Expression Of Cytotoxic Molecules Research Articles

Prognostic and predictive value of NanoString-based immune-related gene signatures in a neoadjuvant setting of triple-negative breast cancer: relationship to tumor-infiltrating lymphocytes.

The prognostic significance of tumor-infiltrating lymphocytes and immune signals has been described previously in triple-negative breast cancer (TNBC). Furthermore, recent studies have shown that immunologic parameters are relevant for the response to neoadjuvant chemotherapy (NAC) in breast cancer as well as for outcomes after adjuvant chemotherapy. However, immune signals are variable, and which signals are important is largely unknown. We, therefore, evaluated the expression of immune-related genes in TNBC treated with NAC. We retrospectively evaluated biopsy tissue from 55 patients with primary TNBC treated with NAC (anthracycline, cyclophosphamide, and docetaxel) against the NanoString nCounter GX Human Immunology Panel (579 immune-related genes). Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, cytokines related to T helper cell type 1 (Th1), and B cell markers was associated with a pathologic complete response (pCR). Higher expression of NFKB1, MAPK1, TRAF1, CXCL13, GZMK, and IL7R was significantly associated with pCR, higher Miller-Payne grade, and lower residual cancer burden class. Expression of NFKB1, TRAF1, and CXCL13genes, in particular, was significantly correlated with a longer disease-free survival rate. Conversely, patients those who failed to achieve a pCR showed increased expression of genes related to neutrophils. Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, Th1-related cytokines, and B cell markers is correlated with pCR and survival in TNBC patients treated with NAC. Our results suggest that the activation status of neutrophils may provide additional predictive information for TNBC patients treated with NAC.

Epstein-Barr virus-positive nodal T/NK-cell lymphoma: an analysis of 15 cases with distinct clinicopathological features.

Nodal peripheral T-cell lymphoma, not otherwise specified, is a heterogeneous entity with variable biologic behavior. We analyze the clinicopathological features of 15 patients with Epstein-Barr virus-positive (EBV+) nodal T/NK-cell lymphoma, including 9 males and 6 females, with a median age of 64 years. All patients presented with multiple lymphadenopathy with common B symptoms (80%, 12/15) at an advanced Ann Arbor stage (III, IV) (87%, 13/15). The International Prognostic Index was high or high/intermediate in 87% (13/15) of patients, and the prognostic index for peripheral T-cell lymphoma was group 3 or 4 in 73% (11/15). Spleen and liver involvement was observed in 73% (11/15) and 60% (9/15) of patients, respectively. In contrast, extranodal involvement was infrequent, with no more than 1 site in 71% (10/15) of patients. Moreover, none had nasal lesions, and only 1 had mucocutaneous involvement. The cell lineage of EBV+ tumor cells was determined to be T cell in all except 1 patient, who was NK-cell lineage. Cytotoxic molecules were expressed in all cases, and 64% (9/14) of patients expressed the αβT-cell receptor. Moreover, most patients (67%, 10/15) showed CD8 positivity, with 2 of them being CD4CD8 double positive; the others were CD4 positive (n = 2) or CD4CD8 double negative (n = 3). The clinical course was very aggressive, with a median survival time of 3.5 months, and 10 patients died within 6 months of diagnosis. Taken together, our data demonstrate that EBV+ nodal T/NK-cell lymphoma is a distinct clinicopathological entity characterized by cytotoxic molecule expression, a frequent CD8-positive αβT-cell lineage, and a very aggressive clinical behavior.

IL-15 Amplifies the Pathogenic Properties of CD4+CD28− T Cells in Multiple Sclerosis

CD4(+)CD28(-) T cells arise through repeated antigenic stimulation and are present in diseased tissues of patients with various autoimmune disorders, including multiple sclerosis (MS). These cells are believed to have cytotoxic properties that contribute to the pathogenic damaging of the target organ. Endogenous cues that are increased in the diseased tissue may amplify the activity of CD4(+)CD28(-) T cells. In this study, we focused on IL-15, a cytotoxicity-promoting cytokine that is increased in the serum and cerebrospinal fluid of MS patients. Using immunohistochemistry, we demonstrate that IL-15 is mainly produced by astrocytes and infiltrating macrophages in inflammatory lesions of MS patients. Moreover, in vitro transmigration studies reveal that IL-15 selectively attracts CD4(+)CD28(-) T cells of MS patients, but not of healthy individuals. IL-15 further induces the expression of chemokine receptors and adhesion molecules on CD4(+)CD28(-) T cells, as investigated using flow cytometry, resulting in enhanced migration over a monolayer of human brain endothelial cells. Finally, flow cytometric analyses revealed that IL-15 increases the proliferation and production of GM-CSF, expression of cytotoxic molecules (NKG2D, perforin, and granzyme B), and degranulation capacity of CD4(+)CD28(-) T cells. Taken together, these findings indicate that increased peripheral and local levels of IL-15 amplify the pathogenic potential of CD4(+)CD28(-) T cells, thus contributing to tissue damage in MS brain lesions.

A novel immunohistochemical classifier to distinguish Hodgkin lymphoma from ALK anaplastic large cell lymphoma

Classical Hodgkin lymphoma and ALK− anaplastic large cell lymphoma share many features like strong CD30 expression and usually loss of B- and T-cell markers. However, their clinical course is dramatically different with curability rates of >90% for classical Hodgkin lymphoma and an unfavorable prognosis for anaplastic large cell lymphoma. Classical Hodgkin lymphoma and ALK− anaplastic large cell lymphoma can usually be distinguished by PAX5 expression in the Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma and expression of cytotoxic molecules in tumor cells of anaplastic large cell lymphoma. However, in some cases the differential diagnosis is difficult owing to absence of established markers. To be able to better classify these cases, we reevaluated gene expression data of microdissected tumor cells of both lymphomas for differentially expressed genes. A classifier was established, comprising four genes strongly expressed in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma (MDC/CCL22, CD83, STAT3, and TUBB2B). Applying this classifier to a test cohort, Hodgkin lymphoma was successfully distinguished from ALK− anaplastic large cell lymphoma with an accuracy of 97% (43/44). MDC/CCL22, CD83, and STAT3 have also been found to be expressed in antigen-presenting cells. Therefore, based on our established classifier, Hodgkin and Reed-Sternberg cells differ from tumor cells of anaplastic large cell lymphoma, which can successfully be applied for practical purposes in histopathologic diagnostics.

High expression of cytotoxic molecules and Th1 bias of circulating T cells in patients with multiple sclerosis under fingolimod treatment

High expression of cytotoxic molecules and Th1 bias of circulating T cells in patients with multiple sclerosis under fingolimod treatment

Ex vivo-expanded natural killer cells kill cancer cells more effectively than ex vivo-expanded γδ T cells or αβ T cells.

Adoptive immunotherapy of cancer is evolving with the development of novel technologies for generating a large number of activated killer cells such as natural killer (NK) cells, γδ T cells, and αβ T cells. We have recently established large-scale culture methods to generate activated NK cells from human peripheral blood, and demonstrated that expanded NK cells have higher cytotoxicity against cancer cells than freshly isolated NK cells. In this study, we compared cultured NK cells with cultured γδ T and αβ T cells that were prepared by conventional culture methods regarding the expression of cytotoxic molecules and cytotoxicity against cancer cells. Natural cytotoxicity receptors such as NKp30, NKp44 and NKp46, and perforin were expressed most exclusively on NK cells. Granzyme A, NKG2D, and interferon-γ were dominantly expressed in NK cells and γδ T cells but not in αβ T cells. Consistent with the expression profiles of the cytotoxic molecules, cultured NK cells from both healthy volunteers and cancer patients demonstrated significantly higher cytotoxicity against cancer cell lines, including MHC class I-positive cell lines, compared with cultured γδ T cells and cultured αβ T cells. Additionally, NK cells, unlike γδ T cells or αβ T cells, expressed high levels of CD16, and showed augmented cytotoxicity when co-administered with an anti-CD20 monoclonal antibody drug, rituximab. These results suggest the excellent efficacy of expanded NK cells for cancer treatment.

Intratumoral CD4+ T Lymphodepletion Sensitizes Poorly Immunogenic Melanomas to Immunotherapy with an OX40 Agonist

Previous studies have shown that the antitumor effects of OX40 agonists depend on the immunogenicity of the tumor and that poorly immunogenic tumors such as B16F10 melanomas do not respond to OX40 agonist treatment. In this study, we have shown that intratumoral CD4+ T lymphodepletion sensitized poorly immunogenic B16F10 melanomas to immunotherapy with an OX40 agonist. CD4+ T lymphodepletion dramatically altered the tumor immune microenvironment, making it more susceptible to the antitumor effects of an OX40 agonist by enhancing the accumulation of CD8+ T cells and natural killer (NK) cells in tumor tissue. However, unexpectedly, the number of CD11b+ Gr-1(+) myeloid-derived suppressor cells (MDSCs) within tumor tissues also significantly increased as a result of CD4+ T lymphodepletion. As a countermeasure against CD8+ T-cell accumulation, CCR2-positive CD11b+ Gr-1(int) (monocytic) MDSCs predominantly increased. Treatment with an OX40 agonist under CD4+ T lymphodepletion neither reduced MDSCs nor increased CD8+ T cells and NK cells, but further enhanced the expression of cytotoxic molecules from tumor-infiltrating effector cells. Our results suggest that combined immunotherapy using both an OX40 agonist and CD4+ T lymphodepletion could be a promising therapeutic strategy for poorly immunogenic tumors and might be more effective if further combined with a therapeutic strategy targeting MDSCs.

IL-17A Promotes Immune Cell Recruitment in Human Esophageal Cancers and the Infiltrating Dendritic Cells Represent a Positive Prognostic Marker for Patient Survival

We previously reported that tumor-infiltrating interleukin (IL)-17A-producing cells play a protective role in human esophageal squamous cell carcinoma (ESCC). However, the potential mechanisms involved remain unclear. In the present study, we investigated the effects of IL-17A on immune cell recruitment and function in ESCC. In vitro chemotaxis assays using the ESCC cell lines EC109 and KYSE30 demonstrated that although IL-17A showed no significant direct effects on the migration of T cells, natural killer (NK) cells as well as dendritic cells (DCs), it could induce ESCC tumor cells to produce inflammatory chemokines, for example, CXCL9, CXCL10 and CCL2, CCL20, which are associated with the migration of T cells, NK cells, and DCs, respectively. In addition, IL-17A enhanced the cytotoxic effects of NK cells against tumor cells by augmenting the expression of cytotoxic molecules, for example, tumor necrosis factor-α, interferon-γ, Perforin, and Granzyme B and activation receptors, for example, NKp46, NKp44, NTB-A, and NKG2D on NK cells. Furthermore, immunohistochemical analysis revealed that the density of IL-17A-producing cells was positively and significantly associated with the density of CD1a DCs in tumor tissues. With the analyses of 181 ESCC patients, we found a correlation of higher number of tumor-infiltrating CD1a DCs with significantly improved overall survival of patients with ESCC. This study provides further understanding of the roles of Th17 cells in ESCC, which may contribute to the development of novel cancer immunotherapy strategies.

Characterization of circulating CD8+T cells expressing skin homing and cytotoxic molecules in active non-segmental vitiligo

Vitiligo is caused by melanocyte depletion. Studies have suggested that skin-homing cytotoxic T lymphocytes that express cutaneous lymphocyte-associated antigen (CLA) are responsible for melanocyte depletion. The characteristics of these skin-homing cytotoxic T cells have not been well established yet. To investigate the frequency of skin-homing CD8(+)T cells (CD8(+)CLA(+)T cells) and their expression of cytotoxic molecules, as well as migration-related molecules in CD8(+)T cell in non-segmental vitiligo patients. The frequency of CD8(+)CLA(+)T cells and their expression of cytotoxic molecules (perforin, granzyme-B and FasL) in peripheral blood of patients with non-segmental vitiligo were assessed using flow cytometry. Levels of chemokine receptors (CCR4, CCR10) on CD8(+)T cells were evaluated. Our results revealed a higher frequency and increased expression of perforin and granzyme-B in circulating CD8(+)CLA(+)T cells from patients with active vitiligo. The expression levels of CCR4 increased in CD8(+)T cells in active vitiligo patients. Patients with active non-segmental vitiligo have a higher frequency of CD8(+)CLA(+)T cells and hyper-activated cytotoxic functions, which may be involved in the pathogenesis of non-segmental vitiligo.

High Proportion of Granzyme B+ Intraepithelial Lymphocytes Contributes to Epithelial Apoptosis in Helicobacter pylori‐Associated Lymphocytic Gastritis

Helicobacter pylori infection has been linked to the development of lymphocytic gastritis (LG) characterized by ≥25 intraepithelial lymphocytes (IELs) per 100 epithelial cells. We hypothesize that the changes in the subpopulation and/or cytotoxicity of IELs leading to epithelial cell apoptosis may be involved in the pathogenesis of H.pylori-associated LG. We examined IEL subpopulations and the expression of cytotoxic molecules by IELs in biopsy specimens from 36 patients with H.pylori-associated LG by immunostainings for CD3, CD4, CD8, T-cell-restricted intracellular antigen-1 (TIA-1), and granzyme B (GrB) and compared the results with those obtained from 49 patients with H.pylori-associated gastritis (HPG). To investigate whether the IEL-mediated cytotoxicity is related to the increase of epithelial apoptosis, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using ApopTag detection kit. Between LG and HPG groups, significant differences in the number of CD3+, CD4+, CD8+, TIA-1+ or GrB+ IELs, and ApopTag indices were found. Among the CD3+ IELs, the proportion of CD8+ IELs or TIA-1+ IELs did not differ between two groups. The LG group showed a selective increase in GrB-positive, phenotypically activated IELs, which was paralleled by an increase in ApopTag indices. In contrast, the HPG group showed more heterogeneous IEL subpopulations with more CD4+ IELs and less GrB+ IELs compared with the LG group, and we did not find any significant variable contributing to the epithelial apoptosis in the HPG group. This study shows that in addition to the numerical increase in the IELs, there are significant changes in the subpopulations and cytotoxicity of IELs between HPG and H.pylori-associated LG. In particular, enhanced GrB-associated cytotoxicity of the IELs in H.pylori-associated LG contributes to an increase in epithelial apoptosis.

Pleural Mesothelial Cells Promote Expansion of IL-17–Producing CD8+ T Cells in Tuberculous Pleural Effusion

IL-17-producing CD8(+) T lymphocytes (Tc17 cells) have recently been detected in many cancers and autoimmune diseases. However, the possible implication of Tc17 cells in tuberculous pleural effusion remains unclarified. In this study, distribution and phenotypic features of Tc17 cells in both tuberculous pleural effusion (TPE) and peripheral blood from patients with tuberculosis were determined. The effects of proinflammatory cytokines and local accessory cells (pleural mesothelial cells) on Tc17 cell expansion were also explored. We found that TPE contained more Tc17 cells than the blood. Compared with IFN-γ-producing CD8(+) T cells, Tc17 cells displayed higher expression of chemokine receptors (CCRs) and lower expression of cytotoxic molecules. In particularly, Tc17 cells in TPE exhibited high expression levels of CCR6, which could migrate in response to CCL20. Furthermore, IL-1β, IL-6, IL-23, or their various combinations could promote Tc17 cell expansion from CD8(+) T cells, whereas the proliferative response of Tc17 cells to above cytokines was lower than that of Th17 cells. Pleural mesothelial cells (PMCs) were able to stimulate Tc17 cell expansion via cell contact in an IL-1β/IL-6/IL-23 independent fashion. Thus this study demonstrates that Tc17 cells marks a subset of non-cytotoxic, CCR6(+) CD8(+) T lymphocytes with low proliferative capacity. The overrepresentation of Tc17 cells in TPE may be due to Tc17 cell expansion stimulated by pleural proinflammatory cytokines and to recruitment of Tc17 cells from peripheral blood. Additionally, PMCs may promote the production of IL-17 by CD8(+) T cells at sites of TPE via cell-cell interactions.

Characterization of CD4+CD28null T cells in patients with coronary artery disease and individuals with risk factors for atherosclerosis

Risk factors for atherosclerosis may contribute to chronic low-grade inflammation. A highly cytotoxic and inflammatory CD4+ cell subset (CD4+CD28null cells) has been associated with inflammatory diseases, including acute coronary syndromes (ACS). The aim of this study was to quantify and characterize CD4+CD28null cells in individuals with risk factors for atherosclerosis and patients with coronary artery disease (CAD). In order to achieve this goal, peripheral blood mononuclear cells (PBMCs) from individuals with risk factors for atherosclerosis and patients with CAD were analyzed using flow cytometry to detect cytotoxic molecules and evaluate the expression of homing receptors and inflammatory cytokines in CD4+ cell subsets. The cells were evaluated ex vivo and after stimulation in culture. We found no differences in the proportions of CD4+CD28null cells among the groups. Compared with the CD4+CD28+ population, the ex vivo CD4+CD28null subset from all groups expressed higher levels of granzymes A and B, perforin, granulysin and interferon-γ (IFN-γ). Individuals with risk factors and patients with ACS showed the highest levels of cytotoxic molecules. After stimulation, tumor necrosis factor-α (TNF-α) expression in the CD4+CD28null subset from these groups increased more than in the other groups. Stimulation with LPS decreased the expression of cytotoxic molecules by CD4+CD28null cells in all groups. In conclusion, our results show that risk factors for atherosclerosis may alter the CD4+CD28null cells phenotype, increasing their cytotoxic potential. Our findings also suggest that CD4+CD28null cells may participate in the early phases of atherosclerosis.

IL-7R low memory CD8+ T cells are significantly elevated in patients with systemic lupus erythematosus

Human effector memory (EM) CD8(+) T cells include IL-7Rα(high) and IL-7Rα(low) cells with distinct cellular characteristics, including the expression of cytotoxic molecules. Both NK cells and the NK cell-associated molecule 2B4 that is expressed on CD8(+) T cells promote cytotoxicity. Here we analysed the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells and its contribution to cytotoxicity. We also analysed the frequency of IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells in patients with SLE or lupus and in healthy individuals given the potential role of cytotoxic CD8(+) T cells in the pathogenesis of lupus. We used flow cytometry to measure the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells as well as the frequency of these cell populations in the peripheral blood of healthy individuals and patients with SLE. Also, 2B4-mediated cytotoxicity was quantitated in IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells using target cells with CD48 antigen. We found that IL-7Rα(high) EM CD8(+) T cells had higher levels of 2B4 expression compared with IL-7Rα(low) EM CD8(+) T cells. Triggering 2B4 enhanced the cytotoxic function of IL-7Rα(low) EM CD8(+) T cells against target cells. We also noticed that patients with SLE had an increased frequency of IL-7Rα(low) EM CD8(+) T cells that correlated with disease manifestation. Our findings show that SLE patients have increased IL-7Rα(low) EM CD8(+) T cells, possibly contributing to tissue damage through 2B4-mediated cytotoxicity.

Hepatocellular carcinoma-associated fibroblasts trigger NK cell dysfunction via PGE2 and IDO

Defects in natural killer (NK) cell function are necessary for tumor immune escape, but the underlying regulatory mechanisms in human cancers remain largely unknown. Here we show that fibroblasts derived from hepatocellular carcinoma (HCC) were significantly superior to foreskin-derived fibroblasts at inducing NK cell dysfunction, which is characterized by low expression of cytotoxic molecules and surface markers for cell activation, impaired production of cytokines, and decreased cytotoxicity against K562 cells in vitro. Our results also indicate that PGE2 and IDO, derived from activated fibroblasts, suppress the activation of NK cells and thereby create favorable conditions for tumor progression.

The blood of healthy individuals exhibits CD8 T cells with a highly altered TCR Vß repertoire but with an unmodified phenotype

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vβ repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vβ repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25-52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vβ families with altered CDR3 length distribution and increased TCR Vβ/HPRT transcript ratios in all individuals tested. The increase in TCR Vβ/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vβ+ T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8+ T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vβ repertoire led to the identification of different types of CD8+ T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.

Cytotoxic molecule-positive classical Hodgkin's lymphoma: a clinicopathological comparison with cytotoxic molecule-positive peripheral T-cell lymphoma of not otherwise specified type

Classical Hodgkin's lymphoma is characterized by Hodgkin and Reed Sternberg cells, which are of B-cell origin in many cases. We recently highlighted the adverse prognostic significance of cytotoxic molecule expression in patients with classical Hodgkin's lymphoma. However, the clinical characteristics of cytotoxic molecule-positive classical Hodgkin's lymphoma remain controversial. We investigated the clinicopathological profiles of 32 patients with cytotoxic molecule-positive Hodgkin's lymphoma, comprising 23 with nodular sclerosis and 9 with mixed cellularity, and compared these profiles with those of 55 patients with cytotoxic molecule-positive nodal peripheral T-cell lymphoma, not otherwise specified and 439 patients with cytotoxic molecule-negative Hodgkin's lymphoma. The patients with cytotoxic molecule-positive Hodgkin's lymphoma consisted of 20 men and 12 women with a median age of 50 years (range, 19 to 81). All these patients had lymphadenopathy at presentation, and 14 showed mediastinal involvement. Physical findings included hepatomegaly and splenomegaly in six patients each. Four patients had a bulky mass, and nine showed stage IV disease. The tumor cells of patients with cytotoxic molecule-positive Hodgkin's lymphoma had a prototypic immunophenotype of CD15(+) CD30(+) CD45RO(-) fascin(+), with positivity for Epstein-Barr virus in 39% of cases. All patients were negative for Pax5. In comparison with patients with cytotoxic molecule-positive nodal peripheral T-cell lymphomas, not otherwise specified, patients with cytotoxic-positive Hodgkin's lymphoma had relatively mild clinical symptoms, similar to those of patients with cytotoxic molecule-negative Hodgkin's lymphoma. Regarding prognosis, the survival of patients with cytotoxic molecule-positive Hodgkin's lymphoma was worse than that of patients with cytotoxic molecule-negative Hodgkin's lymphoma (P = 0.0003) but better than that of patients with cytotoxic molecule-positive peripheral T-cell lymphomas, not otherwise specified (P = 0.002). Cytotoxic molecule-positive Hodgkin's lymphoma is characterized by an unfavorable prognosis, even if its clinicopathological features are within the boundaries of classical Hodgkin's lymphoma. More effective chemotherapy for cytotoxic molecule-positive Hodgkin's lymphoma is clearly required.

The Blood of Healthy Individuals Exhibits CD8 T Cells with a Highly Altered TCR Vb Repertoire but with an Unmodified Phenotype

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vβ repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vβ repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25–52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vβ families with altered CDR3 length distribution and increased TCR Vβ/HPRT transcript ratios in all individuals tested. The increase in TCR Vβ/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vβ+ T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8+ T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vβ repertoire led to the identification of different types of CD8+ T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.

Autoreactive Cytotoxic T Lymphocytes Acquire Higher Expression of Cytotoxic Effector Markers in the Islets of NOD Mice after Priming in Pancreatic Lymph Nodes

Cytotoxic T lymphocytes (CTLs) that cause type 1 diabetes are activated in draining lymph nodes and become concentrated as fully active CTLs in inflamed pancreatic islets. It is unclear whether CTL function is driven by signals received in the lymph node or also in the inflamed tissue. We studied whether the development of cytotoxicity requires further activation in islets. Autoreactive CTLs found in the islets of diabetes-prone NOD mice had acquired much higher expression of the cytotoxic effector markers granzyme B, interferon γ, and CD107a than had those in the pancreatic lymph node (PLN). Increased expression seemed to result from stimulation in the islet itself. T cells held up from migrating from the PLN by administration of the sphingosine-1-phosphate agonist FTY720 did not increase expression of cytotoxic molecules in the PLN. Stimulation did not require antigen presentation or cytokine secretion by the target β cells because it was not affected by the absence of class I major histocompatibility complex expression or by the overexpression of suppressor of cytokine signaling-1. Activation of CD40-expressing cells stimulated increased CTL function and β-cell destruction, suggesting that signals derived from CD40-expressing cells promote the acquisition of cytotoxicity in the islet environment. These data provide in vivo evidence that stimulation of cytotoxic effector molecule expression occurs in inflamed islets and is independent of β cells.

Linkage of expression of chemokine receptors (CXCR3 and CCR4) and cytotoxic molecules in peripheral T cell lymphoma, not otherwise specified and ALK-negative anaplastic large cell lymphoma

Peripheral T cell lymphoma not otherwise specified (PTCL-N) and ALK-negative anaplastic large cell lymphoma (ALCL) are heterogeneous categories with poor diagnostic reproducibility. To clarify the biologic features of these categories, we investigated the expression of two chemokine receptors, type 1 (Th1/Tc1)-associated CXCR3 and type 2 (Th2/Tc2)-associated CCR4 in 110 PTCL-N and 35 ALK-negative ALCL cases, as well as the expression of cytotoxic molecules (CM). CXCR3 and CCR4 were expressed in 69 (63%) and 37 (34%) of PTCL-N, and in 12 (34%) and 6 (17%) of ALK-negative ALCL, respectively. In PTCL-N, type 1 pattern (CXCR3(+)CCR4(-)) was dominant (52%), whereas in ALK-negative ALCL, 54% were negative for both (P < 0.0001). CM was expressed in 38% of PTCL-N and 51% of ALK-negative ALCL. CM-positive PTCL-N consisted mostly of type 1 disease, which shows type 1 phenotype. In contrast, type 2 pattern (CXCR3(-)CCR4(+)) was recognized in the CM-negative group only. Among type 1 disease, CM-positive cases had a higher female ratio and more aggressive clinical features than CM-negative cases and a poorer prognosis (P = 0.006). Multivariate analysis confirmed that the type 1 phenotype with CM expression was an independent prognostic factor. In both PTCL-N and ALK-negative ALCL, CM-positive type 1 disease had an extremely poor prognosis.

The duration of TCR/pMHC interactions regulates CTL effector function and tumor‐killing capacity

Effector CTL contribute to tumoral immunity by killing tumor cells through secretion of cytotoxic granules and cytokines. Activation of CTL requires specific recognition of cognate peptide-MHC-I (pMHC) complexes on the tumor cell surface by the CTL TCR. It has been suggested that the half-life (t(1/2)) of the TCR/pMHC interaction modulates the activation of naïve CD8(+) T cells; however, it remains unknown whether CTL effector function can also be regulated by the TCR/pMHC t(1/2). Here, we have studied CTL activity in response to tumor cells loaded with pMHC that bind the TCR with different t(1/2). We observed that the TCR/pMHC t(1/2) can differentially regulate CTL effector function during the interaction with tumor cells and defines the nature of anti-tumoral CTL responses in vivo. Although prolonged TCR/pMHC t(1/2) promoted only partial expression of cytotoxic molecules, short t(1/2) induced partial polarization of lytic machinery toward target cells. In contrast, intermediate TCR/pMHC t(1/2) induced strong expression of cytotoxic molecules, efficient polarization of lytic machinery and subsequent release of toxic granules by CTL that killed tumor cells. Consistently, efficient in vivo CTL-mediated tumor clearance was only observed for tumors expressing intermediate t(1/2) pMHC ligands. These data suggest that there is an optimal TCR/pMHC t(1/2) for efficient CTL activity.