The Suppression of Peritoneal Cellular Immunity in Women With Endometriosis Could Be Restored After Gonadotropin Releasing Hormone Agonist Treatment

PROBLEM: Our previous study reported that peritoneal natural killer (NK) cytotoxicity and CD3⁺CD25⁺ lymphocyte subpopulation were suppressed in women with advanced endometriosis. Whether these phenomena are general for all stages of endometriosis and whether these alterations could be restored by long-term use of gonadotropin releasing hormone agonist (GnRHa) are further tested in this study. METHOD: Lymphocyte subpopulations (B cells, NK cells, T cells, and T-cell activation markers such as CD69, HLA-DR, and CD25) and NK cell cytotoxicity of peripheral blood and peritoneal fluid by dual-color flow cytometry and ^5lCr release assay in 30 cases of endometriosis were compared with those in 26 controls. We also compared these changes before and after 6-month treatment with GnRHa for advanced endometriosis. RESULTS: Compared with the controls, only those women with advanced endometriosis showed lower NK cytotoxicity in peritoneal fluid mononuclear cells (PFMC). The CD3⁺CD69⁺ lymphocyte subpopulation decreased in peripheral blood mononuclear cells (PBMC) of advanced endometriosis, while the CD3⁺CD25⁺ lymphocyte subpopulation decreased in both PBMC and PFMC of mild and advanced endometriosis. After GnRHa treatment, the CD3⁺CD69⁺ lymphocyte subpopulation increased in both PBMC and PFMC and the CD3⁺CD25⁺ lymphocyte subpopulation increased in PFMC, but not in PBMC. CONCLUSION: Impaired local immunological function in the PF of endometriosis was confirmed by this study and the impairments could be restored after long-term GnRHa therapy.     

Flow Cytometry Analysis of Lymphocyte Subpopulations in Peritoneal Fluid of Women With Endometriosis

PROBLEM: We investigated the lymphocyte subpopulations in peripheral blood (PB) and peritoneal fluid (PF) of women with and without endometriosis to evaluate if the decreased natural killer (NK)-mediated cytotoxicity in women with endometriosis was due to a quantitative defect or not. METHOD: The PB and PF mononuclear cells of 59 women undergoing a diagnostic laparoscopy for pain and/or infertility were analyzed by flow cytometry. RESULTS: The number and concentration of PF mononuclear cells (MC) was increased in women with endometriosis compared to women without endometriosis. The monocyte/macrophage marker (CD14) was expressed on 70.3 and 66.9% of PFMC of women with and without endometriosis, respectively. The CD4/CD8 ratio was inverted in the PF, and this was more pronounced in women with endometriosis. In the PF of women with endometriosis, 41.3% of the lymphocytes were CD8 positive, compared to 34.3% in women without endometriosis. The percentage of NK positive lymphocytes in PF, using three different monoclonal antibodies directed against NK cell markers (CD57, CD 16, and CD56) were not different between women with and without endometriosis. In women with endometriosis, 12.7,9.5, and 28.8% of lymphocytes were CD57, CD16, and CD56 positive, respectively. CONCLUSION: PFMC consisted mainly of phagocytic and human leukocyte antigen (HLA)-restricted or HLA unrestricted cytotoxic cells capable of reacting to various antigens entering the cavity from the lower genital tractus. Furthermore, the decreased NK activity reported in PB and PF of women with endometriosis was not likely to be caused by a quantitative defect, since the percentage of NK positive lymphocytes was not different between women with and without endometriosis.     

Killer immunoglobulin-like receptor and human leukocyte antigen expression as immunodiagnostic parameters for pelvic endometriosis

PROBLEM: We investigated host immunologic responses to endometriosis by comparing immune cell surface antigens in peripheral blood (PB) and peritoneal fluid (PF) from women with endometriosis with those in PB and PF from other patients. METHOD OF STUDY: Japanese women with endometriosis (n = 56) were compared with controls with other laparoscopic diagnoses (n = 68). PB and PF were collected at the time of laparoscopy for flow cytometry. RESULTS: No significant difference in phenotypic parameters of T cells (CD3, CD4, and CD8), B cells (CD19), natural killer (NK) cells (CD56), or monocytes/macrophages (CD14) was seen between women with and without endometriosis. However, increased killer immunoglobulin-like receptor (CD158a) expression by NK cells and decreased human leukocyte antigen (HLA)-ABC and -DR expression by macrophages, all suggesting decreased functional activation were found in endometriosis. These markers showed significant association with endometriosis by odds ratio, logistic regression, and decision tree analyses. CONCLUSIONS: Increased CD158a(+) NK cells in PB and PF indicated decreased NK cell cytotoxicity in endometriosis, while decreased HLA expression on PF macrophages suggested impaired antigen presentation. Thus, aberrant immune responses by NK cells and macrophages may represent risk factors for endometriosis.     

Expression of inhibitory-motif killer immunoglobulin-like receptor, KIR2DL1, is increased in natural killer cells from women with pelvic endometriosis

Problem:  We investigated inhibitory and activation motif expression of killer immunoglobulin-like receptor (KIR) by natural killer (NK) cells, which may be pathogenetically involved in endometriosis.
Method of study:  We compared cells from 24 Japanese women laparoscopically diagnosed with endometriosis, to cells from 25 women with other laparoscopic diagnoses. KIR expression by NK cells was assessed in peripheral blood (PB) and peritoneal fluid (PF) by flow cytometry. Intracellular immunoreceptor tyrosine-based (IT) inhibitory and activation motifs (ITIM and ITAM) of KIR in PB was assessed by Western blotting.
Results:  ITIM-KIR expression by PB NK cells was significantly and similarly greater than ITAM-KIR expression in women with and without endometriosis. Percentages of CD56⁺ NK cells in PB and PF did not differ significantly between women with and without endometriosis; however, the percentage of CD158a⁺ cells among CD56⁺ NK cells in PB and PF was significantly higher in women with than without endometriosis.
Conclusions:  ITIM-KIR expressing NK cells might confer tolerance to peritoneal endometriotic implants.     

Gonadotropin Releasing Hormone (GnRH) Agonist Induces Down-Regulation of the CD3+CD25+ Lymphocyte Subpopulation in Peripheral Blood

PROBLEM : To test whether GnRH agonist could alter in vivo human immune cells and whether the alteration is related to the success of pregnancy in an in vitro fertilization-embryo transfer (IVF-ET) program. METHODS : Thirty-six infertile patients were enrolled under the long protocol of GnRH agonist (buserelin acetate) and superovulation with gonadotropin from our IVF-ET program. Peripheral B cells, NK cells, CD4⁺ and CD8⁺ T cells, and the expression of CD69, CD25, HLA-DR, and CD71 antigens on the T cells were serially examined by dual-color flow cytometry. RESULTS : B cells, NK cells, CD8⁺ T cells, and CD71⁺ T lymphocyte subpopulations were not changed throughout the whole course of treatment. CD4⁺ T cell and CD25⁺ T cell sub-populations were significantly down-regulated when the GnRH agonist was used for approximately 2 wk. CD3⁺CD69⁺, CD3⁺CD25⁺, and CD3⁺DR⁺ lymphocyte subpopulations were increased at 7 days (during implantation) and at 14 days after embryo transfer in pregnant patients, but not in patients who failed to get pregnant. CONCLUSIONS : The GnRH agonist had a transiently immunosuppressive effect on CD4⁺ and CD25⁺ T cells, but CD69⁺, CD25⁺, and HLA-DR⁺ T cells were activated during and after successful implantation.     

3H11, a unique cell surface molecule involved in the function of the CD45RA+subset of CD4+ cells

We have developed a mAb anti-3H11 by immunizing mice with aT cell line derived from the Callithrix Jacchus (common marmoset).anti-3H11 is reactive with 48% of unfractlonated T cells, 62%of CD4⁺ cells and 39% of CD8⁺ cells. Among CD4 cells, anti-3H11preferentially reacts with the CD45RA⁺ T cell subset. The majorityof helper activity for pokeweed mitogen (PWM)-driven B cellIgG synthesis and T cell response to recall antigen such astetanus toxold was found within the 3H11-CD4 cell population,whereas anti-3H11⁺CD4⁺ cells provided poor helper function forPWM-driven B cell IgG synthesis and were more responsive toconcanavalln A and autologous mixed lymphocyte reaction. Biochemicalcharacterization showed that anti-3H11 precipitated a singleprotein band with a relative molecular weight of 32,000 from¹²⁵l-surface labeled cell lysate. Biochemical, phenotyplc andfunctional studies revealed that the 3H11 molecule appearedto be different from previously established molecules on theT cell surface. Interestingly, addition of anti-3H11 to thecombination of CD4 and B cells in the presence of CDS cellsbut not to the combination of CD4 and B cells resulted in enhancementof the suppression of PWM-driven B cell IgG synthesis. Moreover,anti-3H11 had a co-mitogenic effect on T cells via the CD2 andCD3 pathways, and this co-mitogenic activity is restricted tothe CD45RA⁺ T cells. Taken together, our results show that the3H11 molecule is a novel antigen which may play an importantrole in the activation and function of the CD45RA⁺ subset ofT cells.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.     

Differential proliferative responses in subsets of human CD28+cells delineated by BB27 mAb

We report the Identification of a novel 140 kDa disulflde-llnkeddimer expressed by a subset of peripheral blood T lymphocytes.This molecule, which Is recognized by mAb BB27, is also detectedon cells of the myelomonocytlc lineage. In the T cell lineage,Its expression Is positively modulated after lymphocyte activation.A series of double-labeling experiments revealed that BB27 mAbIdentifies new CD4 and CDS cell subsets different from thosedefined by CD45RA, CD45RO, CD26, CD29, CD31, and CD38. Finally,BB27 mAb also subdivides the CD28 subset. Of the utmost interestIs the finding that a proliferative response to CD28 mAb andphorbol myrlstate acetate stimulation is exclusively obtainedin the CD28⁺BB27⁺ cell subset, whereas the CD28⁺BB27^–subset falls to proliferate.     

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (T_reg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of T_reg (CD4⁺CD25^highforkhead box protein 3⁺), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4⁺CD25⁺, CD4⁺CD25^highFoxP3⁺ and CD8⁺CD25⁺ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4⁺CD25^low/negIL‐10⁺ in PB and CD4⁺CD25^low/negIFN‐γ⁺ in PF; meanwhile, CD25^high mainly expressed IL‐10 in both compartments. A high proportion of CD4⁺CD107⁺ and CD8⁺CD107⁺ cells was observed in PF. T_reg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4⁺ and CD8⁺ degranulation responses and decreased CD4⁺IL‐10⁺ cells in PF. Our results demonstrated that in TB pleurisy T_reg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4⁺ and CD8⁺ cells to degranulate in response to M. tuberculosis.     

Inhibition of anti-GD3-ganglioside antibody-induced proliferation of human CD8+ T cells by CD16+ natural killer cells

The ganglioside GD3 has been described as a membrane component of human T cells which is involved in T cell growth. In the present study the activating function of GD3 for human CD4⁺ and CD8⁺ T cells was analyzed by five different monoclonal antibodies (mAb) directed against the GD3 molecule. Three mAb U5, Z21 and R24 induced strong proliferation of peripheral blood mononuclear cells and purified CD8⁺ and CD4⁺ T cells of normal donors containing less than 5% CD16⁺ natural killer (NK) cells. In contrast to CD4⁺ T cells, CD8⁺ T cells proliferated only weakly in the presence of 15% CD16⁺ NK cells. The proliferative response of purified CD4⁺ and CD8⁺ T cells (<5% NK cells) correlated with the antibody-dependent induction of integral and soluble interleukin-2 (IL-2) receptors and was reduced to 20% by an anti-IL-2 receptor antibody. Our results show, that the GD3 molecule represents an activation molecule for both CD4⁺ and CD8⁺ T cells and that CD16⁺ NK cells selectively inhibit anti-GD3 antibody-induced proliferation of CD8⁺ T cells.     

Role of CD4+CD45RA+ T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse

The non-obese diabetic (NOD) mouse spontaneously develops aT cell-mediated autoimmune disease, sharing many features withhuman insulin-dependent diabetes mellltus (IDDM), leading toinsulin-secreting ß cell destruction. The role ofCD4⁺ T cells has been evidenced at two levels. First, CD4⁺ Tcells from diabetic animals are required to transfer diabetesto non-diabetic recipients in conjunction with CD8⁺ effectorT cells. Second, suppressive CD4⁺ T cells have been characterizedin non-diabetic NOD mice. T cells with different functions canthus share the CD4⁺ phenotype. Since CD4⁺ T cells can be dividedinto at least two subgroups on the basis of CD45 isoform expression,we evaluated the distribution of CD4⁺ T cells expressing theCD45RA isoform on NOD mouse thymocytes and peripheral T cells.The percentage of CD45RA⁺ cells was dramatically increased amongthe most mature CD3^bright thymocytes and among CD4⁺ T cellsin lymph nodes of the NOD mouse as compared with control strains.This increase was related to the development of insulitls. Interestingly,the CD45RA isoform was expressed on most CD4⁺ T cells invadingthe islets. In vivo treatment with an antl-CD45RA mAb preventedthe development of insulitls and spontaneous diabetes in femaleanimals but not the transfer of diabetes by T cells collectedfrom diabetic NOD donors. These results indicate that anti-CD45RAmAb is only effective if given before the full commitment ofeffector T cells to the destruction of islet ß cells.ThusCD4⁺CD45RA⁺ T cells play a key role in early activation stepsof anti-islet immunity.     

Expression profiles of peripheral CD160+ lymphocytes during the course of healthy human pregnancy

Citation
Barakonyi A, Weisdorn R, Miko E, Varga P, Bodis J, Szekeres‐Bartho J, Szereday L. Expression profiles of peripheral CD160⁺ lymphocytes during the course of healthy human pregnancy. Am J Reprod Immunol 2011; 66: 137–142 Problem CD160 receptor is expressed by natural killer (NK) and T‐cell subsets, and after activation, it could enhance cytotoxicity or pro‐inflammatory cytokine production on NK cells. Here, we investigated the phenotype of peripheral CD160⁺ cells during healthy pregnancy. Method of study We analyzed the expression of CD69 activation marker, gamma/delta TCR, and NKG2A or NKG2D NK cell receptors on CD160⁺ lymphocytes of non‐pregnant and healthy pregnant women at four different stages of pregnancy by flow cytometry. Results In our hands, CD160 receptor‐positive lymphocytes were present during pregnancy; however, they had different characteristics depending on gestational age. During implantation, CD160⁺ cells showed low activation rate, decreased NK receptor expression while 40% of Vδ2 + T cells expressed CD160 receptor. In turn, all the above parameters increased as pregnancy proceeds. Conclusion Our results indicate that CD160⁺ lymphocytes could be able to play a role in the maintenance of healthy pregnancy.     

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.     

类风湿关节炎CD4+CD28-T细胞与淋巴细胞凋亡的相关性研究

目的: 研究类风湿关节炎(RA)患者外周血CD4⁺CD28^-T细胞比例与淋巴细胞凋亡异常的相关性。方法: 采用流式细胞术三色分析法检测50例患者和50例健康志愿者的外周血淋巴细胞中CD4⁺CD28^-T细胞比例;通过加入PHA孵育检测RA病人外周淋巴细胞和正常对照的淋巴细胞对激活诱导细胞死亡(AICD)易感性差异;分析CD4⁺CD28^-T细胞比例与外周血淋巴细胞凋亡率的相关性。结果: RA组CD4⁺CD28^-T细胞比例的均数明显高于健康对照组(7.79%±3.52% vs 1.89%±1.78%,P<0.05)。RA组病人外周血淋巴细胞的AICD凋亡率低于健康对照组(11.38%±5.73% vs 19.46%±6.32%,P<0.05)。Spearman相关分析结果显示CD4⁺CD28^-T细胞比例与外周血淋巴细胞AICD凋亡率负相关(r=-0.433,P<0.01)。结论: RA患者外周血中CD4⁺CD28^-T细胞比例增多,活化淋巴细胞生存期延长,这可能参与RA的发病机制。     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

In Vivo CD3+CD25+ Lymphocyte Subpopulation Is Down-regulated Without Increased Serum-Soluble Interleukin-2 Receptor (sIL-2R) by Gonadotropin Releasing Hormone Agonist (GnRH-a)

PROBLEM: To test further whether the suppression of the CD3⁺CD25⁺ lymphocyte sub-population by gonadotropin-releasing hormone agonist (GnRH-a) is related to the change in levels of cytokines and soluble interleukin-2 receptor (sIL-2R). METHOD: Twenty-seven infertile patients were enrolled under the long protocol of GnRH-a agonist (buserelin acetate) and superovulation with gonadotropin from our IVF-ET program. Peripheral B cells, NK cells, CD4⁺ and CD8⁺ T cells and the expression of CD69, CD25, HLA-DR, and CD71 antigens on the T cells were serially examined by dual-color flow cytometry. Serum levels of cytokines and sIL-2R were measured. RESULTS: The B cells, NK cells, T cells, CD4⁺, CD8⁺ T cells, CD3⁺DR⁺, and CD3⁺CD71⁺ lymphocyte subpopulations were not changed after the use of GnRH-a. The CD25⁺ T cell subpopulation was significantly down-regulated, but the CD69⁺ T cell subpopulation was increased when the GnRH-a was used for approximately 2 wk. The serum levels of interleukin-lp (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), and sIL-2R were not changed. CONCLUSION: The GnRH-a had a transiently suppressive effect on CD25⁺ T cells, but a stimulatory effect on CD69⁺ T cells. However, the serum level of cytokines or sIL-2R did not change. These immunological modulations seems to be the result of interaction between GnRH-a and estrogen.