Activated but not resting regulatory T cells accumulated in tumor microenvironment and correlated with tumor progression in patients with colorectal cancer

Activated T regulatory (T_reg) cells are potent suppressors that mediate immune tolerance. We investigated the relationship between activated T_reg cells and the progression of human colon cancer. We designed a cross‐sectional study of CD4⁺Foxp3⁺ T cells from peripheral blood, primary tumor and nontumor colon tissue of 42 patients with colon cancer and correlated the percentages of different subgroups of T_reg cells with colon cancer stage. The phenotypes, cytokine‐release patterns and suppression ability of these T_reg cells were analyzed. We found that T_reg cells increased significantly in both peripheral blood and cancer tissue. In addition, the T_reg cells expressed significantly lower levels of CCR7, CD62L and CD45RA in comparison to normal volunteers. Further dividing T_reg cells into subgroups based on Foxp3 and CD45RA expression revealed that both activated T_reg cells (Foxp3^hiCD45RA^?) and nonsuppressive T_reg cells (Foxp3^loCD45RA^?), but not resting T_reg cells (Foxp3^lowCD45RA⁺), increased in the peripheral blood and cancer tissue of patients with colon cancer. Only the activated T_reg cells expressed significantly higher levels of tumor necrosis factor receptor 2 and cytotoxic T‐cell antigen‐4. Activated T_reg cells, however, secreted significantly lower levels of effector cytokines (interleukin‐2, tumor necrosis factor‐α and interferon‐γ) than did resting T_reg cells and nonsuppressive cells upon ex vivo stimulation. Activated, but not resting, T_reg cells in cancer tissue correlated with tumor metastases. In summary, we confirmed that activated T_reg cells are a distinct subgroup with effector memory phenotype and fully functional regulatory activity against human colorectal cancer immunity.     

Dynamic changes in immune cell profile in head and neck squamous cell carcinoma: Immunomodulatory effects of chemotherapy

Tumor cells have evolved sophisticated means of escape from the host immune system. To date, several important immunological phenomena have been revealed in peripheral blood as well as within tumors. In the present study, we first investigated the proportion and activation status of peripheral immune regulatory cells and CD8⁺ T‐cell subsets in patients with head and neck squamous cell carcinoma (HNSCC) using a multicolor flow cytometer, and then evaluated how therapy with docetaxel, cisplatin, and 5‐fluorouracil modulated the immune cell profile in peripheral blood. The proportion of naïve T cells was lower and that of effector memory T cells (T_EM) was higher in HNSCC patients than in healthy donors. Moreover, the proportions of activated T_EM cells and effector T cells (T_EFF) were dramatically increased in patients with advanced stage disease. The proportion of regulatory T cells and CD14⁺HLA‐DR^? myeloid‐derived suppressor cells was elevated in HNSCC patients. Of note, after therapy, in addition to the transient reduction in immune regulatory cells, decreases in central memory T cells and increases in T_EFF cells were observed among CD8⁺ T‐cell subsets, suggesting differentiation from central memory T cells into T_EFF cells. Our results suggested that, despite the immunosuppressive status in HNSCC patients, tumor‐specific immune responses mediated by CD8⁺ T cells might be induced and maintained. Moreover, chemotherapy can trigger not only a transient reduction in immune regulatory cells but also further activation of CD8⁺ T cells.     

Synergistic effects of CTLA‐4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo

Anti‐CTLA‐4 monoclonal antibodies (mAb) that block the interaction of CTLA‐4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de‐repress tumor‐specific cellular immunity. Addition of the fully human anti‐CTLA‐4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead‐mediated elimination of CD4⁺ CD25⁺ regulatory T cells (T_reg) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4⁺ CD25⁺ T_reg and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T‐cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA‐4 expression in effector cells as a result of T_reg elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2^?/? IL‐2Rγ^?/? mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T_reg cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T‐cell compartment. In addition, T_reg depletion and CTLA‐4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV‐transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T_reg depletion.     

High frequencies of less differentiated and more proliferative WT1‐specific CD8+ T cells in bone marrow in tumor‐bearing patients: An important role of bone marrow as a secondary lymphoid organ

In tumor‐bearing patients, tumor‐associated antigen (TAA)‐specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti‐tumor immunity. Wilms’ tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan‐TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1‐specific CD8⁺ T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1‐specific CD8⁺ T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7⁺ CD45RA⁺), central memory (CCR7⁺ CD45RA?), effector‐memory (CCR7? CD45RA^?), and effector (CCR7? CD45RA⁺) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector‐memory and effector subsets of the WT1‐specific CD8⁺ T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide‐specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide‐specific proliferation also showed that WT1‐specific CD8⁺ T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor‐bearing patients. Preferential residence of WT1‐specific CD8⁺ T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1‐specific CD8⁺ T cells in BM, compared to those in PB. These results should provide us with an insight into WT1‐specific immune response in tumor‐bearing patients and give us an idea of enhancement of clinical response in WT1 protein‐targeted immunotherapy. (Cancer Sci 2010; 101: 848–854)     

Bone marrow contains melanoma-reactive CD8+ effector T cells and, compared with peripheral blood, enriched numbers of melanoma-reactive CD8+ memory T cells

Circulating melanoma-specific T cells can be frequently detected in patients with melanoma. Effective T-cell immunity and tumor surveillance, however, requires the presence of specific T cells in tissues populated by tumor cells. The bone marrow (BM) is a compartment frequently harboring micrometastatic tumor cells. Here, we compared directly ex vivo in peripheral blood (PB) and BM frequencies and differentiation phenotypes of T cells reactive with the melanoma-associated antigen tyrosinase and with autologous melanoma cells. Using intracellular cytokine and tetramer staining, we detected tyrosinase- and melanoma-reactive CD3+CD8+ T cells in the BM in similar or enhanced frequencies as in PB. Additional characterization of the differentiation subset using CD45RA and CCR7 revealed the presence of specific effector and memory T cells in the BM in all five patients analyzed. Remarkably, the frequency of tyrosinase- and melanoma-specific memory T cells was significantly increased in BM compared with PB. Thus, the BM may be an important compartment for tumor surveillance harboring a tumor-specific memory T-cell pool in addition to effector T cells.     

Correlation between CD4+CD25+Treg cells and CCR4 in nasopharyngeal carcinoma

Objective  

CD4⁺CD25⁺ T regulatory (T_reg) cells are a population of T cells which suppress an overactive immune system. CCR4 is a chemokine receptor involved in the recruitment of lymphocytes. Nasopharyngeal carcinoma (NPC) is resistant to immunosurveillance, owing to the increased number of tumor-infiltrating T_reg cells which are recruited to the tumor by CCR4.     

Memory CD4<Superscript>+</Superscript> T cell subsets in tumor draining lymph nodes of breast cancer patients: A focus on T stem cell memory cells

Background

The compartments of memory T cells play a fundamental role in the immune system by substantiating specific and acquired immunity. A new subset of memory cells, T stem cell memory (T_SCM) cells, with stem cell-like properties, a high capacity to proliferate, a long survival, and an ability to differentiate into all effector and memory cells has recently been introduced. In the present study, we aimed to determine the frequency of CD4⁺ T_SCM and other T memory cell subsets in tumor draining lymph nodes of breast cancer patients.

Materials and methods

Mononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with breast cancer (BC) and stained with fluorochrome conjugated anti-CD4, ?CCR7, ?CD45RO and -CD95 antibodies to detect different subtypes of memory cells in CD4⁺ lymphocyte populations. Data were acquired using a four-color FACSCalibur flow cytometer and analyzed using CellQuest Pro software.

Results

We found that >70% of CD4⁺ lymphocytes in draining lymph nodes of BC patients exhibited a memory phenotype of which 7.04 ± 1.04% had a T_SCM phenotype (CD4⁺CCR7⁺CD45RO^?CD95⁺). The frequency of T_SCM cells was significantly higher in tumor positive lymph nodes compared to tumor negative lymph nodes (p = 0.026) as well as among those patients who had at least one affected lymph node (p = 0.012). Moreover, we found that the total frequency of central memory T cells (T_CM) with a low expression of CD45RO was significantly higher among these patients. The percentage of CD45RO^Low T_CM cells was also found to increase with tumor progression from stage I to stage III (p = 0.020). On the other hand, we found that the percentage of CD95^Hi effector memory T cells (T_EM) was significantly decreased in involved lymph nodes (p = 0.009).

Conclusion

Our data suggest that following long-term exposure to putative tumor antigens, T_SCM cells proliferate to generate a pool of committed memory and effector T cells. As the tumor progresses, the immunosuppressive milieu induced by tumor cells may slow down the differentiation of CD45RO^Low T_CM cells to more functional sub-populations.

     

Good syndrome presenting with CD8+ T-Cell large granular lymphocyte leukemia

Good Syndrome is an adult-onset combined immunodeficiency defined by hypogammaglobulinemia, low or absent number of B cells, T cell deficiency and thymic tumor. We have characterized CD8+ T cells from a patient with Good syndrome that presented with CD8+T-cell large granular lymphocytic leukemia (LGL). Characterization of peripheral blood CD8+ T cells revealed that majority of CD8+ T cells were terminally differentiated effector memory phenotype (T_EMRA; CD8+CCR7-CD45RA+), and were PD-1^high (CD279), ICOS^low (CD278), and granzyme^high. Almost all CD8+ T cells were IFN-γ+. CD8 Treg (CD8+CD183+CCR7+CD45RA-) were decreased. T_EMRA phenotype along with CD279^high, demonstrates that these are exhausted CD8+ T cells. This phenotype along with CD278^low may also explain severe T cell functional deficiency in our patient. In the present patient, T-LGL appears to be a clonal expansion of CD279+granzyme+IFN-γ+CD8+T_EMRA cells. To best of our knowledge this is the first case of CD8+T-cell LGL leukemia associated with Good syndrome.     

Levels of circulating regulatory CD4+CD25+ T cells are decreased in breast cancer patients after vaccination with a HER2/neu peptide (E75) and GM-CSF vaccine

SummaryPurpose We are conducting clinical trials in breast cancer (BrCa) patients to test the HER2/neu peptide vaccine (E75). We have investigated the impact of this vaccine on circulating levels of regulatory T cells (T_reg) and the resulting effects on antitumor responses.Experimental design Twenty-two blood samples from healthy individuals and from 22 BrCa patients including pre- and post-vaccination samples from seven vaccinated HLA-A2⁺ patients were stained for CD4, CD25, and CD69 as well as CD8 and E75:HLA-A2 Ig dimer and quantified by flow cytometry. Cytotoxic activity against HER2/neu ⁺ tumors was measured by ⁵¹Cr-release. Serum from BrCa patients and normal subjects were analyzed for TGF-β levels.Results BrCa patients have a greater percentage of circulating T_reg (CD4⁺CD25⁺, 4.45% versus 2.96%; p = 0.007) than normal subjects. HLA-A2⁺ BrCa patients had more T_reg compared to the HLA-A2^– BrCa patients (CD4⁺CD25⁺, 5.63% versus 3.28%; p = 0.001). E75 vaccination increased circulating activated CD4⁺ T cells post-vaccination (CD4⁺CD69⁺, 1.23 versus 3.81%; p = 0.03). However, T_reg were significantly reduced after vaccination (CD4⁺CD25⁺, 5.31–1.81%; p < 0.0001). Furthermore, activated T_reg also decreased (CD4⁺CD25⁺CD69⁺, 0.23% versus 0.08%; p = 0.06). Importantly, post-vaccination decreases in T_reg were temporally associated with increased E75 vaccine-specific CD8⁺ T cells and corresponding HER2/neu ⁺ tumor cytotoxicity. Serum TGF-β levels were significantly elevated in BrCa patients compared to normals (3548 pg/ml versus 1007 pg/ml; p = 0.007). Four of seven vaccinated patients showed decreased serum TGF-β levels post-vaccination.Conclusions T_reg, are increased in BrCa patients along with serum levels of TGF-β. E75 vaccination resulted in CD4⁺ recruitment but was associated with a significant decrease in circulating T_reg and TGF-β levels in the majority of the vaccinated patients. Successful cancer vaccination strategies may require the alteration of complex immune interactions.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense     

Regulatory T cells predict the time to initial treatment in early stage chronic lymphocytic leukemia

BACKGROUND:

Early stage chronic lymphocytic leukemia is characterized by a highly variable course of disease. Because it is believed that regulatory T cells (T_regs) are potent suppressors of antitumor immunity, the authors hypothesized that increased T_regs may favor disease progression.

METHODS:

T_reg levels (cluster of differentiation 3 [CD3]‐positive, [CD4]‐positive, CD25‐positive, and CD127‐negative) in peripheral blood from 102 patients were analyzed by flow cytometry. Statistical analysis was used to evaluate correlations with clinical data.

RESULTS:

The relative T_reg numbers in CD4‐positive T cells were significantly greater in patients with chronic lymphocytic leukemia compared with the numbers in a control group of 170 healthy individuals (P = .001). Patients were divided into 2 groups using a median T_reg value of 9.7% (the percentage of CD4‐positive T cells). Patients with higher T_reg levels had a significantly shorter time to initial treatment (median, 5.9 years) compared with patients who had lower T_reg levels (median, 11.7 years; log‐rank P = .019). Furthermore, T_reg levels (the percentage of CD4‐positive T cells) had significant prognostic power to predict the time to initial treatment in univariate analysis (P = .023) and in multivariate Cox regression analysis that included the variables Rai stage, immunoglobulin heavy‐chain variable region gene mutational status, chromosomal aberrations, and CD38 expression (P = .028).

CONCLUSIONS:

Higher T_reg levels had significant and independent prognostic power for predicting the time to initial treatment in patients with low to intermediate stage chronic lymphocytic leukemia. Cancer 2011. © 2010 American Cancer Society.     

Enhanced suppressive capacity of tumor‐infiltrating myeloid‐derived suppressor cells compared with their peripheral counterparts

Although the main site of action for myeloid‐derived suppressor cells (MDSCs) is most likely the tumor microenvironment, so far the study of these cells has been largely restricted to spleen‐derived MDSCs. In this study, we compared the suppressive capacity of splenic and tumor‐derived MDSCs in different subcutaneous mouse tumor models. We investigated which suppressive mechanisms were involved. Finally, we investigated whether MDSCs and regulatory T cells (T_reg) cooperate in the suppression of T‐cell responses. In all models, splenic granulocytic MDSCs (grMDSC) strongly suppress CD4⁺ T‐cell proliferation while the suppressive effect on CD8⁺ T cells is less pronounced. Splenic monocytic MDSCs (moMDSC) have a lower suppressive capacity, compared to grMDSC, on both CD4⁺ and CD8⁺ T‐cell proliferation. Both grMDSC and moMDSC isolated from the tumor have a much stronger suppressive activity compared to MDSCs isolated from the spleen of tumor‐bearing mice, associated with a higher NO₂^? production by the tumor‐derived moMDSC and arginase activity for both subsets. The expression of CD80 is also elevated on tumor‐derived grMDSC compared with their peripheral counterparts. Direct contact with tumor cells is required for the upregulation of CD80 and CD80⁺ MDSCs are more suppressive than CD80^? MDSCs. Coculture of T_reg and MDSCs leads to a stronger suppression of CD8⁺ T‐cell proliferation compared to the suppression observed by T_reg or MDSCs alone. Thus, we showed that tumor‐infiltrating MDSCs possess a stronger suppressive capacity than their peripheral counterparts and that various suppressive mechanisms account for this difference.     

Generation and application of human induced‐stem cell memory T cells for adoptive immunotherapy

Adoptive T‐cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen‐specific stem cell memory T (T_SCM) cells is expected to overcome this shortcoming as T_SCM cells are close to naïve T cells, but are also highly proliferative, long‐lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into T_SCM‐like cells (iT_SCM) by coculturing with OP9 cells expressing Notch ligand, Delta‐like 1 (OP9‐hDLL1). Here we show the methodological parameters of human CD8⁺ iT_SCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti‐CD3/CD28 antibodies or by antigen‐presenting cells, human iT_SCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9‐hDLL1 cells, interleukin (IL)‐7 and IL‐15 (but not IL‐2 or IL‐21) could efficiently generate iT_SCM cells. Epstein–Barr virus‐specific iT_SCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed‐tumor model mice. Thus, adoptive T‐cell therapy with iT_SCM offers a promising therapeutic strategy for cancer immunotherapy.     

Tumor-induced changes in the phenotype of blood-derived and tumor-associated T cells of early stage breast cancer patients

Tumor‐induced immune suppression has mainly been studied in patients with advanced cancer. Despite the fact that they are most likely to benefit from immunotherapy, patients with early stage cancers were under‐represented in these studies. We analyzed blood and tumor‐derived T cells from patients with stage 1 (n = 20), stage 2 (n = 23) or stage 3 (n = 1) breast cancer and found that, even early stage tumors induced T cell differentiation. Breast cancer patients had significantly more circulating CD8+ memory and fewer CD8+ naïve T cells than healthy controls (n = 10). Up‐regulation of CD69 and PD1 on cancer patient T cells suggests previous activation, and increased expression of the chemokine receptors CCR5 and CXCR3 on CD8+ T cells indicates that their homing capacity differs from that of healthy individuals. Comparison of blood‐derived and tumor‐associated T cells from patients with different metastatic status and tumor grades revealed that tumor progression and aggressiveness seem to favor the expansion of memory T cells over naive T cells. We have previously shown that immunosuppression in this patient population is stronger in the tumor than in the blood. Here, we report signs of exhaustion, such as loss of CD28, on tumor‐associated as compared to blood‐derived CD8+ T cells, despite the fact that tumor‐associated T cells are predominantly effector memory cells and express high levels of CD69. The finding that the presence of a tumor potentially induces immunosenescence early during tumorigenesis indicates that efficient immunotherapy might be difficult even in patients with early stage cancer due to T cell exhaustion and tolerance.     

CD4+ T cells support polyfunctionality of cytotoxic CD8+ T cells with memory potential in immunological control of tumor

Polyfunctionality/multifunctionality of effector T cells at the single cell level has been shown as an important parameter to predict the quality of T cell response and immunological control of infectious disease and malignancy. However, the fate of polyfunctional CD8⁺ CTLs and the factors that control the polyfunctionality of T cells remain largely unknown. Here we show that the acquisition of polyfunctionality on the initial stimulation is a sensitive immune correlate of CTL survival and memory formation. CD8⁺ T cells with high polyfunctionality, assessed with γ‐interferon and tumor necrosis factor‐α production and surface mobilization of the degranulation marker CD107a, showed enhanced Bcl‐2 expression, low apoptosis, and increased CD127^highKLRG1^low memory precursor phenotype. Consistent with these observations, CD8⁺ T cells were found to acquire high frequency of cells with polyfunctionality when stimulated in conditions known to enhance memory formation, such as the presence of CD4⁺ T cells, interleukin (IL)‐2, or IL‐21. Utilizing T‐cell receptor (TCR) transgenic mouse‐derived CD8⁺ T cells that express a TCR specific for a tumor‐derived neoantigen, we showed that polyfunctional tumor‐specific CTLs generated in the presence of CD4⁺ T cells showed long persistence in vivo and induced enhanced tumor regression when adoptively transferred into mice with progressing tumor. Acquisition of polyfunctionality thus impacts CTL survival and memory formation associated with immunological control of tumor.     

Interleukin‐37 is highly expressed in regulatory T cells of melanoma patients and enhanced by melanoma cell secretome

Immune suppression is one of the 10 hallmarks of cancer. Interleukin‐37 (IL‐37), a member of the IL‐1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL‐37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL‐37 in age‐ and sex‐matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL‐37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL‐37 expression, we discovered that IL‐37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (T_reg) cells in healthy individuals, and that IL‐37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, T_reg cells from melanoma patients exhibited the highest IL‐37 gene expression levels. We provided evidence that melanoma‐conditioned media induces IL‐37 mRNA and protein expression in multiple lymphocyte populations, particularly in T_reg cells. We further confirmed that the IL‐1‐mediated secretome from human melanoma cells, specifically transforming growth factor‐β, induces IL‐37 mRNA expression in human T_reg cells. Our results suggest a potential immunosuppressive role for IL‐1 and IL‐37 in melanoma tumorigenesis. Highly elevated IL‐37 in specific lymphocyte populations could serve as a biomarker for tumor‐induced immunosuppression.     

Forkhead box P3 regulatory T cells coexisting with cancer associated fibroblasts are correlated with a poor outcome in lung adenocarcinoma

Recently, an association between tumor infiltrating Forkhead box P3 regulatory T cells (T_reg) and an unfavorable prognosis has been clinically shown in some cancers, but the mechanism of T_reg induction in the tumor microenvironment remains uncertain. The aims of the present study were to examine the relationship between T_reg and patient outcome and to investigate whether T_reg induction is influenced by the characteristics of cancer‐associated fibroblasts (CAF) in lung adenocarcinoma. The numbers of T_reg in both the tumor stroma and the tumor nest were counted in 200 consecutive pathological stage I lung invasive adenocarcinoma specimens. To examine whether the characteristics of CAF influence T_reg induction, we selected and cultured CAF from low T_reg and high T_reg adenocarcinoma. The number of T_reg was much higher in the stroma than in the nest (P < 0.01). Patients with high T_reg had a significantly poorer prognosis than those with low T_reg (overall survival: P = 0.03; recurrence‐free survival: P = 0.02; 5‐year overall survival: 85.4% vs 93.0%). Compared with the CAF from low T_reg adenocarcinoma, culture supernatant of the CAF from high T_reg adenocarcinoma induced more T_reg (P = 0.01). Also, CAF from high T_reg adenocarcinoma expressed significantly higher mRNA levels of transforming growth factor‐β (P = 0.01) and vascular endothelial growth factor (P = 0.01), both of which are involved in T_reg induction. Our studies suggest the possibility that CAF expressing immunoregulatory cytokines may induce T_reg in the stroma, creating a tumor‐promoting microenvironment in lung adenocarcinoma that leads to a poor outcome.     

Novel soluble HLA‐A2/MELAN‐A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma

Multimeric MHC I‐peptide complexes containing phycoerythrin‐streptavidin are widely used to detect and investigate antigen‐specific CD8+ (and CD4+) T cells. Because such reagents are heterogeneous, we compared their binding characteristics with those of monodisperse dimeric, tetrameric and octameric complexes containing linkers of variable length and flexibility on Melan‐A‐specific CD8+ T cell clones and peripheral blood mononuclear cells (PBMC) from HLA‐A*0201⁺ melanoma patients. Striking binding differences were observed for different defined A2/Melan‐A_26‐35 complexes on T cells depending on their differentiation stage. In particular, short dimeric but not octameric A2/Melan‐A_26‐35 complexes selectively and avidly stained incompletely differentiated effector‐memory T cells clones and populations expressing CD27 and CD28 and low levels of cytolytic mediators (granzymes and perforin). This subpopulation was found in PBMC from all six melanoma patients analyzed and proliferated on peptide stimulation with only modest phenotypic changes. By contrast influenza matrix_58‐66 ‐specific CD8+ PBMC from nine HLA‐A*0201⁺ healthy donors were efficiently stained by A2/Flu matrix_58‐61 multimers, but not dimer and upon peptide stimulation proliferated and differentiated from memory into effector T cells. Thus PBMC from melanoma patients contain a differentiation defective sub‐population of Melan‐A‐specific CD8+ T cells that can be selectively and efficiently stained by short dimeric A2/Melan‐ A_26‐35 complexes, which makes them directly accessible for longitudinal monitoring and further investigation.     

The effects of trastuzumab on the CD4+CD25+FoxP3+ and CD4+IL17A+ T-cell axis in patients with breast cancer

In addition to the direct targeting effects on HER2-positive cells, trastuzumab may have a therapeutic role modulating the activity of the cellular immune system in patients with breast cancer. To investigate this further, the balance of T-regulatory (T_reg), Th17, natural killer (NK) and NK T (NKT) cells before, during and after trastuzumab therapy was investigated. Sequential frequencies of circulating T_reg cells, Th17 cells, NK and NKT cells were measured in peripheral blood of breast cancer patients and normal controls throughout therapy. Individuals with breast cancer had significantly higher T_reg frequencies of peripheral blood compared with healthy controls (9.2 or 8.6 vs 6%; P<0.05), and no significant differences in T_reg frequencies were observed between HER2-positive and HER2-negative individuals. The number of Th17 cells was lowest in HER2-positive patients compared with both healthy controls and HER2-negative patients (0.31 vs 0.75% or 0.84%; P=0.01). There appeared to be an inverse relationship between T_reg and Th17 frequencies in metastatic breast cancer (MBC) with T_reg levels significantly reduced during treatment with trastuzumab (P=0.04), whereas Th17 frequencies were concomitantly increased (P=0.04). This study supports earlier data that T_reg cells are present at higher frequencies in breast cancer patients compared with healthy individuals. For the first time, we show that HER2-positive individuals with breast carcinomas have reduced numbers of circulating Th17 cells, which appear, in turn to have an inverse relationship with T_reg frequency in MBC. The change in balance of the T_reg : Th17 ratio appears to characterise the cancer state, and furthermore, is disrupted by trastuzumab therapy.     

CD8+Foxp3+ tumor infiltrating lymphocytes accumulate in the context of an effective anti‐tumor response

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3‐expressing CD8⁺ T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti‐tumor response. CD8⁺Foxp3⁺ T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM‐CSF secreting HER‐2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen‐specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3⁺ and Foxp3^? CD8⁺ T cells have similar IFN‐γ production and antigen‐specific degranulation after stimulation with RNEU_420–429, the immunodominant HER‐2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU_(420–429)‐specific effector T cells into neu‐N mice (a model that results in immune tolerance to neu), confirm that CD8⁺Foxp3⁺ T cells are present in tumors only if there is an existing pool of tumor‐rejecting effector T cells. CD8⁺Foxp3⁺ TILs mark the presence of tumor‐rejecting antigen‐specific T cells and their accumulation serves as a marker for an effective T cell response.