Impact of CD68/(CD3+CD20) Ratio at the Invasive Front of Primary Tumors on Distant Metastasis Development in Breast Cancer

Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68⁺), T-cells (CD3⁺) and B-cells (CD20⁺) at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs) and their inhibitors (TIMPs) either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20) ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2). In addition, a high CD68/(CD3+CD20) ratio (>0.05) was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20) ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23–5.24), p<0.01). Therefore, CD68/(CD3+CD20) ratio at the invasive front could be used as an important prognostic marker.     

Enrichment of Murine CD68+CCR2+ and CD68+CD206+ Lung Macrophages in Acute Pancreatitis-Associated Acute Lung Injury

Acute lung injury (ALI) is an important cause of mortality in critically ill patients. Acute pancreatitis (AP) is one of the risk factors for developing this syndrome. Among the inflammatory cells, macrophages have a key role in determining the severity of the acute lung injury. In the lungs, macrophages constitute a heterogeneous cell population distributed in different compartments. Changes in not only the macrophage count, but also in their phenotype have been seen during the course of lung injury. A murine ductal ligation model of acute pancreatitis showed substantial morphological changes in the pancreas and lungs. Immunohistochemistry showed neutrophil recruitment into both organs after 9 hours and later on. F4/80⁺ cells in the pancreas increased in the ligated animals, though there was not a significant difference in their number in the lungs as compared to sham operated animals. Flow cytometry analysis of lung macrophages demonstrated an enrichment of F4/80⁻ CD68⁺CCR2⁺ and F4/80⁻ CD68⁺CD206⁺ lung macrophages in ligated animals (AP) as compared to the sham operated group. The level of interleukin-6 in plasma increased 3 hours after ligation compared to the sham operated group, as a first indicator of a systemic inflammatory response.This study suggests a role for F4/80⁻ CD68⁺ macrophages in the pathogenesis of acute lung injury in acute pancreatitis. Studying lung macrophages for different phenotypic markers, their polarization, activation and recruitment, in the context of acute lung injury, is a novel area to potentially identify interventions which may improve the outcome of acute lung injury.     

The Clinical Significance of the CD163+ and CD68+ Macrophages in Patients with Hepatocellular Carcinoma

Our previous study has found that the abundance of peritumoral CD68⁺ macrophages was associated with poor prognosis in hepatocellular carcinoma (HCC) after resection. However, CD68 staining could not discriminate the protumoral or tumoricidal subpopulations from pan-macrophages. CD163 is a marker of alternatively activated macrophages. In this study, the clinical significance of CD163⁺ cells in tumors and peritumoral liver tissues was evaluated in a cohort of 295 patients with HCC after curative resection. We found that the density of CD163⁺ cells was well correlated with that of CD68⁺ cells in both tumors and peritumoral liver tissues but was much more. Immunostaining on consecutive sections and flow cytometry assay on surgical resected specimens further supported the findings that the CD163⁺ cells was more abundant than CD68⁺ cells. The density of peritumoral CD68⁺ cells was associated with poor recurrence-free survival (RFS) and poor overall survival (OS) (P = 0.004 and P = 0.001, respectively), whereas the CD163⁺ cells have no prognostic values either in tumors or in peritumoral liver tissues. In another cohort of 107 HCC patients, preoperative plasma concentration of soluble form of CD163 (sCD163) was associated with active hepatitis-related factors but not associated with the markers of tumor invasion. In conclusion, both the CD163⁺ cells local infiltration and plasma sCD163 were of limited significance in HCC, and they were more likely markers related to active hepatitis rather than tumor progression.     

A Potential Inhibitory Profile of Liver CD68+ Cells during HCV Infection as Observed by an Increased CD80 and PD-L1 but Not CD86 Expression

AimThe lack of potent innate immune responses during HCV infection might lead to a delay in initiating adaptive immune responses. Kupffer cells (KCs) and liver-infiltrating monocytes/macrophages (CD68⁺ cells) are essential to establish effective anti-HCV responses. They express co-stimulatory molecules, CD80 and CD86. CD86 upregulation induces activator responses that are then potentially regulated by CD80. The relative levels of expression of CD80, CD86 and the inhibitory molecule, PD-L1, on CD68⁺ cells modulate T cell activation. A few studies have explored CD80 and PD-L1 expression on KCs and infiltrating monocytes/macrophages in HCV-infected livers, and none investigated CD86 expression in these cells. These studies have identified these cells based on morphology only. We investigated the stimulatory/inhibitory profile of CD68⁺ cells in HCV-infected livers based on the balance of CD80, CD86 and PD-L1 expression.MethodsCD80, CD86 and PD-L1 expression by CD68⁺ cells in the lobular and portal areas of the liver of chronic HCV-infected (n = 16) and control (n = 14) individuals was investigated using double staining immunohistochemistry.ResultsThe count of CD68⁺ KCs in the lobular areas of the HCV-infected livers was lower than that in the control (p = 0.041). The frequencies of CD68⁺CD80⁺ cells and CD68⁺PD-L1⁺ cells in both lobular and total areas of the liver were higher in HCV-infected patients compared with those in the control group (p = 0.001, 0.031 and 0.007 respectively). Moreover, in the lobular areas of the HCV-infected livers, the frequency of CD68⁺CD80⁺ cells was higher than that of CD68⁺CD86⁺ and CD68⁺PD-L1⁺ cells. In addition, the frequencies of CD68⁺CD80⁺ and CD68⁺CD86⁺ cells were higher in the lobular areas than the portal areas.ConclusionsOur results show that CD68⁺ cells have an inhibitory profile in the HCV-infected livers. This might help explain the delayed T cell response and viral persistence during HCV infection.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection

Liver macrophages internalize circulating bloodborne parasites. It remains poorly understood how this process affects the fate of the macrophages and T cell responses in the liver. Here, we report that infection by Trypanosoma brucei induced depletion of macrophages in the liver, leading to the repopulation of CXCL16-secreting intrahepatic macrophages, associated with substantial accumulation of CXCR6⁺CD4⁺ T cells in the liver. Interestingly, disruption of CXCR6 signaling did not affect control of the parasitemia, but significantly enhanced the survival of infected mice, associated with reduced inflammation and liver injury. Infected CXCR6 deficient mice displayed a reduced accumulation of CD4⁺ T cells in the liver; adoptive transfer experiments suggested that the reduction of CD4⁺ T cells in the liver was attributed to a cell intrinsic property of CXCR6 deficient CD4⁺ T cells. Importantly, infected CXCR6 deficient mice receiving wild-type CD4⁺ T cells survived significantly shorter than those receiving CXCR6 deficient CD4⁺ T cells, demonstrating that CXCR6⁺CD4⁺ T cells promote the mortality. We conclude that infection of T. brucei leads to depletion and repopulation of liver macrophages, associated with a substantial influx of CXCR6⁺CD4⁺ T cells that mediates mortality.     

Impairment in the telocyte/CD34+ stromal cell network in human rheumatoid arthritis synovium

Telocytes (TCs)/CD34⁺ stromal cells have recently emerged as peculiar interstitial cells detectable in a variety of organs throughout the human body. TCs are typically arranged in networks establishing unique spatial relationships with neighbour cells and likely contributing to the maintenance of tissue homeostasis by both cell-to-cell contacts and releasing extracellular vesicles. Hence, TC defects are being increasingly reported in different pathologies, such as chronic inflammatory and fibrotic conditions. In this regard, TCs/CD34⁺ stromal cells have been shown to constitute an intricate interstitial network in the subintimal area of the normal human synovial membrane, but whether they are altered in chronic synovitis has yet to be explored. We therefore undertook a morphologic study to compare the distribution of TCs/CD34⁺ stromal cells between normal synovium and chronically inflamed synovium from patients with rheumatoid arthritis (RA) by using CD34 immunohistochemistry and CD31/CD34 double immunofluorescence. CD34 immunostaining revealed that, at variance with normal synovium, the inflamed and hyperplastic RA synovial tissue was nearly or even completely devoid of TCs/CD34⁺ stromal cells. Double immunofluorescence confirmed that almost all CD34⁺ tissue components detectable in RA synovium were blood vessels coexpressing CD31, while a widespread network of CD31⁻/CD34⁺ TCs was clearly evident in the whole sublining layer of normal synovium. In the context of the emerging diverse roles of TCs/CD34⁺ stromal cells in the regulation of tissue homeostasis and structure, the remarkable impairment in their networks herein uncovered in RA synovium may suggest important pathophysiologic implications that will be worth investigating further.     

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.     

A Higher Frequency of CD14+CD169+ Monocytes/Macrophages in Patients with Colorectal Cancer

Objective

Monocytes and macrophages can infiltrate into tumor microenvironment and regulate the progression of tumors. This study aimed at determining the frequency of different subsets of circulating monocytes and tumor infiltrating macrophages (TIMs) in patients with colorectal cancer (CRC).

Methods

The frequency of different subsets of circulating monocytes was characterized in 46 CRC patients and 22 healthy controls (HC) by flow cytometry. The frequency of different subsets of macrophages was analyzed in TIMs from 30 tumor tissues and in lamina propria mononuclear cells (LPMCs) from 12 non-tumor tissues. The concentrations of plasma cytokines and carcinoembryonic antigen (CEA) were determined. The potential association of these measures with the values of clinical parameters was analyzed.

Results

In comparison with that in the HC, the percentages of circulating CD14⁺CD169⁺, CD14⁺CD169⁺CD163⁺ and CD14⁺CD169⁺CD206⁺ monocytes and TIMs CD14⁺CD169⁺ as well as IL-10⁺CD14⁺CD169⁺, but not IL-12⁺ CD14⁺CD169⁺ macrophages were significantly increased, accompanied by higher levels of plasma IL-10 in the CRC patients. The percentages of CD14⁺CD169⁺ circulating monocytes and TIM macrophages were associated with the stage of disease and correlated positively with the levels of plasma IL-10 and CEA in CRC patients.

Conclusion

Our data suggest that an increase in the frequency of CD14⁺CD169⁺ cells may be associated with the development and progression of CRC and is concomitant rise of both, pro-tumor (M2-like, IL-10 producing) and anti-tumor (M1-like, IL-12 producing) monocytes and infiltrating macrophages. The frequency of CD14⁺CD169⁺ circulating monocytes and infiltrating macrophages may serve as a biomarker for evaluating the pathogenic degrees of CRC.     

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3⁺CD56⁺ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3⁺CD56⁺ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3⁺CD56⁺ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3⁺CD56⁺ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3⁺CD56⁺ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3⁺CD56⁺ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3⁺CD56⁺ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3⁺CD56⁺ cells in the PB before treatment. These observations suggest that the normalization of the CD3⁺CD56⁺ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.     

Parasite Fate and Involvement of Infected Cells in the Induction of CD4+ and CD8+ T Cell Responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4⁺ and CD8⁺ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4⁺ and CD8⁺ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4⁺ and CD8⁺ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4⁺ and CD8⁺ T cell responses.     

CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4⁺ CD8⁺ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8⁺ T cells and MHV68 epitope specific CD8⁺ T cells to the lungs—despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4⁺ CD8⁺ T cell expansion, eliminated MHV68-induced fibrosis—further implicating the activated Vβ4⁺ CD8⁺ T cell population in the induction of fibrosis. We further addressed the role that CD8⁺ T cells play in the induction of fibrosis by depleting CD8⁺ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4⁺ CD8⁺ T cells as mediators of fibrotic disease in IFNγR-/- mice.     

CD83+CCR7+ NK cells induced by interleukin 18 by dendritic cells promote experimental autoimmune uveitis

Natural killer (NK) cells have been reported to play a pathological role in autoimmune uveitis. However, the mechanisms regarding NK cells in uveitis and factors that affect NK‐cell activation in this condition remain unclear. Here, we report that the number of CD3^‐NK1.1⁺CD83⁺CCR7⁺ cells is increased in the inflamed eyes within a mouse model of experimental autoimmune uveitis (EAU), and these cells express elevated levels of NKG2D, CD69 and IFN‐γ. Adoptively transferring CD83⁺CCR7⁺NK cells aggravates EAU symptoms and increases the number of CD4⁺IFN‐γ⁺T cells and dendritic cells (DCs) within the eye. These CD83⁺CCR7⁺NK cells then promote the maturation of DCs and IFN‐γ expression within T cells as demonstrated in vitro. Furthermore, IL‐18, as primarily secreted by DCs in the eyes, is detected to induce CD83⁺CCR7⁺NK cells. In EAU mice, anti‐IL‐18R antibody treatment also decreases retinal tissue damage, as well as the number of infiltrating CD83⁺CCR7⁺NK cells, T cells and DCs in the inflamed eyes and spleens of EAU mice. These results suggest that CD83⁺CCR7⁺NK cells, as induced by IL‐18 that primarily secreted by DCs, play a critical pathological role in EAU. Anti‐IL‐18R antibody might serve as a potential therapeutic agent for uveitis through its capacity to inhibit CD83⁺CCR7⁺NK cells infiltration.     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4⁺ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4⁺ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4⁺ T cells leading to enhanced transmigration of CXCR4⁺ total CD4⁺ T cells and CXCR3⁺ effector/memory CD4⁺ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4⁺ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4⁺ T-cell transmigration in vitro as well as migration of CD4⁺ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3⁺ CD4⁺ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4⁺ T-cell populations during immune surveillance and liver inflammation.     

TRAIL suppresses tumor growth in mice by inducing tumor-infiltrating CD4+CD25+ Treg apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) but did not affect CD4⁺CD25⁺Foxp3⁺ Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4⁺CD25⁺Foxp3⁺ Tregs, but not of CD4⁺CD25^? T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4⁺CD25⁺Foxp3⁺ Tregs, but not on naïve CD4⁺CD25⁺Foxp3⁺ Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4⁺CD25⁺Foxp3⁺ Tregs but also increased the number of tumor-specific CD8⁺ CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.     

CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

CD4+NKG2D+ T Cells Exhibit Enhanced Migratory and Encephalitogenic Properties in Neuroinflammation

Migration of encephalitogenic CD4⁺ T lymphocytes across the blood-brain barrier is an essential step in the pathogenesis of multiple sclerosis (MS). We here demonstrate that expression of the co-stimulatory receptor NKG2D defines a subpopulation of CD4⁺ T cells with elevated levels of markers for migration, activation, and cytolytic capacity especially when derived from MS patients. Furthermore, CD4⁺NKG2D⁺ cells produce high levels of proinflammatory IFN-γ and IL-17 upon stimulation. NKG2D promotes the capacity of CD4⁺NKG2D⁺ cells to migrate across endothelial cells in an in vitro model of the blood-brain barrier. CD4⁺NKG2D⁺ T cells are enriched in the cerebrospinal fluid of MS patients, and a significant number of CD4⁺ T cells in MS lesions coexpress NKG2D. We further elucidated the role of CD4⁺NKG2D⁺ T cells in the mouse system. NKG2D blockade restricted central nervous system migration of T lymphocytes in vivo, leading to a significant decrease in the clinical and pathologic severity of experimental autoimmune encephalomyelitis, an animal model of MS. Blockade of NKG2D reduced killing of cultivated mouse oligodendrocytes by activated CD4⁺ T cells. Taken together, we identify CD4⁺NKG2D⁺ cells as a subpopulation of T helper cells with enhanced migratory, encephalitogenic and cytotoxic properties involved in inflammatory CNS lesion development.     

Characterization of cells containing factor XIII subunita in benign and malignant buccal lesions

Summary In the present study, the distribution pattern and characteristics of cells containing Factor XIII subunita (FXIII A) have been studied in benign and malignant lesions of human buccal mucosa. Tissues from four irritation fibromas and three squamous cell carcinomas were studied by means of double immunofluorescent staining techniques in which the detection of FXIII A was combined with a reaction with CD14 (recognizing a monocyte/macrophage differentiation marker antigen), Mac 387 (reacting with a special subset of macrophages), anti-HLA-DR, Ki-M7 (labelling phagocytosing macrophages) or Ki-67 (visualizing a nuclear antigen associated with cell proliferation) monoclonal antibodies. FXIII A was detected in cells of the connective tissue stroma in both benign and malignant buccal lesions. The number of these FXIII A-reactive cells (FXIII A⁺ cells) increased considerably in the tumour tissues, in particular in those surrounding tumour cell clusters. FXIII A⁺ cells scattered in the fibromatous tissues were spindle-shaped, whereas in the tumour stroma, large stellate cells predominated, and round cells were likewise labelled around blood vessels. FXIII A⁺ cells were labelled with CD14 and Ki-M7 in both fibromatous and tumoural buccal mucosa; however, they failed to show any reaction with Ki-67. FXIII A⁺ cells accumulated in the tumour stroma reacted for HLA-DR as well. These results indicate that in both the benign and malignant buccal lesions FXIII A is contained in a subpopulation of tissue macrophages, which represents a monocyte-derived (CD14⁺) and phagocytosing (KiM7⁺) cell population. The accumulation of the FXIII A⁺ cells in the tumour stroma is believed to be a result of direct migration from the circulating blood. The FXIII A⁺ cells of the tumour stroma may be actively involved in both antigen presentation and matrix remodelling during tumour progression.     

Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice

Indirubin, a traditional Chinese medicine, is used to treat autoimmune diseases in clinics. However, the effects of indirubin on the immunosuppressive CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) have not been addressed. Thus, we aimed to investigate the effects of indirubin on CD4⁺CD25⁺Treg cells in immune thrombocytopenia (ITP) CBA mice, which were established by immunization with Wistar rat platelets. 50 mg/kg indirubin treatment daily for 4 weeks significantly decreased anti-platelet antibody production and prevented the decrease of platelets caused by immunization in ITP mice. Consistently, indirubin significantly enhanced the percentage and cell number of CD4⁺CD25⁺Foxp3⁺Treg cells in the peripheral blood, spleen and lymph nodes. We also observed a significant increase of the frequency and cell number of CD4⁺CD25⁺Foxp3⁺Treg cells in the thymus upon indirubin treatment. Furthermore, CD4⁺CD25⁺Treg cells from indirubin-treated mice showed similar immunosuppression on T effector cells as compared to those from control mice. Altogether, indirubin ameliorates ITP by enhancing CD4⁺CD25⁺Foxp3⁺Treg cell level with preserving immunosuppressive function.