The Ratios of CD8+ T Cells to CD4+CD25+ FOXP3+ and FOXP3- T Cells Correlate with Poor Clinical Outcome in Human Serous Ovarian Cancer

Ovarian cancer is an immune reactive malignancy with a complex immune suppressive network that blunts successful immune eradication. This suppressive microenvironment may be mediated by recruitment or induction of CD4⁺ regulatory T cells (Tregs). Our study sought to investigate the association of tumor-infiltrating CD4⁺CD25⁺FOXP3⁺ Tregs, and other immune factors, with clinical outcome in serous ovarian cancer patients. We performed immunofluorescence and quantification of intraepithelial tumor-infiltrating triple positive Tregs (CD4⁺CD25⁺FOXP3⁺), as well as CD4⁺CD25⁺FOXP3^-, CD3⁺ and CD8⁺ T cells in tumor specimens from 52 patients with high stage serous ovarian carcinoma. Thirty-one of the patients had good survival (i.e. > 60 months) and 21 had poor survival of < 18 months. Total cell counts as well as cell ratios were compared among these two outcome groups. The total numbers of CD4⁺CD25⁺FOXP3⁺ Tregs, CD4⁺CD25⁺FOXP3^-, CD3⁺ and CD8⁺ cells were not significantly different between the groups. However, higher ratios of CD8⁺/CD4⁺CD25⁺FOXP3⁺ Treg, CD8⁺/CD4⁺ and CD8/CD4⁺CD25⁺FOXP3^- cells were seen in the good outcome group when compared to the patients with poor outcome. These data show for the first time that the ratios of CD8⁺ to both CD4⁺CD25⁺FOXP3⁺ Tregs and CD4⁺CD25⁺FOXP3^- T cells are associated with disease outcome in ovarian cancer. The association being apparent in ratios rather than absolute count of T cells suggests that the effector/suppressor ratio may be a more important indicator of outcome than individual cell count. Thus, immunotherapy strategies that modify the ratio of CD4⁺CD25⁺FOXP3⁺ Tregs or CD4⁺CD25⁺FOXP3^- T cells to CD8⁺ effector cells may be useful in improving outcomes in ovarian cancer.     

Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

Background

Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma.

Methodology/Principal Findings

Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25^highFOXP3⁺CD127^low putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4⁺ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8⁺granzyme B⁺ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4⁺ T cells in the tumor, while frequencies of CD4⁺CCR4⁺ lymphocytes were significantly increased.

Conclusions/Significance

This study shows that CCR4⁺CTLA4^hi Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols.     

Human CD8+ T lymphocytes can be divided into CD45RA+ and CD45RO+ cells with different requirements for activation and differentiation.

The functional distinction between CD45RA+ and CD45RO+ cells within the human CD4+ T cell subset is well established. This study was undertaken to investigate whether a similar division can be made within the CD8+ T cell population. A quantitative comparison was made of the requirements for activation and differentiation of CD8+CD45RA+ and CD8+CD45RO+ cells. Stimulation of T lymphocytes with anti-CD3 mAb immobilized at high-density induced strong proliferation and CTL activity in both CD45RA+ and CD45RO+ cells. Suboptimal TCR/CD3 triggering, in contrast, induced substantially higher levels of proliferation and CTL activity in CD8+CD45RO+ cells compared with their CD45RA+ counterparts. Lymphokine secretion (i.e., Il-2 and TNF-alpha) was under any condition more readily induced in CD8+CD45RO+ cells. Markedly, proliferation of both CD8+CD45RA+ and CD8+CD45RO+ T cells initiated by anti-CD3 mAb immobilized at high densities was not inhibited by addition of anti-CD25 mAb, in contrast to proliferation induced by suboptimal anti-CD3 mAb concentrations. These findings show that a functional division between CD45RA+ and CD45RO+ T cells with distinct requirements for activation and differentiation may also be made in the CD8+ subset.     

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

Prognostic Impact of FoxP3+ Regulatory T Cells in Relation to CD8+ T Lymphocyte Density in Human Colon Carcinomas

Background

T-lymphocyte infiltration into colon carcinomas can influence clinical outcome, and interactions among T cell subsets may be more informative than either subset alone. Our objective was to examine the prognostic impact of tumor-infiltrating FoxP3⁺ regulatory T cells (Tregs) in relation to cytotoxic CD8⁺ T lymphocytes in patients with colon carcinomas characterized by DNA mismatch repair (MMR) status who participated in adjuvant chemotherapy trials.

Methods

FoxP3⁺ and CD8⁺ densities in tumor epithelial and stromal compartments were analyzed by immunohistochemistry and quantified in resected, stage II and III colonic carcinomas (N = 216). Immune marker density was dichotomized at the median and categorized as high vs low. MMR status was classified as MMR deficient (dMMR) or proficient (pMMR). Cox models were adjusted for age, stage, and tumor grade.

Results

The density of FoxP3+ infiltration was similar in tumor stroma and epithelia, whereas CD8+ was higher in stroma. The prognostic impact of FoxP3+ and CD8+ T cell infiltration was stronger in stroma vs epithelia, and the density of each marker in stroma was independently associated with improved overall survival (OS). However, the impact of FoxP3+ on survival was dependent upon CD8+ density (P interaction  = .040). Among CD8+_low tumors, FoxP3+_high cases had significantly improved OS compared to FoxP3+_low cases after adjustment for covariates (hazard ratio 0.43; 95% confidence interval 0.19 to 0.95; P = .030). In contrast, FoxP3+ was not prognostic among CD8+_high tumors. FoxP3+ remained prognostic in CD8+_low tumors after further adjustment for MMR or BRAF ^V600E mutation status. Additionally, these immune markers identified a pMMR subgroup with a similarly favorable OS as for dMMR tumors.

Conclusions

The prognostic impact of FoxP3+ and CD8+ T cell density are inter-dependent, whereby FoxP3+ exerts a favorable influence on survival only in colon cancers with low CD8+ infiltration.     

Development of Virus-Specific CD4+ and CD8+ Regulatory T Cells Induced by Human Herpesvirus 6 Infection

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4⁺ and CD8⁺ HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4⁺ and CD8⁺ Treg cells secreted gamma interferon (IFN-γ) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4⁺ effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.     

CD4+FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome

Objective

We investigated whether the frequency, phenotype, and suppressive function of CD4⁺FOXP3⁺ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls.

Design and Methods

Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4–35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (–) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4⁺CD25^bright were co-cultured with autologous CD4⁺CD25⁻ target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function.

Results

Despite a lower frequency of CD4⁺ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3⁺ Tregs among CD4⁺ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3⁺, and CCR4⁺CCR6⁺ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4⁺CD25⁻ T cells was significantly impaired in the TS (–) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group.

Conclusions

The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4⁺ T cells.     

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells. When cultured together, CCR4(+)CCR6(+)Th17 cells suppressed the lytic function, proliferation, and cytokine secretion of both Ag-specific and CD3/CD28/CD2-stimulated autologous CD8(+) T cells. In contrast, CCR4(-)CCR6(+) CD4(+) T cells, which also secrete IL-17, did not affect the CD8(+) T cells. Suppression of CD8(+) T cells by CCR4(+)CCR6(+)Th17 cells was partially dependent on TGF-β, because neutralization of TGF-β in cocultures reversed their suppressor function. In addition, we also found an increase in the frequency of CCR4(+)CCR6(+), but not CCR4(-)CCR6(+) Th17 cells in peripheral blood of hepatocellular carcinoma patients. Our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in hepatocellular carcinoma. This has important implications when studying the mechanisms of carcinogenesis, as well as designing effective immunotherapy protocols for patients with cancer.     

Decreased effector memory CD45RA+ CD62L- CD8+ T cells and increased central memory CD45RA- CD62L+ CD8+ T cells in peripheral blood of rheumatoid arthritis patients

Although a role for CD8+ T cells in the pathogenesis of rheumatoid arthritis (RA) has been suggested, the precise nature of their involvement is not fully understood. In the present study we examined the central and effector memory phenotypes of CD4+ and CD8+ T cells in the peripheral blood of patients with RA and systemic lupus erythematosus. Terminally differentiated effector memory CD45RA+CD62L-CD8+ T cells were significantly decreased in RA patients, whereas the central memory CD45RA-CD62L+ CD8+ T-cell population was increased as compared with levels in healthy control individuals. Na?ve and preterminally differentiated effector memory CD45RA-CD62L- CD8+ T cells did not differ between RA patients and control individuals. The CD45RA-CD62L+ central memory CD4+ T-cell subpopulation was increased in RA patients, whereas the na?ve and effector memory phenotype of CD4+ T cells did not differ between RA patients and control individuals. In patients with systemic lupus erythematosus the distribution of na?ve/memory CD4+ and CD8+ T cells did not differ from that in age- and sex-matched control individuals. These findings show that peripheral blood CD8+ T cells from RA patients exhibit a skewed maturation phenotype that suggests a perturbation in the homeostasis of these cells. The central memory CD45RA-CD62L+ CD4+ and CD8+ T-cell numbers were increased in RA, suggesting an accelerated maturation of na?ve T cells. The decreased numbers of terminally differentiated CD45RA+CD62L- effector memory CD8+ T cells in peripheral blood of RA patients may reflect increased apoptosis of these cells or enhanced migration of these cells to sites of inflammation, which may play a role in the pathogenesis of RA.     

Frequencies of circulating cytolytic,CD45RA+CD27-, CD8+ T lymphocytes depend on infection with CMV

Viral infections may cause serious disease unless the adaptive immune system is able to clear the viral agents through its effector arms. Recent identification and functional characterization of subpopulations of human CD8(+) T cells has set the stage to study the correlation between the appearance of particular subsets and common viral infections during childhood, i.e., EBV, CMV, varicella-zoster virus (VZV), and the attenuated measles-mumps-rubella (MMR) vaccine strains. In a cohort of 220 healthy children we analyzed lymphocytes and subpopulations of CD4(+) and CD8(+) T cells. The presence of the cytolytic CD45RA(+)CD27(-) subset of CD8(+) T cells correlated with prior CMV infection as defined by seroconversion (p < 0.0001). The number of this CD8(+) T cell subset remained stable during follow-up over 3 years in 40 children. The CD45RA(+)CD27(-) subset of CD8(+) T cells first appeared during acute CMV infection and subsequently stabilized at an individual set-point defined by age and immunocompetence. The functional importance of these cells in CMV surveillance was reflected by their increased numbers in immunosuppressed pediatric kidney transplant patients. Preferential expansion of CD8(+)CD45RA(+)CD27(-) cytolytic T cells seems unique for CMV.     

Interleukin-7 influences FOXP3+CD4+ regulatory T cells peripheral homeostasis

Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance.     

Glioma-Derived ADAM10 Induces Regulatory B Cells to Suppress CD8+ T Cells

CD8⁺ T cells play an important role in the anti-tumor activities of the body. The dysfunction of CD8⁺ T cells in glioma is unclear. This study aims to elucidate the glioma cell-derived ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in the suppression of CD8⁺ effector T cells by the induction of regulatory B cells. In this study, glioma cells were isolated from surgically removed glioma tissue and stimulated by Phorbol myristate acetage (PMA) in the culture. The levels of ADAM10 in the culture were determined by enzyme-linked immunosorbent assay. Immune cells were assessed by flow cytometry. The results showed that the isolated glioma cells express ADAM10, which was markedly up regulated after stimulated with PMA. The glioma-derived ADAM10 induced activated B cells to differentiate into regulatory B cells, the later suppressed CD8⁺ T cell proliferation as well as the induced regulatory T cells, which also showed the immune suppressor effect on CD8⁺ effector T cell proliferation. In conclusion, glioma cells produce ADAM10 to induce Bregs; the latter suppresses CD8⁺ T cells and induces Tregs.