Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Compensatory T-Cell Regulation in Unaffected Relatives of SLE Patients, and Opposite IL-2/CD25-Mediated Effects Suggested by Coreferentiality Modeling

In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.

Broadened T-cell Repertoire Diversity in ivIg-treated SLE Patients is Also Related to the Individual Status of Regulatory T-cells

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4 + Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density.

Short Peptide Vaccine Induces CD4+ T Helper Cells in Patients with Different Solid Cancers

Previous cancer vaccination trials often aimed to activate CD8(+) cytotoxic T-cell (CTL) responses with short (8-10mer) peptides and targeted CD4(+) helper T cells (TH) with HLA class II-binding longer peptides (12-16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8(+) T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4(+) TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4- and HLA-DR-restricted TH1 cells. Some short peptide-reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4(+) T-cell repertoire in many patients, facilitating a strong combined CD4(+)/CD8(+) T-cell response. Cancer Immunol Res; 4(1); 18-25. ©2015 AACR.

Lymphocytes and mast cells,

Mode of access: Internet.

Mapping the determinants in owl monkey CD4 and CCR5 that allow entry of early-stage HIV-1 Env variants

Thesis (Master's)--University of Washington, 2016-06

Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine

Advanced metastatic melanoma is incurable by standard treatments, but occasionally responds to immunotherapy. Recent trials using dendritic cells (DC) as a cellular adjuvant have concentrated on defined peptides as the source of antigens, and rely on foreign proteins as a source of help to generate a cell-mediated immune response. This approach limits patient accrual, because currently defined, non-mutated epitopes are restricted by a small number of human leucocyte antigens. It also fails to take advantage of mutated epitopes peculiar to the patient's own tumour, and of CD4(+) T lymphocytes as potential effectors of anti-tumour immunity. We therefore sought to determine whether a fully autologous DC vaccine is feasible, and of therapeutic benefit. Patients with American Joint Cancer Committee stage IV melanoma were treated with a fully autologous immunotherapy consisting of monocyte-derived DC, matured after culture with irradiated tumour cells. Of 19 patients enrolled into the trial, sufficient tumour was available to make treatments for 17. Of these, 12 received a complete priming phase of six cycles of either 0.9X10(6) or 5X10(6) DC/intradermal injection, at 2-weekly intervals. Where possible, treatment continued with the lower dose at 6-weekly intervals. The remaining five patients could not complete priming, due to progressive disease. Three of the 12 patients who completed priming have durable complete responses (average duration 3 5 months +), three had partial responses, and the remaining six had progressive disease (WHO criteria). Disease regression was not correlated with dose or with the development of delayed type hypersensitivity responses to intradermal challenge with irradiated, autologous tumour. However, plasma S-100B levels prior to the commencement of treatment correlated with objective clinical response (P = 0.05) and survival (log rank P < 0.001). The treatment had minimal side-effects and was well tolerated by all patients. Mature, monocyte-derived DC preparations exposed to appropriate tumour antigen sources can be reliably produced for patients with advanced metastatic melanoma, and in a subset of those patients with lower volume disease their repeated administration results in durable complete responses.

A major quantitative trait locus for CD4-CD8 ratio is located on chromosome 11

CD4-CD8 ratio is an important diagnostic measure of immune system functioning. In particular, CD4-CD8 ratio predicts the time taken for progression of HIV infection to acquired immune deficiency syndrome (AIDS) and the long-term survival of AIDS patients. To map genes that regulate differences between healthy individuals in CD4-CD8 ratio, we typed 757 highly polymorphic microsatellite markers at an average spacing of similar to5 cM across the genome in 405 pairs of dizygotic twins at ages 12, 14 and 16. We used multipoint variance components linkage analysis to test for linkage between marker loci and CD4-CD8 ratio at each age. We found suggestive evidence of linkage on chromosome 11p in 12-year-old twins (LOD=2.55, P=0.00031) and even stronger evidence of linkage in the same region at age 14 (LOD 3.51, P=0.00003). Possible candidate genes include CD5 and CD6, which encode cell membrane proteins involved in the positive selection of thymocytes. We also found suggestive evidence of linkage at other areas of the genome including regions on chromosomes 1, 3, 4, 5, 6, 12, 13, 15, 17 and 22.

Virus-specific CD8+ T lymphocytes within the normal human liver

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (Valpha24/Vbeta11) was also observed, a finding which may be relevant to the regulation of the antiviral population:.

Immunohistological analysis of Tannerella forsythia-induced lesions in a murine model

Tannerella forsythia has been implicated as a defined periodontal pathogen. In the present study a mouse model was used to determine the phenotype of leukocytes in the lesions induced by subcutaneous injections of either live (group A) or nonviable (group B) T. forsythia. Control mice (group C) received the vehicle only. Lesions were excised at days 1, 2, 4, and 7. An avidin-biotin immunoperoxidase method was used to stain infiltrating CD4(+) and CD8(+) T cells, CD14(+) macrophages, CD19(+) B cells, and neutrophils. Hematoxylin and eosin sections demonstrated lesions with central necrotic cores surrounded by neutrophils, macrophages and lymphocytes in both group A and group B mice. Lesions from control mice exhibited no or only occasional solitary leukocytes. In both groups A and B, neutrophils were the dominant leukocyte in the lesion 1 day after injection, the numbers decreasing over the 7-day experimental period. There was a relatively low mean percent of CD4(+) and CD8(+) T cells in the lesions and, whereas the percent of CD8(+) T cells remained constant, there was a significant increase in the percent of CD4(+) T cells at day 7. This increase was more evident in group A mice. The mean percent of CD14(+) macrophages and CD19(+) B cells remained low over the experimental period, although there was a significantly higher mean percent of CD19(+) B cells at day 1. In conclusion, the results showed that immunization of mice with live T. forsythia induced a stronger immune response than nonviable organisms. The inflammatory response presented as a nonspecific immune response with evidence of an adaptive (T-cell) response by day 7. Unlike Porphyromonas gingivalis, there was no inhibition of neutrophil migration.

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autommume encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4(+) T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4(+), CD8(+), CD14(+) (monocytes) and CD56(+) NK cells but not to CD19(+) B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn 10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn 10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.

Reduction of β-Endorphin-Containing Immune Cells in Inflamed Paw Tissue Corresponds with a Reduction in Immune-Derived Antinociception: Reversible by Donor Activated Lymphocytes

The functional integrity of the immune system is essential for peripheral antinociception. Previous studies have demonstrated that immune cells elicit potent antinociception in inflamed tissues and that corticotropin-releasing factor-induced antinociception is significantly inhibited in animals that have undergone cyclosporin A (CsA)-induced immunosuppression. In this study, we examined the effect of a single bolus of CsA on inflammatory nociception. CsA-treated rats had substantially increased nociception compared with nonimmunosuppressed rats, consistent with a reduction in circulating and infiltrating lymphocytes. Furthermore, CsA-treated rats had inhibition of corticotropin-releasing factor-induced immune-derived antinociception, which was dose-dependently reversed by IV injection of concanavalin A-activated donor lymphocytes (1.0-7.0 X 10(6) cells/0.1 mL). In conclusion, our findings provided further evidence that opioid-containing immune cells are essential for peripheral analgesia. It is evident from these findings that control of inflammatory pain relies heavily on a functioning immune system.

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes

The efficient in vitro expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8(+) cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

Analysis of the effect of different NKT cell subpopulations on the activation of CD4 and CD8 T cells, NK cells, and B cells

Objective. NKT cells have diverse immune regulatory functions including activation of cells involved in Th1- and Th2-type immune activities. Most previous studies have investigated the functions of NKT cells as a single family but more recent evidence indicates the distinct functional properties of NKT cell subpopulation. This study aims to determine whether NKT cell subpopulations have different stimulatory activities on other immune cells that may affect the outcome of NKT cell-based immunotherapy. Methods. NKT cells and NKT cell subpopulations (CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(+)) were cocultured with PBMC and their activities on immune cells including CD4(+) and CD8(+) T cells, NK cells, and B cells were assessed by flow cytometry. The production of cytokines in culture was measured by enzyme-linked immunsorbent assay. Results. The CD4(+)CD8(-) NKT cells demonstrated substantially greater stimulatory activities on CD4(+) T cells, NK cells, and B cells than other NKT cell subsets. The CD4(-)CD8(+) NKT cells showed the greatest activity on CD8(+) T cells, and were the only NKT cell subset that activated these immune cells. The CD4(-)CD8(-) NKT cells showed moderate stimulatory activity on CD4(+) T cells and the least activity on other immune cells. Conclusion. The results here suggest that NKT cell subpopulations differ in their abilities to stimulate other immune cells. This highlights the potential importance of manipulating specific NKT cell subpopulations for particular therapeutic situations and of evaluating subpopulations, rather than NKT cells as a group, during investigation of a possible role of NKT cells in various disease settings. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.