Development and composition of lymphoid lesions in the spleens of Marek's disease virus-infected chickens: Association with virus spread and the pathogenesis of Marek's disease

Changes in lymphocyte distribution in spleens of Marek's disease virus (MDV) infected White Leghorn chickens of line 72 (MD susceptible) and line 61 (MD resistant) were studied by immunocytochemistry. Lymphocytes expressing the MDV antigen pp38 (predominantly B cells) were detected from 4 to 6 days post-inoculation (d.p.i.) but not at or after 8 d.p.i., and were more numerous in line 72. In line 61, infection resulted in depletion of B lymphocytes and an increase in T lymphocytes from 3 to 6 d.p.i., but no change in distribution of these cells. From 8 d.p.i., the B-dependent tissue began to recover and the T cells decreased in number. In line 72, infection caused a dramatic change in lymphocyte distribution, with formation of 'lymphoid lesions'. Diffuse, irregular patches of B lymphocytes, around the capillaries, became surrounded by large aggregates of TCRαβ1⁺ CD8⁺ and CD4⁺ lymphocytes, bordered by a band of TCRγδ⁺ lymphocytes. From 8 d.p.i., the B-dependent areas partially recovered, while TCRαβ1⁺ CD4⁺ and CD8⁺ lymphocytes, potentially transformed, became extensively scattered throughout the spleen. We conclude that in line 72, replication and spread of MDV is more efficient and T cell responses in early infection are greater, favouring the tumour stage of the disease.     

Hepatitis B e-antigen Persistency is Associated with the Properties of HBV-Specific CD8 T Cells in CHB Patients

To investigate the correlation between the HBeAg and the properties of HBV-specific CD8 T cells, as well as liver injury, serum HBV markers, liver histology, the frequency and phenotypic characteristics of CD4⁺CD25⁺ Treg and HBV-pentamer⁺ CD8 T cells were measured. No significant differences between the median serum ALT levels, the frequency and Foxp3 expression of CD4⁺CD25⁺ Treg, and liver fibrosis were observed. Higher HBV DNA levels in HBeAg⁺ patients were observed, while liver necroinflammation was more severe in HBeAg⁻ patients. Differences in HBV-pentamer⁺ T cell frequency were not significant, but increased PD-1 and CTLA-4 expression on HBV-specific CD8 T cells was seen in the HBeAg⁺ group. HBV-peptide stimulation with anti-PD-L1 and anti-CTLA-4 significantly increased the proliferation in PBMCs from both groups, but enhanced IFN-γ production only in the HBeAg⁺ patients. Therefore, HBeAg persistency in CHB patients probably increased the expression of PD-1 and CTLA-4 on HBV-specific CD8 T cells, which may be associated with the low intensity of T cell responses and high HBV DNA load.     

Anti-viral and anti-tumor effects induced by an attenuated Marek’s disease virus in CD4- or CD8-deficient chickens

Summary.  By vaccination with an attenuated Marek’s disease virus (MDV), strain CVI988, chickens are protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8⁺ T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4⁺ or CD8⁺ T cell-depleted chickens. These results suggest that specific CD8⁺ T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV. Received Februray 15, 1999 Accepted May 7, 1999     

Accumulation of CD5<Superscript>+</Superscript>CD19<Superscript>+</Superscript> B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4⁺ cells (70.3 %) than on CD8⁺ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19⁺ B lymphocytes (6.5 %), while CD5⁺CD19⁺ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5⁺CD19⁺ B cells (16.5 %) than on CD19⁺ B cells (3.5 %) and on CD4⁺ T cells (20 %) than on CD8⁺ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5⁺CD19⁺ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.     

Re-isolation of Marek’s disease virus from T cell subsets of vaccinated and non-vaccinated chickens

Summary.  To know the effect of Marek’s disease (MD) vaccines, we analyzed the distribution of MD virus (MDV) among T cell subsets from chickens vaccinated or non-vaccinated with MD vaccine and subsequently challenged with a virulent MDV. The challenged MDV was reisolated preferentially from CD4⁺ T cells, and the average titers of challenged MDV rescued were significantly lower in vaccinated chickens compared to that of non-vaccinated chickens. In addition, it was also shown that different serotypes of MDV, CVI988 and SB-1, have remarkable difference in recovery rates of viruses from CD4+ and CD8+ T cells, though both CVI988 and SB-1 can reduce the infection rates of virulent MDV to splenocytes. Received April 14, 1998 Accepted August 15, 1998     

Soluble PD-1 rescues the proliferative response of simian immunodeficiency virus-specific CD4 and CD8 T cells during chronic infection

Phenotypic and functional studies of the programmed death-1 (PD-1) molecule on CD4⁺ and CD8⁺ T cells were performed on peripheral blood mononuclear cells from uninfected and simian immunodeficiency virus (SIV)-infected rhesus macaques. These data demonstrated a rapid upregulation of PD-1 expression on tetramer-positive CD8⁺ T cells from MamuA.01⁺ SIV-infected macaques upon infection. Upregulation of PD-1 on total CD8⁺ T cells was not detectable. In contrast, CD4⁺ T-cell PD-1 expression was markedly higher in total CD4⁺ T cells during chronic, but not acute, infection and there was a correlation between the level of PD-1 expression on naive and central memory CD4⁺ T cells and the levels of viral loads. Such association was emphasized further by a marked decrease of PD-1 expression on tetramer-positive CD8 T cells as well as on CD4⁺ T cells on longitudinal samples collected before and after the initiation of antiretroviral therapy and downregulation of viral replication in vivo. Cloning of PD-1 and its two ligands from several non-human primate species demonstrated > 95% conservation for PD-1 and PD-L2 and only about 91% homology for PD-L1. Functional studies using soluble recombinant PD-1 protein or PD-1–immunoglobulin G fusion proteins induced marked increases in the SIV-specific proliferative responses of both CD4⁺ and CD8⁺ T cells from rhesus macaques. The results of these studies serve as a foundation for future in vivo trials of the use of rMamu-PD-1 to potentially enhance and/or restore antiviral immune responses in vivo.     

B7-H1 on myeloid-derived suppressor cells in immune suppression by a mouse model of ovarian cancer

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and are associated with immune suppression. Here, we described high level of expression of B7-H1 (CD274), PD-1 (CD279) and CTLA4 (CD152) by Gr-1⁺CD11b⁺ MDSCs obtained from both ascites and spleens of mice bearing the 1D8 ovarian carcinoma, whereas B7-DC (CD273), CD40 and CD86 were absent. In contrast, B7-H1, PD-1 and CTLA-4 expression was not detected on Gr-1⁺CD11b⁺ cells from naive mice. Expression of B7-H1 by Gr-1⁺CD11b⁺ cells from naive mice could be induced by co-culture with 1D8 ovarian carcinoma cells. Gr-1⁺CD11b⁺ cells derived from 1D8 tumor-bearing mice markedly suppressed antigen-specific immune responses, whereas Gr-1⁺CD11b⁺ cells from naive mice did not. siRNA-mediated knockdown of B7-H1 in Gr-1⁺CD11b⁺ cells of 1D8 tumor-bearing mice alleviated suppression of antigen-specific immune responses. Suppression of antigen-specific immune responses via B7-H1 on Gr-1⁺CD11b⁺ myeloid cells was mediated by CD4⁺CD25⁺ Foxp3⁺ T regulatory cells and required PD-1. Antibody blockade of either B7-H1 or PD-1 retarded the growth of 1D8 tumor in mice. This suggests that expression of B7-H1 on Gr-1⁺CD11b⁺ myeloid cells triggered by the 1D8 mouse model of ovarian carcinoma suppresses antigen-specific immunity via interaction with PD-1 on CD4⁺CD25⁺ Foxp3⁺ regulatory T cells.     

Systemic Immunomodulatory Effects of Combinatorial Treatment of Thalidomide and Dexamethasone on T Cells and Other Immune Cells

PurposeIn organ transplantation, the need for immune modulation rather than immune suppression has been emphasized. In this study, we investigated whether combinatorial treatments of with thalidomide (TM) and dexamethasone (DX) might be new approaches to induce systemic immunomodulation on T cells and other immune cells that regulate the expression of co-inhibitory molecules.Materials and MethodsNaïve splenic T cells from C57BL/6 mice were sort-purified and cultured in vitro for CD4⁺ T cell proliferation and regulatory T cell (Treg) conversion in the presence of TM or/and DX. Expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) in proliferated and converted T cells was quantified by flow cytometry. We also quantified in vivo expression of CTLA-4 and PD-1 on splenic CD4⁺ T cells and other immune cells isolated from TM- or/and DX-treated mice. Mixed lymphocytes reactions (MLR) were performed to evaluate the capacity of immune cells in carrying out immune responses.ResultsCTLA-4 expressions in effector T cells in vivo and in Tregs in vivo/vitro significantly increased upon TM/DX combinatorial treatment. Corresponding to increased CTLA-4 expression in T cells, the expression of ligand molecules for CTLA-4 significantly increased in splenic dendritic cells in TM/DX-treated groups. In addition, MLR results demonstrated that splenocytes isolated from TM/DX-treated mice significantly suppressed the proliferation of T cells isolated from other strains.ConclusionBased on these results, we suggest that TM/DX combinatorial treatments might be efficient immunomodulatory methods for regulating T cell immunity.     

Effects of cold stress and Salmonella Heidelberg infection on bacterial load and immunity of chickens

We analysed the effects of cold stress (19?±?1°C, 6?h /day, from the first to the seventh day of life) applied to specific pathogen free (SPF) chickens. On experimental Day 1 (ED1), chicks were divided into four groups: C (not infected and kept under thermoneutral condition); CS (not infected and cold stressed); PC (Salmonella Heidelberg (SH) infected and kept under thermoneutral condition) and PCS (SH infected and cold stressed). High concentrations of corticosterone were found in the cold stressed birds on ED7 and ED21, with a greater increase in birds of the PCS group. Stress or non-stressed SH-infected birds had high levels of norepinephrine on ED21. On ED21, an increased percentage and number of SH were found in birds of the PCS group. On ED7, a decrease in macrophages presenting MHCII, CD8⁺ and CD8⁺ γδ cells was observed in the chickens of the CS group. Decrease was observed in CD3⁺ cells in the birds of the PCS group and increase in macrophages presenting MHCII cells and of the CD4⁺/CD8⁺ ratio in chickens of the CS group on ED21. There was a decrease in CD8⁺ γδ cells in birds of the CS group on ED21 and in the CD3⁺ and CD8⁺cell numbers in chickens of the PCS group on ED21. Our results suggest that cold stress applied to chickens in the first 7 days of life increases both the hypothalamus pituitary adrenal axis and the sympathetic nervous system activities, leading to long-term immune cell dysfunction, thus allowing increased SH invasion and persistence within the birds’ body.     

Upregulation of Immune Checkpoint Molecules,PD-1 and LAG-3, on CD4+ and CD8+ T Cells after Gastric Cancer Surgery

Background

Surgery has been reported to suppress cell-mediated immunity; however, the detailed mechanisms responsible for this remain unclear. This study determined the expression of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in lymphocytes following surgery for gastric cancer.

Methods

LAG-3 and PD-1 expression on both CD4+ and CD8+ T cells obtained pre- and post-operatively from gastric cancer patients were evaluated by multicolor flow cytometry.

Results

The total lymphocyte count decreased rapidly from preoperative levels, reaching a minimum on postoperative day 1 and remaining significantly decreased on days 3 and 7. PD-1⁺CD4⁺ T cells significantly increased, reaching a maximum on postoperative day 1 and remaining significantly elevated on day 3. PD-1⁺CD8⁺ T cells significantly increased and reached a maximum on day 7 before returning to the preoperative level on day 30. There were no statistically significant differences in the frequency of LAG-3⁺CD4⁺ or LAG-3⁺CD8⁺ T cells after surgery. There were significant positive correlations between PD-1 and LAG-3 expression on both CD4⁺ andCD8⁺ T cells.

Conclusion

PD-1 and LAG-3 expression on both CD4⁺ and CD8⁺ T cells was up-regulated and might be related to impaired cell-mediated immunity after surgery for gastric cancer.     

Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes

CTLA-4 (CD152) is a T cell surface receptor with sequence homology to the co-stimulatory molecule CD28. The molecule, which is essential for the inhibitory regulation of the immune response, becomes transiently expressed on mature T cells after stimulation in vitro. In situ, CTLA-4⁺ T cells are enriched in the light zones of the germinal centers in human peripheral lymphoid organs. In this study we have studied expression of CTLA-4 in human thymus in situ. CTLA-4 was expressed on about one third of CD4⁺/CD8^–/CD1^– medullary thymocytes. CTLA-4 was acquired by a subset of immature (CD1⁺) thymocytes and lost from the mature (CD1^–) subpopulation within 48 h of cell culture, suggesting that the expression on medullary thymocytes is transient. The demonstration of CTLA-4 on a substantial subpopulation of mature CD4⁺ thymocytes adds a new dimension to the understanding of this important molecule. When contemplating application of anti-CTLA-4 for therapy its potential influence on T cell maturation has to be taken into account. Received: 5 January 1998     

LncRNA XLOC_003810 promotes T cell activation and inhibits PD-1/PD-L1 expression in patients with myasthenia gravis-related thymoma

This study aimed to investigate the effect of long non-coding RNA XLOC_003810 on the activation of CD4⁺ T cells and expression of PD-1/PD-L1 in patients with myasthenia gravis-related thymoma (MG-T). Thymus specimens and thymic mononuclear cells were obtained from MG and MG-T patients or cardiac surgery patients undergoing thoracotomy who were selected as negative controls (NC). XLOC_003810 expression was examined using quantitative real-time PCR (qRT-PCR). Frequency of CD4⁺ T cells and proportion of CD4⁺PD-1⁺ T cells and CD14⁺PD-L1⁺ monocytes were quantified by flow cytometry. The release of inflammatory cytokines was measured by qRT-PCR and enzyme-linked immunosorbent assay. Compared with the NC group, expression of XLOC_003810, frequency of CD4⁺ T cells and the production of inflammatory cytokines were increased in patients with MG and MG-T. XLOC_003810 overexpression significantly increased the frequency of CD4⁺ T cells, facilitated the production of inflammatory cytokines and decreased the proportion of CD4⁺PD-1⁺ T cells and CD14⁺PD-L1⁺ monocytes in the thymic mononuclear cells. In contrast, XLOC_003810 knockdown exerted the opposite effect. Together, XLOC_003810 promotes T cell activation and inhibits PD-1/PD-L1 pathway in patients with MG-T.     

Programmed death-1 (PD-1)-dependent functional impairment of CD4+ T cells in recurrent genital papilloma

Genital papilloma is caused by human papilloma virus (HPV) infection and recurs frequently. Although T cells are known to play a critical role in the control of HPV infection and papilloma development, the function and phenotype of these cells in the lesion remain to be elucidated. In the present study, we examined the function and phenotype of CD4⁺ T cells isolated from the lesions of primary (n = 9) and recurrent (n = 11) genital papillomas. In recurrent papillomas, the frequency of proliferating (Ki-67⁺) CD4⁺ T cells was significantly reduced compared with primary papillomas. Cytokine production was evaluated by intracellular cytokine staining in anti-CD3/anti-CD28-stimulated CD4⁺ T cells. CD4⁺ T cells from recurrent lesions showed impaired production of IL-2, IFN-γ, and TNF-α. Of interest, the frequency of cytokine-producing CD4⁺ T cells significantly correlated with the frequency of Ki-67⁺CD4⁺ T cells. We also studied expression of programmed death-1 (PD-1), a T-cell exhaustion marker. The frequency of PD-1⁺CD4⁺ T cells was significantly increased in recurrent lesions and inversely correlated with the frequency of cytokine-producing CD4⁺ T cells. The functional significance of PD-1 expression was determined in blocking assays with anti-PD-L1, which restored cytokine production of CD4⁺ T cells from recurrent lesions. Taken together, in recurrent genital papilloma lesions, proliferation, and cytokine production by CD4⁺ T cells are impaired and the PD-1/PD-L1 interaction is responsible for the functional impairment of CD4⁺ T cells.     

Apoptosis and CD8-down-regulation in the thymus of chickens infected with Marek's disease virus

Summary Marek's disease virus (MDV)-infected chickens show thymic atrophy during the acute phase of infection. We examined whether the thymic atrophy by MDV-infection was mediated by apoptosis. Apoptosis-specific DNA ladderings were clearly observed in thymocytes one week after MDV-infection. Histological and flow cytometry studies revealed that immature CD4⁺CD8⁺ thymocytes underwent apototic cell death. In addition, the expression level of CD8 molecules on both CD4^–CD8⁺ and CD4⁺CD8⁺ thymocyte populations was down-regulated in the infected chickens. These thymic changes might be involved in the pathogenesis of Marek's disease.     

Function of T regulatory type 1 cells is down-regulated and is associated with the clinical presentation of coronary artery disease

T regulatory type 1 (Tr1) cells can promote tolerance and suppress inflammation. Atherosclerosis may be induced by the proinflammatory activation of cells in the vasculature and the immune system. Hence, we wondered whether defects in Tr1 function were a contributing factor to coronary artery disease (CAD). Data showed that the frequency of IL-10⁺ Tr1 cells was significantly lower in CAD patients than in controls. Compared to healthy controls, Tr1 cells from CAD patients presented lower CTLA-4 but higher PD-1 expression, in addition to lower IL-10 secretion. When co-incubated with Tconv cells, the CD4⁺CD49b⁺LAG-3⁺CD45RO⁺ Tr1 cells presented IL-10-dependent inhibitory effects, and those from CAD patients presented significantly lower suppression capacity than those from healthy controls. Interestingly, the characteristics of Tr1 cells were associated with clinical features of CAD patients. The frequency of Tr1 cells and the IL-10 and LAG-3 expression by Tr1 cells were negatively correlated with the BMI of the CAD patients. In addition, the Tr1 frequency and the LAG-3 and CTLA-4 expression on Tr1 cells were lower in CAD patients with higher numbers of narrowed vessels. Together, these results suggest that in CAD, Tr1 cells present multiple defects, which are associated with the clinical presentation of the disease.     

Combination of pegylated interferon-alpha and nucleos(t)ide analogue treatment enhances the activity of natural killer cells in nucleos(t)ide analogue experienced chronic hepatitis B patients

A combination of pegylated interferon-alpha (peg-IFN-α) and nucleos(t)ides analogue (NA) therapy can effectively reduce hepatitis B surface antigen (HBsAg), especially in NA-experienced chronic hepatitis B (CHB) patients. However, the immune mechanism of this therapy is unclear. Forty NA-experienced CHB patients were enrolled into this study. The frequencies of peripheral blood natural killer (NK) cells, dendritic cells (DCs), CD4⁺ T cells, CD8⁺ T cells, T helper (Th) cells, regulatory T cells (T_reg), B cells and follicular T helper (Tfh) cells were evaluated by flow cytometry. Seven of the 40 patients converted to peg-IFN-α combined with NA treatment, while the other 33 continued to NA therapy. The decrease in HBsAg was more pronounced in the combination treatment group, and only patients receiving combination treatment achieved HBsAg loss. The frequency and absolute number of CD56^bright NK cells in the combination treatment group increased significantly compared with the NA treatment group, whereas the CD56^dim NK cells were decreased. In the NA treatment group, the proportions of CD4⁺ T_N, CD8⁺ T_N, CD19⁺ B and cytotoxic T lymphocyte antigen-4 (CTLA-4)⁺CD4⁺ T cells were increased, while the proportions of CD4⁺ T_EM, CD8⁺ T_EM, CD25⁺CD4⁺ T_reg, CD25^highCD4⁺ T_reg, CD127^lowCD25⁺ T_reg, programmed cell death 1 (PD-1)⁺CD4⁺ T, PD-1⁺CD8⁺ T, CTLA-4⁺CD8⁺ T, CCR4⁺CD25⁺ T_reg and CCR4⁺CD25^high T_reg cells were decreased after therapy. For NA-experienced CHB patients who achieved low HBsAg levels, combination treatment is more likely to result in HBsAg decline and HBsAg clearance by increasing the activity of CD56^brightNK cells.