CD56bright NK cells after hematopoietic stem cell transplantation are activated mature NK cells that expand in patients with low numbers of T cells

We studied early NK‐cell recovery in 29 allografted patients undergoing different lymphoreductive regimens. Already at 2 wk after graft take, the number of NK cells had reached (supra)normal levels but NK‐cell subsets were skewed. The number of CD56^dimCD16^bright NK cells was low and correlated strongly with the level of hematopoiesis, whereas the number of the more abundant NK cells expressing high levels of CD56 did not. Post‐transplant CD56^bright NK cells (ptCD56^bright) differed from CD56^bright NK cells in normal controls (CD56^bright) in being HLA‐DR‐ and perforin‐positive, CCR7^?, CD27^?, CD127^? and mostly c‐kit^?. CD56^bright from normal controls stimulated by IL‐15 in vitro (NK^IL‐15) acquired all the characteristics distinguishing CD56^bright from ptCD56^bright. IL‐2 exerted similar effects. Moreover, when cultured without cytokines, ptCD56^bright, CD56^bright and NK^IL‐15 responded similarly by upregulating CD127 and c‐kit but not CCR7. IL‐12 stimulated IFN‐γ production in ptCD56^bright, whereas CD56^bright responded only to IL‐12 plus IL‐15. Hence, ptCD56^bright have all the features of cytokine‐stimulated CD56^bright. Because only patients with low numbers of T cells had high numbers of ptCD56^bright, we conclude that ptCD56^bright are activated CD56^bright that expand while competing with T cells for the elevated post‐transplant level of IL‐15.     

Expression of killer immunoglobulin-like receptors on peripheral blood NK cell subsets of women with recurrent spontaneous abortions or implantation failures

PROBLEM: Decidual natural killer (NK) cells express inhibitory receptors (killer immunoglobulin-like receptors, KIRs), which bind to ligands on trophoblast cells (human leucocyte antigen, HLA-C). This interaction appears to block NK cytotoxicity against trophoblast cells. In this study, we investigated the expression of inhibitory and activating receptors in peripheral blood NK cells of women with recurrent spontaneous abortion (RSA) or implantation failures. METHOD OF STUDY: CD56(dim)/CD16(+), CD56(bright)/CD16(-) NK cells and CD56(+)/CD3(+) NKT cells of women with RSA or in vitro fertilization (IVF) failures and normal controls were analyzed for the expression of CD158a, CD158b inhibitory KIRs or CD161-activating receptors, by flow cytometric analysis. RESULTS: CD158a and CD158b inhibitory receptor expression by CD56(dim)/CD16(+) and CD56(bright)/CD16(-) NK cells were significantly decreased, and CD161-activating receptor expression by CD56(+)/CD3(+) NKT cells was significantly increased in women with implantation failures when compared with normal controls. CONCLUSIONS: An imbalance between inhibitory and activating receptor expression was found in NK cells of women with implantation failures. This imbalance may explain the adverse reproductive outcome.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)‐DR4+ monozygotic twins discordant for rheumatoid arthritis

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell‐ and B cell‐epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4‐related (antigen DR4^? HLA‐DR4)⁺ woman with early RA, her healthy monozygotic twin and an unrelated HLA‐DR3⁺ woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7‐aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)?17/IL‐4/IL‐10/IL‐6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261‐273), the K264 carbamylated T cell epitope (carT261–273), the native B cell epitope (B359–369) or the R360 citrullinated B cell epitope (citB359–369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4⁺ twins, but not in the DR3⁺ RA. The collagen‐specific activation of CD4⁺ T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL‐17 production in the RA twin, but a greater IL‐4 and IL‐10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL‐6/IL‐10 ratio in the RA twin compared to a greater IL‐10 production in the healthy twin. Our data suggest that circulating collagen‐specific T and B cells are found in HLA‐DR4⁺ subjects, but only RA activated cells express co‐stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.     

Increased natural killer cell subsets with inhibitory cytokines and inhibitory surface receptors in patients with recurrent miscarriage and decreased or normal subsets in kidney transplant recipients late post‐transplant

Patients with recurrent miscarriage (RM) show up‐regulated cytotoxic natural killer (NK) cells that are suspected to play a causal role in abortion. In the present study, we investigated counter‐regulating inhibitory mechanisms and compared the results in RM patients with those of healthy controls (HC), patients with end‐stage renal disease (ESRD) and kidney transplant recipients late post‐transplant (TX). NK, NK T and T cell subsets were analysed in the peripheral blood of 31 RM, 14 female ESRD and nine female TX patients as well as 21 female HC using eight‐colour fluorescence flow cytometry. Compared with HC, RM patients showed significantly higher absolute numbers of CD56⁺ NK cells co‐expressing the phenotype interferon (IFN)‐γR⁺, IL‐4⁺, transforming growth factor (TGF)‐β⁺, IL‐4⁺ human leucocyte antigen D‐related (HLA‐DR)⁺, TGF‐β⁺HLA‐DR⁺, IL‐4⁺TGF‐β⁺, IL‐4⁺TGF‐β^–, IFN‐γ⁺ and/or IL‐10^–IFN‐γ⁺ (all P ≤ 0·01), more IL‐17⁺CD56^bright (P = 0·028) NK cells and more CD56^dimCD16⁺ NK cells co‐expressing IFN‐γR, IFN‐γ, IL‐4 and/or TGF‐β (all P ≤ 0·01). When the same cell subsets were analysed in ESRD or TX patients, cytokine‐producing NK cell subsets were not significantly different from those of HC. RM patients showed significantly higher absolute numbers of CD158a⁺, CD158b⁺, CD158a^–CD158e⁺ (all P < 0·05), NKG2D⁺NKG2A⁺, NKG2D ⁺NKG2A^–, NKG2D⁺ and/or NKG2A⁺ (all P ≤ 0·01) CD56⁺ NK cells and higher CD158a⁺, CD158b⁺ (all P < 0·05), NKG2D⁺ and/or NKG2A⁺ (all P < 0·01) CD56^dim+CD16⁺ NK cells than HC. In contrast, ESRD patients had normal and TX recipients had lower CD158a⁺ and NKG2D⁺NKG2A^–CD56⁺ NK cells and lower CD158a⁺CD56^dim+CD16⁺ NK cells (all P < 0·05) than HC. RM patients have abnormally high circulating NK cells expressing inhibitory cytokines and inhibitory surface receptors which might contribute to the pathogenesis of RM.     

Expression of natural cytotoxicity receptors and a2V-ATPase on peripheral blood NK cell subsets in women with recurrent spontaneous abortions and implantation failures

PROBLEM: Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. a2V-ATPase is expressed on subsets of PBMC and regulates the extracellular environment, which facilitates NK cytotoxicity or cytokine secretion. In this study, we aim to investigate the expression of NCRs and a2V-ATPase in peripheral blood NK cells of women with recurrent spontaneous abortions (RSA) or implantation failures. METHOD OF STUDY: Peripheral blood NK cells (CD56(dim) and CD56(bright) were analyzed for the expression of NCRs (NKp46, NKp44 and NKp30) and a2V-ATPase using 3-color flow cytometry in women with RSA (n=24), implantation failures (n=19) or normal healthy women (n=13). RESULTS: CD56+/NKp46+ cells were markedly decreased (P<0.05) and CD56(bright)/a2V-ATPase+ cells were significantly increased (P<0.05) in women with RSA as compared to those of normal controls. In women with RSA or implantation failures, expression of NKp46, NKp44, NKp30, and a2V-ATPase on CD56(bright) NK cells was significantly up-regulated as compared with those of CD56(dim) NK cells. CONCLUSION: The differential expression of NCRs and a2V-ATPase in NK cell subsets may suggest dysregulation of NK cytotoxicity and cytokine production in women with RSA and implantation failures.     

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Expression of the lymph node homing and CC‐chemokine receptor 7 (CCR7), with L‐selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T‐cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62L^high CD44^low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7^high CD62L^high CD44^high (central memory) and CCR7^low CD62L^low CD44^high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short‐term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7^high CD4 T cells produced predominantly interleukin (IL)‐2, whereas CCR7^low CD4 T cells produced both IL‐2 and interferon‐γ (IFN‐γ). However, in contrast to previously published reports, the CCR7^high CD8 T‐cell subpopulation produced both IFN‐γ and IL‐2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down‐regulated CCR7 only after multiple cell divisions, and this coincided with the down‐regulation of CD62L and production of IL‐4 and IFN‐γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL‐2‐ and IFN‐γ‐producing cells are CCR7^low, while few cytokine‐expressing CCR7^high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T‐cell subpopulations, but indicate additional complexity in that CCR7^high CD8 T cells also may produce IFN‐γ.     

HIV-1感染者淋巴细胞活化与第二受体表达的研究

目的：了解HIV－1感染者体内淋巴细胞的活化情况及表达第二受体CCR5、CXCR4的淋巴细胞活化状态，分析这些指标与疾病严重程度的关系，探讨HIV感染的免疫基础。方法：用三色标记法流式细胞术检测24例HIV－1感染者及13例健康对照的抗凝血标本，分析活化标志物HLA－DR及第二受体CCR5、CXCR4的表达等指标。结果：HIV－1感染者CD8^ T淋巴细胞的HLA－DR表达高于健康对照（P＜0．001）；HIV－1感染者表达CCR5、CXCR4的CD8^ T淋巴细胞活化明显高于健康对照（P＜0．001）；表达CCR5CD4^ 、CD8^ T淋巴细胞与表达CXCR4相比HL－DR表达均明显增高（P＜0．001）；CD4^ 、CD8^ T淋巴细胞的活化状态与CD4百分率的变化明显关系。结论：HIV－1感染者CD8^ T淋巴细胞及表达不同第二受体的CD8^ T淋巴细胞活化程度明显增高，活化程度与疾病进程相关。     

A distinct cellular profile is seen in the human endocervix during Chlamydia trachomatis infection

Problem The endocervix is a major target of Chlamydia trachomatis infection, but little is known about the immune repertoire in this tissue, or its response to these common bacteria. Method of study Using a cytobrush, we isolated cells from the endocervix of 20 women during C. trachomatis infection, and post‐antibiotic treatment. Endocervical swabs and blood were taken in parallel. Endocervical cells were enumerated, and endocervical and blood T cells immunophenotyped. Chlamydia trachomatis was genotyped by sequence analysis of the OmpA gene, and quantified by culture. Results Chlamydia trachomatis genotypes were D, E, F and Ia, and infectious burden varied considerably. Endocervical T cell and neutrophil numbers were highly elevated during infection, with both CD4 and CD8 T‐cell subsets accumulating. Regardless of the presence or absence of infection, the endocervical cell infiltrate was dominated by effector memory T cells, and the numbers of CCR5 and CD103 expressing T cells was significantly higher than in the blood. Human leukocyte antigen (HLA‐DR) expression by endocervical T cells was significantly increased during infection. Conclusion The human endocervix exhibits a distinct cellular response to C. trachomatis infection that can be longitudinally evaluated by cytobrush sampling. Infecting organisms can be sampled and analyzed in parallel.     

Increasing circulating T-cell activation markers are linked to subsequent implantation failure after transfer of in vitro fertilized embryos

PROBLEM: Implantation determines success of in vitro fertilization (IVF) and embryo transfer (ET) cycles. Data are accumulating to support a role of the immune system in implantation. Most of the literature addresses the importance of natural killer (NK) cells in this process. The purpose of the current study is to examine the role of circulating T cells in implantation failure. METHOD OF STUDY: Blood from 22 women undergoing IVF/ET during November, 2001, was drawn on cycle day 9 and analyzed for the percentage of circulating T cells expressing the activation markers CD69+ and human leukocyte antigen (HLA)-DR and the suppressor marker CD11b using immunofluorescence and flow cytometry. These results were compared with total percentage circulating CD3, CD4 and CD8 cells as well as NK cells and pregnancy outcome that cycle. RESULTS: Infertile women had significantly greater expression of the activation marker of CD69+ among CD8+ and CD4+ T cells and HLA-DR among CD4 cells than fertile women. No difference in expression of T cell suppressor marker of CD11b was noted when infertile and fertile women were compared. No correlations were observed when activated T cells were compared with circulating CD3+, CD4+, CD8+, activated NK cells and NK cytotoxicity. CD3+ 4+ HLA-DR+ was expressed significantly less among successfully pregnant compared with unsuccessfully pregnant women. CONCLUSION: T-cell activation markers CD 69+ and HLA-DR+ are associated with increased implantation failure after IVF/ET.     

Dendritic cells from human mesenteric lymph nodes in inflammatory and non‐inflammatory bowel diseases: subsets and function of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDC) in mesenteric lymph nodes (MLN) may be important regulators of both inflammatory and non‐inflammatory mucosal immune responses but human studies are rare. Here we compare pDC from human MLN and peripheral blood (PB) by phenotype and function. MLN from patients with or without inflammatory bowel disease (IBD) undergoing colon surgery and PB from patients with IBD and from controls were used to isolate mononuclear cells. The pDC were analysed by flow cytometry for the expression of CD40, CD80, CD83, CD86, CCR6, CCR7, CX3CR1, CD103 and HLA‐DR. Purified pDC from MLN and PB were stimulated with staphylococcus enterotoxin B (SEB), CpG‐A, interleukin‐3 (IL‐3), SEB + IL‐3, CpG‐A + IL‐3 or left unstimulated, and cultured alone or with purified allogeneic CD4⁺ CD45RA⁺ HLA‐DR‐ T cells. Subsequently, concentrations of IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, interferon‐α (IFN‐α), IFN‐γ and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by multiplex bead array. The PB pDC from IBD patients exhibited an activated and matured phenotype whereas MLN pDC and control PB pDC were less activated. CpG‐A and CpG‐A + IL‐3‐stimulated MLN pDC secreted less IL‐6 and TNF‐α compared with PB pDC from controls. Compared with co‐cultures of naive CD4 T cells with PB pDC, co‐cultures with MLN pDC contained more IL‐2, IL‐10 and IFN‐γ when stimulated with SEB and SEB + IL‐3, and less IFN‐α when stimulated with CpG‐A. MLN pDC differ phenotypically from PB pDC and their pattern of cytokine secretion and may contribute to specific outcomes of mucosal immune reactions.     

The induction of autoimmune hepatitis in the human leucocyte antigen‐DR4 non‐obese diabetic mice autoimmune hepatitis mouse model

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti‐smooth muscle actin and/or anti‐nuclear, anti‐liver kidney microsomal type 1 (anti‐LKM1) and anti‐liver cytosol type 1 (anti‐LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)‐DR3, ‐DR7 and ‐DR13. HLA‐DR4 has the second strongest association with adult AIH, after HLA‐DR3. We investigated the role of HLA‐DR4 in the development of AIH by immunization of HLA‐DR4 (DR4) transgenic non‐obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti‐LKM1/anti‐LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (T_regs), which had decreased programmed death (PD)‐1 expression. Splenic T_regs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8⁺ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild‐type (WT) NOD mice. Our results demonstrate that HLA‐DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of T_regs and reduced PD‐1 expression may result in spontaneous activation of key immune cell subsets, such as antigen‐presenting cells and CD8⁺ T effectors, facilitating the induction of AIH and persistent liver damage.     

Preserved immune system in long-term asymptomatic vertically HIV-1 infected children

The objective of this study was to study immune system status in long‐term asymptomatic (LTA) HIV‐1‐infected children. A cross‐sectional study was used, involving HIV‐1‐infected children over 7 years of age who were rated into two groups according to their clinical and immunological classification: (a) LTA: 7 asymptomatic HIV‐1‐infected children in A1; (b) Rapid progressor (RP): 14 age‐matched C3 HIV‐1‐infected children. The control group consisted of 17 age‐matched uninfected children. The characterization of CD4+ T‐cell subsets was determined by three‐colour flow cytometry. The proliferative response and cytokine production by activated peripheral blood T‐cells were also measured. IL‐7 levels were measured in serum. Thymic production of T‐cells was quantified by TCR rearrangement excision circles (TRECs). The LTA children showed similar proliferative responses to PHA, PWM and anti‐CD3+ anti‐CD28, but lower responses to tetanus toxoid and streptokinase, in comparison with the controls but always higher responses in comparison with the RP group. The production of TNF‐α and IFN‐γ was similar in the LTA and control groups, and both were higher than the levels in the RP group. The LTA group showed a lower percentage of memory CD4+ T‐cells (CD4+ CD45RO+, CD4+ CD45RA‐CD62L+) than the control and RP groups. The LTA group also showed lower percentages of CD4+ CD7‐ cells than the controls. As for naïve CD4+ T‐cells (CD4+ CD45RA+ CD62L+), CD4+ CD45RA+ and CD4+ CD62L+ cells, the LTA group showed higher values than the control and RP groups. The LTA group showed higher percentages of CD4+ HLA‐DR+ CD38+ than the controls, but lower values than the RP group. In contrast, the LTA group had percentages of CD4+ HLA‐DR‐CD38+ T‐cells higher than both the control and RP groups, whereas CD4+ CD38+ levels were only higher in the LTA group in comparison with the controls. CD4+ HLA‐DR+ CD38‐ and CD4+ HLA‐DR+ cell numbers were lower in the LTA group in comparison with the RP group. We found almost normal values of TRECs and IL‐7 in the LTA group, but lower values in the RP group. Moreover, we found an inverse relation between TREC levels and IL‐7 in plasma from HIV‐infected children. Asymptomatic HIV‐1 infected children have a well preserved immune system similar to that of control uninfected children in spite of HIV‐infection for more than 7 years. Moreover, our results identified new markers of HIV disease, such as TRECs and IL‐7, that could be used to monitor disease.     

The repertoire of HLA-Cw-specific NK cell receptors CD158 a/b (EB6 and GL183) in individuals with different HLA phenotypes.

Over the last few years, natural killer (NK) cells have been shown to express major histocompatibility complex (MHC) molecules recognizing receptors that are thought to function primarily as negative signalling receptors. Much attention has been focused on the NK cell receptors CD158a (EB6) and CD158b (GI 183), which recognize two alternative epitopes on the HLA-Cw locus. In order to investigate whether HLA type affects the CD158a/b repertoire, expressed as percentage positive cells for a particular receptor and mean expression on this population of NK cells, peripheral blood lymphocytes of 47 HLA-typed donors were examined. Peripheral blood samples were examined by flow cytometric analysis to investigate the expression of CD158a and CD158b receptors on the surface of NK cells. In parallel, we determined each individual's HLA phenotype. There was a great heterogeneity in CD158 expression; nevertheless all individuals had NK cells belonging either to the CD158 a+b-, a-b+ or a-b- populations. No positive or inverse correlations could be shown between either receptor expression intensity or proportion of positive cells, and presence of the appropriate ligand. Thus no association between an individual's NK receptor repertoire and HLA serotype could be demonstrated. It is concluded that CD158 is expressed on NK cells in a highly redundant fashion. Our data do not support either a positive selection mechanism or the receptor calibration model.     

Phenotype of NK Cells and Cytotoxic/Apoptotic Mediators Expression in Ectopic Pregnancy

Citation Laskarin G, Redzovic A, Vukelic P, Veljkovic D, Gulic T, Haller H, Rukavina D. Phenotype of NK cells and cytotoxic/apoptotic mediators expression in ectopic pregnancy. Am J Reprod Immunol 2010 Problem The expression of cytotoxic/apoptotic mediators and the phenotype characteristics of uterine NK cells (uNK) in tubal ectopic pregnancy (EP) were investigated. Method of study Samples of uterine decidua and tubal mucosa as well as peripheral blood (PB) of the same women with EP were used for phenotype characterization of NK cells and detection of cytotoxic/apoptotic mediators and IL‐15. Results In tubal mucosa, perforin, FasL, granulysin and IL‐15 were almost completely absent, but they were present in normal and EP uterine deciduas. TRAIL was present on trophoblast and tubal mucosa, contrary to its lack in normal and EP uterine decidua. CD16^?CD56^dim NK cells, mostly CD94^? and NKG2A^?, predominate in tubal mucosa, whereas CD16^?CD56^bright NK cells, predominantly CD94⁺ and NKG2A⁺ prevail in EP uterine decidua. NK cells at the EP implantation site express lower percentages of perforin and granulysin, but they express a higher percentage of TRAIL than do EP uterine decidual and PB NK cells. Lower percentage of TNF‐α‐expressing and IL‐4‐expressing NK cells were found at the implantation site compared to EP uterine decidua. Conclusions Authentic uNK cell population seems to be insufficient to restrict trophoblast invasion because of low expression of cytotoxic/apoptotic mediators.     

Presence of HLA‐DR Molecules and HLA‐DRB1 mRNA in Circulating CD4+ T Cells

The human major histocompatibility complex class II isotype HLA‐DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA‐DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA‐DR protein to the presence of associated mRNA (HLA‐DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA‐DR observed on CD4⁺ T cells was almost exclusively cell surface‐associated, while for autologous CD19⁺ B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA‐DRB1 were found in CD4⁺ T cells, using an HLA‐DRB1 allele‐specific qPCR assay. Finally, the presence of HLA‐DR was not confined to activated CD4⁺ and CD8⁺ T cells, as evaluated by the co‐expression of CD25. The functional role of the HLA‐DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA‐DR on T cells from HLA‐DR in the context of antigen‐presenting cells, which is a well‐known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re‐evaluation of the HLA‐DR as a T cells activation marker is warranted.     

Correlation Between Natural Cytotoxicity Receptors and Intracellular Cytokine Expression of Peripheral Blood NK Cells in Women with Recurrent Pregnancy Losses and Implantation Failures

Problem Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We investigated whether women with recurrent pregnancy losses (RPLs) and implantation failures have aberrant correlation between NCRs and intracellular cytokine expression of NK cells. Method of study Peripheral blood NK cells (CD56^dim and CD56^bright) were analyzed for NCRs (NKp46, NKp44 and NKp30) and cytokine expression (TNF‐α, IFN‐γ, IL‐4, IL‐10) using flow cytometry in RPL (n = 22), implantation failures (n = 23) or controls (n = 15). Results In type 1 cytokine studies, CD56^bright/NKp30⁺ cells in controls (r = 0.696, P < 0.05) were positively correlated with CD56^bright/IFN‐γ⁺/TNF‐α⁺ cells. CD56^bright/NKp46⁺ cells in implantation failures (r = ?0.76, P < 0.01) were negatively correlated with CD56^bright/IFN‐γ⁺/TNF‐α^? cells. RPL did not have any correlation. In type 2 cytokine studies, CD56⁺/NKp46⁺ cells (r = 0.758, P < 0.01) and CD56⁺/NKp30⁺ cells (r = 0.637, P < 0.05) were positively correlated with CD56^bright/IL‐4⁺/IL‐10⁺ cells in controls. CD56⁺/NKp30⁺ cells in implantation failures (r = ?0.778, P < 0.05) were negatively correlated with CD56^bright/IL‐10⁺/IL‐4⁺ cells. There were no correlations in RPL. Conclusion Recurrent pregnancy losses and implantation failures have lack of, or negative correlation between NCRs and intracellular cytokines expression. This observation suggests that excessive pro‐inflammatory cytokine expression in NK cells in RPL and implantation failures may be exerted through the NCRs or interruption of signal transduction processes.     

Relationship of CD146 expression to secretion of interleukin (IL)‐17, IL‐22 and interferon‐γ by CD4+ T cells in patients with inflammatory arthritis

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4⁺ T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146⁺CD4⁺ and interleukin (IL)‐17⁺CD4⁺ T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146⁺CD4⁺ T cells were enriched for secretion of IL‐17 [alone or with IL‐22 or interferon (IFN)‐γ] and for some putative Th17‐associated surface markers (CD161 and CCR6), but not others (CD26 and IL‐23 receptor). CD4⁺ T cells producing IL‐22 or IFN‐γ without IL‐17 were also present in the CD146⁺ subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4⁺ helper T cells.     

Natural killer and dendritic cells collaborate in the immune response induced by the vaccine against uterine cervical cancer

Virus‐like particles (VLPs) of human papillomavirus (HPV) are used as a vaccine against HPV‐induced cancer, and recently we have shown that these VLPs are able to activate natural killer (NK) cells. Since NK cells collaborate with dendritic cells (DCs) to induce an immune response against viral infections and tumors, we studied the impact of this crosstalk in the context of HPV vaccination. NK cells in the presence of HPV‐VLPs enhanced DC‐maturation as shown by an upregulation of CD86 and HLA‐DR and an increased production of IL‐12p70, but not of the immunosuppressive cytokine IL‐10. This activation was bidirectional. Indeed, in the presence of HPV‐VLPs, DCs further activated NK cells by inducing the upregulation of cell surface activation markers (CD69 and HLA‐DR). The function of NK cells was also improved as shown by an increase in IFN‐γ secretion and cytotoxic activity against an HPV⁺ cell line. This crosstalk between NK cells and DCs needed CD40 interaction and IL‐12p70 secretion, whereas NKG2D was not implicated. Our results provide insight into how VLPs interact with innate immune cells and how NK cells and DCs play a role in the immune response induced by this vaccine agent.