute to HIV persistence through different mechanisms.     

Enhanced expression of CTLA-4 (CD152) on CD4+ T cells in HIV infection

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Down-regulation of CD28 predominantly on CD8+ T cells has been described in HIV infection, but analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on peripheral blood mononuclear cells (PBMC) during HIV infection. CTLA-4 was expressed only on T cells. Expression levels were significantly increased selectively on CD4+ T cells during all stages of HIV infection, while CTLA-4 expression on CD8+ T cells was always low. In contrast, after stimulation with the mitogen phytohaemagglutinin (PHA), CTLA-4 levels were strongly increased on T cells from controls but in T cells from HIV patients this response was severely impaired. Our data suggest that in HIV infection CD4+ and CD8+ T cells may be less responsive to B7 costimuli due to two different mechanisms: increase in CTLA-4 expression by CD4+ cells and down-regulation of CD28 by CD8+ cells.     

CD4+CD25+调节性T细胞的发育与胸腺CD4-CD25+细胞关联性的探讨

目的:探讨CD4 CD25 调节性T细胞(CD4 CD25 regulatoryTcells,CD4 CD25 Tr)的发育及其与胸腺CD4-CD25 细胞的关系。方法:以流式细胞术检测小鼠从出生至发育成熟过程中,胸腺、脾脏、淋巴结和外周血中CD4 CD25 Tr比例变化,以及胸腺CD4-CD25 细胞比例变化;通过磁激活细胞分选(MACS)从小鼠淋巴结纯化CD4 CD25 T和CD4 CD25-T细胞,经CFDA-SE标记,以多种刺激形式诱导增殖。结果:小鼠出生1d到10周的发育过程中,胸腺CD4 CD25 Tr比例一直比较恒定,但在脾脏、淋巴结和外周血,随鼠龄增加而不断升高,从1d龄到1周时升高最迅速,其后的升高速度逐渐减慢,10周龄时达平台期。胸腺中CD4-CD25 细胞在出生1d的小鼠比例非常高,1d龄到1周龄期间迅速下降,10周龄时达平台期。ConA不能诱导CD4 CD25 Tr和CD4 CD25-T细胞增殖,但CD4 CD25 Tr出现一过性细胞增大;佛波醇酯加离子霉素能诱导CD4 CD25 Tr和CD4 CD25-T细胞增殖;包被的抗CD3抗体加可溶性抗CD28抗体能刺激CD4 CD25-T细胞增殖,但CD4 CD25 Tr不增殖,加入高浓度IL-2,CD4 CD25-T细胞增殖更强,CD4 CD25 Tr出现增殖。结论:胸腺CD4-CD25 细胞很有可能是CD4 CD25 Tr的前体。     

CD8+ T cells promote HIV latency by remodeling CD4+ T cell metabolism to enhance their survival,quiescence, and stemness

1. Download : Download high-res image (203KB)
2. Download : Download full-size image

     

Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation

Activation of naive CD4⁺ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4⁺ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4⁺ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4⁺ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4⁺ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.     

Increased frequency of CD56Bright NK‐cells,CD3−CD16+CD56− NK‐cells and activated CD4+T‐cells or B‐cells in parallel with CD4+CDC25High T‐cells control potentially viremia in blood donors with HCV

A detailed phenotypic analysis of major and minor circulating lymphocyte subsets is described in potential blood donors with markers of hepatitis C virus (HCV), including non‐viremic and viremic groups. Although there were no changes in the hematological profile of either group, increased the levels of pre‐NK cells (CD3^?CD16⁺CD56^?) and a lower frequency of mature NK cells (CD3^?CD16⁺CD56⁺) characterized innate immunity in the non‐viremic group. Both non‐viremic and viremic groups displayed significantly increased levels of CD56^Bright NK cells. Furthermore, this subset was significantly elevated in the viremic subgroup with a low viral load. In addition, an increase in the NKT2 subset was observed only in this subgroup. An enhanced frequency of activated CD4⁺ T‐cells (CD4⁺HLA‐DR⁺) was a characteristic feature of the non‐viremic group, whereas elevated CD19⁺ B‐cells and CD19⁺CD86⁺ cell populations were the major phenotypic features of the viremic group, particularly in individuals with a low viral load. Although CD4⁺CD25^High T‐cells were significantly elevated in both the viremic and non‐viremic groups, it was particularly evident in the viremic low viral load subgroup. A parallel increase in CD4⁺CD25^High T‐cells, pre‐NK, and activated CD4⁺ T‐cells was observed in the non‐viremic group, whereas a parallel increase in CD4⁺CD25^High T‐cells and CD19⁺ B‐cells was characteristic of the low viral load subgroup. These findings suggest that CD56^Bright NK cells, together with pre‐NK cells and activated CD4⁺ T‐cells in combination with CD4⁺CD25^High T‐cells, might play an important role in controlling viremia. Elevated CD56^Bright NK cells, B‐cell responses and a T‐regulated immunological profile appeared to be associated with a low viral load. J. Med. Virol. 81:49–59, 2009. © 2008 Wiley‐Liss, Inc.     

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon (IFN) in response to virus. Although type I IFN is a natural killer (NK) cell modulator, a direct role for pDC in coordinating NK cell functions has not yet been elucidated in detail, especially in humans. Here we report that human pDC, following engagement of Toll-like receptor (TLR) 9, not only activate autologous NK cells, as indicated by the induction of CD69 expression, but also enhance their effector functions, especially cytotoxicity. Moreover, they can induce selective proliferation of CD56bright CD16- NK cells. This activity can be strongly augmented by the addition of autologous CD4+ CD25- T helper cells in an IL-2-dependent fashion. Strikingly, CD4+ CD25hi T regulatory (Treg) cells completely abrogate this IL-2-dependent proliferation of NK cells, while they are not able to influence NK cell activation or proliferation solely induced by pDC. Our data show that TLR9-engaged pDC represent a critical stimulus for human NK cells and that CD4+ Th cells and CD4+ CD25hi Treg cells play an important role in modulating human NK cell responses.     

Uncoupling activation-induced modulation of CD16 and CD69 in CD56+ cells during AIDS

The immune system of HIV+ patients is chronically activated, which has been associated with a detrimental effect on both innate and acquired immunity during AIDS. We analyzed the expression and modulation of the triggering markers CD69 and CD16 in CD56+ cells from 18 asymptomatic HIV+ individuals and 8 AIDS patients, compared with 21 seronegative subjects. We observed a diminished PMA-induced CD16 downregulation in AIDS patients (p<0.01), associated with low numbers of CD4+ cells (p<0.02). Furthermore, an enhanced unstimulated expression of CD69 in asymptomatic HIV+ patients (p<0.05) was shown. AIDS patients could not efficiently upregulate PHA-dependent CD69 expression (p<0.05), which correlated with low CD4+ counts (p< 0.05). These abnormalities in CD16 and CD69 modulation were recorded in patients under highly active antiretroviral therapy (HAART). Our results demonstrate an altered modulation of two functionally relevant receptors in CD56+ cells from AIDS patients, contributing to our understanding of the immunopathogeny of NK cell dysfunction during disease progression.     

NK1.1+ CD4+ CD8- thymocytes with specific lymphokine secretion

CD4+8^? or CD4^?8⁺ thymocytes have been regarded as direct progenitors of peripheral T cells. However, recently, we have found a novel NK1.1⁺ subpopulation with skewed T cell antigen receptor (TcR) Vβ family among heat-stable antigen negative (HSA^?) CD4⁺8^? thymocytes. In the present study, we show that these NK1.1⁺ CD4+8^? thymocytes, which represent a different lineage from the major NK1.1^? CD4⁺8^? thymocytes or CD4⁺ lymph node T cells, vigorously secrete interleukin (IL)-4 and interfron (IFN)-γ upon stimulation with immobilized anti-TcR-αβ antibody. On the other hand, neither NK1.1^? CD4+8^?thymocytes nor CD4⁺ lymph node T cells produced substantial amounts of these lymphokines. A similar pattern of lymphokine secretion was observed with the NK1.1⁺ CD4⁺ T cells obtained from bone marrow. The present findings elucidate the recent observations that HSA^? CD4⁺8^? thymocytes secrete a variety of lymphokines including IFN-γ, IL-4, IL-5 and IL-10 before the CD4⁺8^? thymocytes are exported from thymus. Our evidence indicates that NK1.1⁺ CD4⁺8^? thymocytes are totally responsible for the specific lymphokine secretions observed in the HSA- CD4⁺8^? thymocytes.