Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

The Fas (CD95)/Fas ligand (CD178) system plays an important role in epithelial damage during the acute respiratory distress syndrome. The goal of this study was to determine whether proximal and distal human lung epithelial cells differ in their sensitivity to Fas ligand (rh-sFasL), and whether the response of lung epithelium to Fas ligation is modulated by proinflammatory cytokines. Although the expression of both Fas message and protein was similar in proximal and distal lung epithelial cells, only distal cells became apoptotic when exposed to serial dilutions of rh-sFasL. Stimulation with tumor necrosis factor-alpha, interleukin-1beta, or interferon-gamma significantly increased the sensitivity of proximal cells to rh-sFasL, and exposure to either tumor necrosis factor-alpha or interferon-gamma enhanced the sensitivity of distal cells to Fas ligation. These findings suggest that in normal human lungs, the responses of the epithelium to Fas ligation become more pronounced from proximal to distal locations. Furthermore, proinflammatory cytokines sensitize lung epithelium to Fas-induced death. These findings are relevant for understanding the role of the Fas/FasL system in acute lung injury, in which epithelial damage occurs primarily in distal airway and alveolar epithelium, whereas sFasL is present throughout the airspaces.

As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNF alpha interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p<0.0001) and the lipofuscin autofluorescence in lymphocytes (p<0.0001) were significantly increased. The expression of several genes regulating autophagy did not significantly vary with the age of the culture. Forty-eight hours after each stimulation, the percentage of induced cell death rose while, in the remaining living cells, the percentage of lymphocytes with autophagic vacuoles (p<0.05), with beta-galactosidase activity and the lipofuscin autofluorescence (p<0.001) significantly decreased, suggesting the preferential death of cells with autophagy. Our data support the view that the accumulation of autolysosomes in senescent lymphocytes might aggravate cellular fragility, leading to apoptosis and necrosis mainly induced by lymphocyte activation.

CD4(+)CD25(++)Foxp3(+) regulatory T cells (Tregs) control self-reactive cells to maintain peripheral tolerance. Treg homeostasis has to be controlled tightly to ensure balanced Treg-mediated suppression. One mechanism that regulates the CD4(+) T cell pool is activation-induced cell death (AICD). This is mimicked in vitro by TCR restimulation-induced expression of the death ligand CD95L (FasL/APO-1L/CD178) in expanded T cells. These cells express the death receptor CD95 (Fas/APO-1), and binding of CD95L to CD95 results in AICD. In contrast, Tregs do not undergo AICD upon TCR (re)stimulation in vitro despite a functional CD95 cell death pathway. In this study, we show that human and murine Tregs express low levels of CD95L upon stimulation. Knockdown of the transcriptional repressor Foxp3 partially rescues CD95L expression and AICD in human Tregs. Moreover, upon stimulation Foxp3-mutant Tregs from Scurfy mice express CD95L similar to conventional T cells. We further addressed whether exogenous CD95 stimulation provides a mechanism of Treg homeostatic control in vivo in mice. Triggering of CD95 reduced Treg numbers systemically as reflected by in vivo imaging and decreased GFP(+) Treg numbers ex vivo. Our study reveals that Foxp3 negatively regulates CD95L expression in Tregs and demonstrates that Tregs are susceptible to homeostatic control by CD95 stimulation.

Cancer development relies on a variety of mechanisms that facilitate tumor growth despite the presence of a functioning immune system. Understanding these mechanisms may foster novel therapeutic approaches for oncology and organ transplantation. By expression of the apoptosis-inducing protein CD95L (FasL, APO-1L, CD178), tumors may eliminate tumor-infiltrating lymphocytes and suppress anti-tumor immune responses, a phenomenon called "tumor counterattack". On the one hand, preliminary evidence of tumor counterattack in human tumors exists, and CD95L expression can prevent T-cell responses in vitro. On the other hand, CD95L-expressing tumors are rapidly rejected and induce inflammation in mice. Here, we summarize and discuss the consequences of CD95L expression of tumor cells and its contribution to immune escape.

Mutation of the Foxp3 transcription factor in Scurfy (Sf) mice results in complete absence of the CD4+Foxp3+ regulatory T cells (Tregs), severe multiorgan autoimmune syndrome, and early death at 4 wk of age. However, Sf mice simultaneously bearing the Il2-/- (Sf.Il2-/-) or Faslpr/lpr gene (Sf.Faslpr/lpr) have extended lifespan despite totally lacking Tregs, indicating a role of IL-2 and CD95 (Fas) signaling pathways in the multiorgan autoimmune syndrome beyond the Treg checkpoint. IL-2 has been implicated in regulating lymphoproliferation and CD178 (FasL) expression. However, Sf.Il2-/- mice have increased lymphoproliferation and FasL expression. Importantly, the pattern of organ-specific autoimmune response of Sf.Il2-/-mice resembled IL-2 knockout mice whereas that of Sf.Faslpr/lpr was similar to Sf mice, indicating that the distinct and weakened autoimmune manifestation in IL-2 knockout mice was not caused by the residual Tregs. Our study demonstrated a novel role of IL-2 in regulating multiorgan autoimmune inflammation beyond the Treg checkpoint and indicated that both Il2-/- and Faslpr/lpr genes prolong the lifespan of Sf mice but by different mechanisms.

The FS7-associated cell surface antigen (Fas, also named CD95, APO-1 or TNFRSF6) attracted considerable interest in the field of apoptosis research since its discovery in 1989. The groups of Shin Yonehara and Peter Krammer were the first reporting extensive apoptotic cell death induction upon treating cells with Fas-specific monoclonal antibodies.1,2 Cloning of Fas3 and its ligand,4,5 FasL (also known as CD178, CD95L or TNFSF6), laid the cornerstone in establishing this receptor-ligand system as a central regulator of apoptosis in mammals. Therapeutic exploitation of FasL-Fas-mediated cytotoxicity was soon an ambitous goal and during the last decade numerous strategies have been developed for its realization. In this chapter, we will briefly introduce essential general aspects of the FasL-Fas system before reviewing its physiological and pathophysiological relevance. Finally, FasL-Fas-related therapeutic tools and concepts will be addressed.

Abnormal proliferation and/or persistence of synoviocytes and inflammatory cells has long been described in inflammatory arthritis conditions, but only relatively recently has substantial attention been drawn to the relevance of abnormal apoptotic processes in disease pathogenesis and treatment. This review summarizes a current understanding of the Fas (CD95)-Fas ligand (CD178) apoptotic system, which has most predominantly been examined in rheumatoid arthritis. There, synovial inflammation is often characterized by a unique resistance to Fas-related apoptosis, and agonistic therapeutic interventions upon Fas have consistently been found beneficial in both animal and human disease models. Therefore, modulation of the Fas pathway will hopefully be of both pathogenic and therapeutic interest in the study of inflammatory arthritis conditions in general.

To determine whether the Fas/Fas ligand (FasL) (CD95/CD178) system contributes to the development of an inflammatory response in vivo, 2.5 microg of bacterial lipopolysaccharide (LPS; endotoxin) per g was administered intranasally to healthy mice (C57BL/6) and mutant mice deficient in either Fas (lpr mice) or FasL (gld mice). Sustained LPS-induced neutrophilic inflammation in the lungs was attenuated in both lpr and gld mice. These observations provide further evidence of a proinflammatory role for the Fas/FasL system in the lungs.

Fas ligand (FasL, CD95L, APO-1L, CD178, TNFSF6, APT1LG1) is the key death factor of receptor-triggered programmed cell death in immune cells. FasL/Fas-dependent apoptosis plays a pivotal role in activation-induced cell death, termination of immune responses, elimination of autoreactive cells, cytotoxic effector function of T and NK cells, and the establishment of immune privilege. Deregulation or functional impairment of FasL threatens the maintenance of immune homeostasis and defense and results in severe autoimmunity. In addition, FasL has been implicated as an accessory or costimulatory receptor in T cell activation. The molecular mechanisms underlying this reverse signaling capacity are, however, poorly understood and still controversially discussed. Many aspects of FasL biology have been ascribed to selective protein-protein interactions mediated by a unique polyproline region located in the membrane-proximal intracellular part of FasL. Over the past decade, we and others identified a large number of putative FasL-interacting molecules that bind to this polyproline stretch via Src homology 3 or WW domains. Individual interactions were analyzed in more detail and turned out to be crucial for the lysosomal storage, the transport and the surface appearance of the death factor and potentially also for reverse signaling. This review summarizes the work in the framework of the Collaborative Research Consortium 415 (CRC 415) and provides facts and hypotheses about FasL-interacting proteins and their potential role in FasL biology.

High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.

HIV infection and the progression to AIDS are characterized by the depletion of CD4(+) T cells through apoptosis of the uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated in part by the human immunodeficiency virus, type 1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells and CD178 gene expression, which is critically involved in T cell apoptosis. The differing ability of HIV strains to induce death of infected and uninfected cells may play a role in the clinical and biological differences displayed by HIV strains. We chemically synthesized the 86-residue truncated short variant of Tat and its full-length form. We show that the trans-activation ability of Tat at the long terminal repeat does not correlate with T cell apoptosis but that the ability of Tat to up-regulate CD178 mRNA expression and induce apoptosis in T cells is critically dependent on the C terminus of Tat. Moreover, the greater 86-residue Tat-induced apoptosis is via the extrinsic pathway of CD95-CD178.

Antithymocyte globulins (ATGs), the immunoglobulin G (IgG) fraction of sera from rabbits or horses immunized with human thymocytes or T-cell lines, are used in conditioning regimens for bone marrow transplantation, in the treatment of acute graft-versus-host disease, in the prevention or treatment of acute rejection in organ transplantation, and in severe bone marrow aplasia. In nonhuman primates, ATGs induce rapid, dose-dependent, T-cell depletion in peripheral lymphoid tissues, where apoptotic cells can be demonstrated in T-cell zones. We show here that increasing ATG concentrations in vitro resulted in reduced lymphocyte proliferative responses, associated with a rapid increase in the percentage of apoptotic cells. Apoptosis did not require prior exposure to interleukin-2, nor did it result in CD178/CD95 or tumor necrosis factor/tumor necrosis factor receptor (TNF/TNF-R) interactions; it was therefore clearly different from activation-induced cell death. Cytochrome c release, caspase-9, and caspase-3 activation were not implicated, excluding a direct involvement of the intrinsic mitochondrial pathway. The cysteine protease inhibitor E64d and cathepsin-B-specific inhibitors conferred significant protection, whereas apoptosis was associated with the release of active cathepsin B into the cytosol. These data demonstrate a role for cathepsin B in T-cell apoptosis induced by ATGs at concentrations achieved during clinical use.

BACKGROUND

Obesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (<25 kg/m(2)) and obese (>30 kg/m(2)). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme(+) and IFN-γ(+) NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined.

RESULTS

As opposed to similar NK cell counts, the number of CD3(+)CD56(+) cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells.

CONCLUSIONS

NK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge.

Chronic lymphocytic leukemia (CLL) B cells become sensitive to Fas (CD95)-mediated apoptosis 3 to 5 days after CD40 ligation. However, CD4+ cytotoxic T lymphocytes (CTLs) can kill CLL B cells via a Fas-ligand (CD178)-dependent process within 24 hours after CD40 cross-linking, when ligation of CD95 alone is insufficient to induce apoptosis. In addition to CD95, CD40-activated CLL cells also express DR5, a receptor for tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that is expressed by CD4+ CTL. In addition, CD40 ligation in vitro and in vivo induces CLL cells to express the proapoptotic protein, BH3 interacting domain death agonist (Bid), which can facilitate crosstalk between mitochondrial-dependent, apoptosis-inducing pathways and death receptors, such as death receptor 5 (DR5). To evaluate whether ligation of CD95 and/or DR5 can induce apoptosis of CD40-activated CLL cells, we generated artificial cytotoxic effector cells that express both human TRAIL and CD178 (Chinese hamster ovary [CHO]-CD178/TRAIL) or only TRAIL (CHO-TRAIL) or CD178 (CHO-CD178). CHO-CD178/TRAIL cells were significantly more effective in killing CD40-activated CLL cells than either CHO-TRAIL or CHO-CD178 and, unlike the latter, could kill CLL cells 24 hours after CD40 ligation. We conclude that CD40 ligation induces CLL cells to express the proapoptotic molecule Bid and the death receptors CD95 and DR5, the latter of which can act synergistically to induce caspase-dependent apoptosis of CD40-activated CLL B cells.

Interactions between the TNF-family receptor Fas (CD95) and Fas Ligand (FasL, CD178) can efficiently induce apoptosis and are critical for the maintenance of immunological self-tolerance. FasL is kept under strict control by transcriptional and posttranslational regulation. Surface FasL can be cleaved by metalloproteases, resulting in shed extracellular domains, and FasL can also traffic to secretory lysosomes. Each form of FasL has distinct biological functions. Fas is more ubiquitously expressed, but its apoptosis-inducing function is regulated by a number of mechanisms including submembrane localization, efficiency of receptor signaling complex assembly and activation, and bcl-2 family members in some circumstances. When apoptosis is not induced, Fas-FasL interactions can also trigger a number of activating and proinflammatory signals. Harnessing the apoptosis-inducing potential of Fas for therapy of cancer and autoimmune disease has been actively pursued, and despite a number of unexpected side-effects that result from manipulating Fas-FasL interactions, this remains a worthy goal.

The contribution of Fas (CD95/APO-1) to cell death mechanisms of differentiated neurons is controversially discussed. Rat cerebellar granule neurons (CGNs) express high levels of Fas in vitro but are resistant to FasL (CD95L/APO-1L/CD178)-induced apoptosis. We here show that this resistance was mediated by a phosphatidylinositol 3-kinase (PI 3-kinase)-Akt/protein kinase B (PKB)-dependent expression of lifeguard (LFG)/neuronal membrane protein 35. Reduction of endogenous LFG expression by antisense oligonucleotides or small interfering RNA lead to increased sensitivity of CGNs to FasL-induced cell death and caspase-8 cleavage. The inhibition of PI 3-kinase activity sensitized CGNs to FasL-induced caspase-8 and caspase-3 processing and caspase-dependent fodrin cleavage. Pharmacological inhibition of PI 3-kinase, overexpression of the inhibitory protein IkappaB, or cotransfection of an LFG reporter plasmid with dominant-negative Akt/PKB inhibited LFG reporter activity, whereas overexpression of constitutively active Akt/PKB increased LFG reporter activity. Overexpression of LFG in CGNs interfered with the sensitization to FasL by PI 3-kinase inhibitors. In contrast to CGNs, 12 glioma cell lines, which are sensitive to FasL, did not express LFG. Gene transfer of LFG into these FasL-susceptible glioma cells protected against FasL-induced apoptosis. These results demonstrate that LFG mediated the FasL resistance of CGNs and that, under certain circumstances, e.g., inhibition of the PI 3-kinase-Akt/PKB pathway, CGNs were sensitized to FasL.

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naïve HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using Chi2 test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p = 0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.

BACKGROUND

In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory.

RESULTS

Based on a combination of differential and density gradient centrifugation steps, we provide a protocol to enrich intact SL from expanded hematopoietic cells, here T lymphocytes and Natural Killer cells. Individual fractions were initially characterized by Western blotting using antibodies against an array of marker proteins for intracellular compartments. As indicated by the presence of LAMP-3 (CD63) and FasL (CD178), we obtained a selective enrichment of SL in one of the resulting organelle fractions. The robustness and reproducibility of the applied separation protocol was examined by a high-resolution proteome analysis of individual SL preparations of different donors by 2D difference gel electrophoresis (2D-DIGE).

CONCLUSIONS

The provided protocol is readily applicable to enrich and isolate intact secretory vesicles from individual cell populations. It can be used to compare SL of normal and transformed cell lines or primary cell populations from healthy donors and patients with lysosomal storage or transport diseases, or from corresponding mutant mice. A subsequent proteome analysis allows the characterization of molecules involved in lysosomal maturation and cytotoxic effector function at high-resolution.

Inflammatory responses initiate rapid production of IL-1 family cytokines, including IL-18. This cytokine is produced at high levels in inflammatory diseases, including allergy and autoimmunity, and is known to induce IgE production in mice. Here we provide evidence that IL-18 is directly coupled to induction of self-reactive IgM and IgG antibody responses and recruitment of innate B2 B cells residing in the marginal zone of the spleen. Moreover, the data suggest that the B-cell activation occurs predominantly in splenic extrafollicular plasma cell foci and is regulated by natural killer T (NKT) cells that prevent formation of mature germinal centers. We also find evidence that NKT cells control this type of B-cell activation via cytotoxicity mediated by both the perforin and CD95/CD178 pathways. Thus, NKT cells regulate innate antibody responses initiated by an inflammatory stimulus, suggesting a general mechanism that regulates B-cell behavior in inflammation and autoreactivity.

Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL(+) killer B cells, showing they are enriched in the IgM(high)CD5(+)CD1d(high) B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL(+) B cells, but most FasL(+) B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL(+) B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL(+) cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4(+) T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL(+) B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders.

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8(+)CD122(+) cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8(+)CD122(-) population, were previously shown to include cells with regulatory activity and could be separated into CD49d(low) cells and CD49d(high) cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122(+) cells. Regulatory activity was observed in CD8(+)CD122(+)CD49d(low) but not in CD8(+)CD122(+)CD49d(high) cells, indicating that the regulatory cells in the CD8(+)CD122(+) population could be narrowed down to CD49d(low) cells. CD8(+)CD122(-) cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122(+) Tregs. CD122(+) Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8(+)CD122(-) cells, indicating that the regulation by CD122(+) Tregs is Fas/FasL-dependent. CD122(+) Tregs taken from IL-10-deficient mice could regulate CD8(+)CD122(-) cells as equally as wild-type CD122(+) Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122(+) Tregs in vitro. CD122(+) Tregs also regulated CD4(+) cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122(+) Tregs that kill activated T cells to maintain immune homeostasis.

To define the effect of estrogen and progesterone concentrations achieved during hormonal contraceptive therapy (HCT) on cell-mediated immunity (CMI) of HIV-infected and uninfected subjects, peripheral blood mononuclear cells (PBMCs) from varicella-zoster virus (VZV)-seropositive individuals were treated with 0.1 ng/mL of estradiol, 33 ng/mL of norgestrel, and 13 ng/mL of dexamethasone and tested for VZV CMI. Estrogen and progesterone decreased VZV lymphocyte proliferation and T helper (Th) 1/inflammatory cytokine secretion, albeit less than dexamethasone. Progesterone decreased the expression of CD69 activation marker on CD8 and CD14 cells and increased the expression of Fas ligand (CD178) on CD14 monocytes, suggesting that induction of apoptosis may contribute to the inhibitory effect of this hormone. Cytokine production of separated CD4, CD8, and CD14 cells confirmed the effect of progesterone on all 3 cellular types, whereas the effect of estrogen was restricted to CD14 monocytes. The estrogen- and progesterone-mediated inhibition of Th1/inflammatory cytokines was greater in HIV-infected subjects (35% decrease for both hormones) compared with uninfected subjects (12% and 19% for estrogen and progesterone, respectively), whereas the effect on proliferation and PBMC phenotype did not differ by HIV status. Overall, HCT concentrations of estrogen and progesterone downregulated ex vivo VZV CMI of HIV-infected and uninfected subjects.

Oral mucosal lamina propria progenitor cells (OMLP-PCs) are a novel, clonally derived PC population of neural crest origin with the potential to differentiate down both mesenchymal and neuronal cell lineages. In this study we aimed to determine the immunological properties of OMLP-PCs and to establish whether they would be suitable candidates for allogeneic tissue engineering and in the treatment of immune-related diseases. OMLP-PCs demonstrated no inherent immunogenicity with insignificant expression of costimulatory molecules (CD40, CD80, CD86, CD154, and CD178) or human leukocyte antigen (HLA) class II. OMLP-PCs required 7 days of stimulation with interferon-γ (IFN-γ) to induce cell surface expression of HLA II. Mixed lymphocyte cultures and mitogen stimulation demonstrated the potent immunosuppressive capability of OMLP-PCs in a contact-independent manner. Complete inhibition of lymphocyte proliferation was seen at doses as low as 0.001% OMLP-PCs to responder lymphocytes, while annexin V staining confirmed that this immunosuppressive effect was not due to the induction of lymphocyte apoptosis. These data demonstrate, for the first time, that OMLP-PC immunomodulation, unlike that for mesenchymal stem cells, occurs via a dose- and HLA II-independent mechanism by the release of immunosuppressive soluble factors and suggests these cells may have wide ranging potential in future immune-related therapies.