CD8 Alpha Beta Research Articles

Implications on pathogenesis and risk of oral lichen planus neoplastic transformation: an ex-vivo retrospective immunohistochemical study.

To evaluate OPN, MCM7, Ki-67, p53, Bcl-2 and 53BP1 presence, together with the abnormal adaptive CD4 and CD8 T-cell response markers expression in a series of oral lichen planus (OLP) affected patients and assess their combined contribution for a more objective disease classification. In this ex-vivo retrospective analysis, biopsy specimens from 28 adults with a clinical diagnosis of OLP at different progression degree (16 reticular, 2 plaque-like, 1 erosive and 9 mixed type) were collected. Sections were immunohistochemically investigated for the proinflammatory cytokine osteopontin (OPN), alpha-beta CD4 and CD8 positive T cells, DNA replication licensing factor (MCM7), proliferating cell marker (Ki-67), apoptotic and tumor antigen (p53), apoptosis modulator (Bcl-2) and cellular response regulator to double-strand breaks tumor suppressor p53-binding protein 1 expression. Statistical analysis revealed that 53BP1 is highly represented among the OLP study patients (p<0.05). Moreover, on the basis of the quantification results of the highly expressed parameters, two illness categories with different severity were evidenced. The classification hypothesis was confirmed by i) OLP lesion persistence, ii) the development of oral severe lesions in the patients belonging to high grade activity OLP group (HGA-OLPs) and iii) the ascertainment of the same evidence both in the oral squamous cell tumor controls (OSCC) and in HGA-OLP cases. This study completes the scenario with respect to early detection, thanks to a more precise histological analysis, for rationalizing the clinical and histological findings toward a sharable international disease scoring system.

Regeneration of Tumor Antigen Specific CTLs Utilizing iPSC Technology for Off-the-Shelf Immunotherapy

In the tumor immunotherapy field tumor antigen specific T cell therapy is currently attracting attention. However, a critical issue still awaits resolution; it is difficult to efficiently expand tumor antigen specific T cells without exhaustion. To solve this problem, we are now utilizing induced pluripotent stem cell (iPSC) technology. When iPSCs are established from tumor antigen specific cytotoxic T cells (CTLs), T cells regenerated from these iPSCs (T-iPSCs) are expected to express the same T cell receptors (TCRs) as the original CTLs. Therefore, it will become possible to obtain de novo generated tumor antigen specific CTLs almost unlimitedly. In line with this concept, we have succeeded in establishing iPSCs from CTLs specific for the melanoma antigen MART-1 and regenerating MART-1 specific CTLs (Vizcardo et al, Cell Stem Cell, 2013).The strategy mentioned above was supposed to be applied as autologous transfusion. Autologous transfusion setting, however, has some problems; i) high cost to establish T-iPSCs from each patient, ii) heterogeneity of the quality as iPSCs, iii) difficulty in getting T-iPSCs with high affinity TCRs. To solve these problems, we are now planning to apply this method to the allogeneic transfusion setting. In allogeneic setting, we can select high quality T-iPSC clones with high affinity TCRs and such clones can be utilized to many patients with the same HLA type. In this context, it is preferable to establish T-iPSCs from HLA haplotype homozygous donors, since CTLs generated from these T-iPSCs can be administered to the patients carrying the same HLA haplotype at least on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured.Now we are trying to establish T-iPSCs from CTLs specific for various tumor antigen from healthy donors. We succeeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific cytotoxicity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to lyse some leukemia cell lines and primary leukemia cells which express endogenous WT1 protein. Administration of regenerated CTLs prolonged the survival of mice inoculated with leukemia cell line in in vivo xenograft model. In addition, we succeeded in transducing iPSCs derived from non-T cells with WT1-specific TCR genes (TCR-iPSCs) and generating CTLs from them. Generated CTLs from TCR-iPSCs exhibited comparable TCR specific cytotoxicity as regenerated CTLs from T-iPSCs. Using this method, it would be much easier to produce CTLs with defined high affinity TCRs specific for various tumor antigen.We are currently endeavoring to apply this method clinically. In the future, we intend to establish an allogeneic transfusion system, in which various tumor antigen specific T-iPSCs/TCR-iPSCs from a wide range of HLA haplotype homozygous donors will be lined up as a "T-iPS/TCR-iPSCs cell bank", with the aim of making off- the-shelf tumor immunotherapy a reality. DisclosuresKataoka:Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kawamoto:Regcel: Research Funding.

Abstract B126: Regeneration of tumor antigen specific CTLs utilizing iPSC technology

Abstract Current cancer immunotherapies mainly aim to activate and expand tumor antigen specific cytotoxic T lymphocytes (CTLs) in vitro or in vivo, but these methods are not so successuful because of the difficulty in preparing sufficient number of CTLs. We are trying to overcome this problem by utilizing the induced pluripotent stem cell (iPSC) technology. Our strategy is based on the concept that i) when iPSCs are produced from antigen specific T cells, the rearranged configurations of T cell receptor (TCR) genes are inherited to the iPSCs (which we call T-iPSCs), and that ii) when T cells are regenerated from such T-iPSCs, all of them should come to express the same TCR as the original one. Therefore it will become possible to obtain de novo generated tumor specific CTLs almost unlimitedly, because it is possible to make a mass-culture of cells at the iPSC level. In line with this concept, we have succeeded in establishing T-iPSCs from CTLs specific for the melanoma antigen MART-1 derived from tumor infiltarating lymphocytes, and in regenerating MART-1 specific T cells from such T-iPSCs (Vizcardo et al, Cell Stem Cell, 2013). The strategy mentioned above has been conducted in the category of autologous transplantation. Autologous transplantation setting, however, has some problems; i) high cost to establish T-iPSCs from each patients, ii) heterogeneneity of the quality as iPSCs, iii) difficulty in getting high affinity TCR. To solve these problems we are now planing to apply this method to the allogeneic transplantation setting. In allogeneic setting, we can select high quality T-iPSC clone and such a clone can be used to many other patients. It is preferable to produce T-iPSCs from a HLA haplotype homo donor, since CTLs generated from these T-iPSCs can be administerd to the patients carrying the same HLA haplotype on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured. Now we are trying to establish T-iPSCs from CTLs specific for various cancer antigen from healthy donors. We succeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific killing activity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to kill some leukemia cell lines which express endogenous WT1 protein. They can be expanded more than ten thousand fold by repeated TCR stimulation without reducing their killing activity. We are now examining in vivo killing activity of these CTLs using zenograft model. This method could bring about a breakthrough in cancer immunotherapy. Citation Format: Takuya Maeda, Seiji Nagano, Hiroshi Ichise, Kyoko Masuda, Toshio Kitawaki, Akifumi Kondo-Takaori, Hiroshi Kawamoto. Regeneration of tumor antigen specific CTLs utilizing iPSC technology. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B126.

Sickle Hemoglobin Confers Tolerance to Plasmodium Infection

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

Molecular and flow cytometry characterization during the follow‐up of three simultaneous lymphoproliferative disorders: Hairy cell leukemia, monoclonal B‐cell lymphocytosis, and CD4++/CD8+/−dim T‐large granular lymphocytosis—A case report

The simultaneous diagnosis of hairy cell leukemia and monoclonal B-cell lymphocytosis with the characteristics of "indolent" chronic lymphocytic leukemia is rare but not unknown. However, an association with a third clonal lymphoproliferative disorder has not previously been described. We report the simultaneous presence of hairy cell leukemia, monoclonal B-cell lymphocytosis, and alpha beta CD4(++) /CD8(+) T-cell large granular lymphocytosis in a 63-year-old man. After the diagnosis, the three lymphoproliferative disorders (i.e., two of B-cell lineage and one of T-cell lineage) were characterized by analysis of multiple sequential bone marrow and peripheral blood samples using flow cytometry and molecular techniques. We discuss these findings in the context of chronic antigen stimulation, immunosuppression, and apoptotic pathway alterations, which might be implicated in the accumulation of these abnormal clones in the same patient. Because the phenotype of the three clones is compatible with fully differentiated B lymphocytes (consistent with a postgerminal origin) and T-CD4(++) cells, we favor the possibility of an antigen-driven mechanism and a dysregulation of homeostatic apoptosis in this patient.

P74 CD161 expressing CD8+ T-cells; elusive players in viral hepatitis

Hepatitis B (HBV) and C (HCV) -specific CD8+ T-cells are characterised by expression of the NK receptor CD161. CD8+ T cells with high levels of CD161 (CD161++) make up a...

Cytotoxic immunological synapses.

One of the most fundamental activities of the adaptive immune system is to kill infected cells and tumor cells. Two distinct pathways mediate this process, both of which are facilitated by a cytotoxic immunological synapse. While traditionally thought of as innate immune cells, natural killer (NK) cells are now appreciated to have the capacity for long-term adaptation to chemical and viral insults. These cells integrate multiple positive and negative signals through NK cell cytotoxic or inhibitory synapses. The traditional CD8(+)alphabeta T-cell receptor-positive cells are among the best models for the concept of an immunological synapse, in which vectoral signaling is linked to directed secretion in a stable interface to induce apoptotic cell death in an infected cell. Large-scale molecular organization in synapses generated a number of hypotheses. Studies in the past 5 years have started to provide clear answers regarding the validity of these models. In vivo imaging approaches have provided some hints as to the physiologic relevance of these processes with great promise for the future. This review provides an overview of work on cytotoxic immunological synapses and suggests pathways forward in applying this information to the development of therapeutic agents.

Rapid αβ TCR-mediated responses in γδ T cells transduced with cancer-specific TCR genes

Adoptive T-cell transfer of in vitro cultured T cells derived from cancer patients with naturally developed immune responses has met with some success as an immunotherapeutic approach, although only a limited number of patients showed spontaneous immune responses. To find alternative ways, such as cancer-specific T-cell receptor (TCR) gene transfer, in preparation for sufficient numbers of antigen-specific T cells is an important issue in the field of adoptive T-cell therapy. Given the inherent disadvantage of alphabeta TCR transfer to other alphabeta T cells, namely the possible formation of mixed TCR heterodimers with endogenous alpha or beta TCR, we employed gammadelta T cells as a target for retroviral transfer of cancer-specific TCR and examined whether gammadelta T cells were useful as an alternative population for TCR transfer. Although retroviral transduction to gammadelta T cells with TCR alphabeta genes alone, isolated from a MAGE-A4(143-151)-specific alphabeta CD8(+) cytotoxic T lymphocyte (CTL) clone, did not provide sufficient affinity to recognize major histocompatibility (MHC)-peptide complexes due to the lack of CD8 co-receptor, gammadelta T cells co-transduced with TCR alphabeta and CD8 alphabeta genes acquired cytotoxicity against tumor cells and produced cytokines in both alphabeta- and gammadelta-TCR-dependent manners. Furthermore, alphabeta TCR and CD8-transduced gammadelta T cells, stimulated either through alphabeta TCR or gammadelta TCR, rapidly responded to target cells compared with conventional alphabeta T cells, reminiscent of gammadelta T cells. We propose alphabeta TCR-transduced gammadelta T cells as an alternative strategy for adoptive T-cell transfer.

Interferon‐γ down‐regulates NKG2D ligand expression and impairs the NKG2D‐mediated cytolysis of MHC class I‐deficient melanoma by natural killer cells

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of alphabeta CD8(+) T cells. Ligands of NKG2D, the MHC class I chain-related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon-gamma (IFN-gamma) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I-negative and -positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN-gamma treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down-regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT-PCR revealed a decrease of MICA-specific mRNA levels upon IFN-gamma treatment and siRNA experiments pointed to an involvement of STAT-1 in this process. Importantly, IFN-gamma-treated MHC class I-negative melanoma cells were less susceptible to NKG2D-mediated NK cell cytotoxicity. Our study suggests that IFN-gamma, by down-regulating ligand expression, might facilitate escape of MHC class I-negative melanoma cells from NKG2D-mediated killing by NK cells.

Phenotypic and functional alterations of Vgamma2Vdelta2 T cell subsets in patients with active nasopharyngeal carcinoma.

Human Vgamma2Vdelta2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their gammadelta T cell antigen receptors. Like CD4 and CD8 alphabeta T cells, adult peripheral Vgamma2Vdelta2 T cells represent a pool of heterogeneous cells with distinct functional capabilities. The aim of this study was to characterize the phenotypes and functions of various Vgamma2Vdelta2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC. Although similar total percentages of peripheral blood Vgamma2Vdelta2 T cells were found in both NPC patients and normal donors, Vgamma2Vdelta2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vgamma2Vdelta2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vgamma2Vdelta2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vgamma2Vdelta2 T cells (T(EM RA)) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vgamma2Vdelta2 T cells (T(CM)) compared with those in healthy controls. Moreover, T(EM RA) and T(CM) Vgamma2Vdelta2 cells from NPC patients produced significantly less IFN-gamma and TNF-alpha, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T(EM RA) Vgamma2Vdelta2 T cell population but did not correct the impaired production of IFN-gamma and TNF-alpha observed for T(EM RA) Vgamma2Vdelta2 T cells. We have identified distinct alterations in the Vgamma2Vdelta2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of gammadelta T cells is compromised in these patients. Our data suggest that the contribution of Vgamma2Vdelta2 T cells to control NPC may depend on the activation state and differentiation of these cells.

The major histocompatibility complex class Ib molecule HLA‐E at the interface between innate and adaptive immunity

The non-classical major histocompatibility complex (MHC) class I molecule human leucocyte antigen (HLA)-E is the least polymorphic of all the MHC class I molecules and acts as a ligand for receptors of both the innate and the adaptive immune systems. The recognition of self-peptides complexed to HLA-E by the CD94-NKG2A receptor expressed by natural killer (NK) cells represents a crucial checkpoint for immune surveillance by NK cells. However, HLA-E can also be recognised by the T-cell receptor expressed by alphabeta CD8 T cells and therefore can play a role in the adaptive immune response to invading pathogens. The recent resolution of HLA-E in complex with both innate and adaptive ligands has provided insight into the dual role of this molecule in immunity.

Hyperbaric Oxygen Prevents Early Death Caused by Experimental Cerebral Malaria

BackgroundCerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis.Methodology/Principal FindingsC57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O2, 3.0 ATA, 1–2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-α, IFN-γ and IL-10 mRNA levels and percentage of γδ and αβ CD4+ and CD8+ T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB) dysfunction and hypothermia.Conclusions/SignificanceThe data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.

Membrane markers of the immune cells in swine: an update

Besides their breeding value, swine are increasingly used as biomedical models. As reported in three international swine clusters of differentiation (CD) workshops and in the animal homologue section of the last workshop for the determination of human leukocyte differentiation antigens (HLDA 8), characterisation of leukocyte surface antigens by monoclonal antibodies and other molecular studies have determined the cell lineages and blood leukocyte subsets implicated in the immune response, including cell adhesion molecules involved in cell trafficking. This review focusses on the current state of knowledge of porcine leukocyte differentiation and major histocompatibility complex (SLA) molecules. Examples of porcine particularities such as the double-positive T lymphocytes with the phenotype CD(4+)CD8(low) and CD(4-)CD8(low) alphabeta T cell subsets and the persistence of SLA class II after T-lymphocyte activation are illustrated, as well as the shared characteristics of the Artiodactyla group, such as the high proportion of gammadelta TcR (T cell receptor) T cells in blood and other lymphoid tissues. Furthermore, discrepancies between swine and humans, such as CD16 expression on dendritic cells and CD11b (wCD11R1) tissue distribution are outlined. The rapidly growing information should facilitate manipulation of the swine immune system towards improving disease control, and open new avenues for biomedical research using the pig as a model.

Four novel ULBP splice variants are ligands for human NKG2D

UL16-binding proteins [ULBPs, also termed as retinoic acid early transcripts (RAET1) molecules] are frequently expressed by malignant transformed cells and stimulate anti-tumor immune responses mediated by NKG2D-positive NK cells, CD8(+) alphabeta T cells and gammadelta T cells in vitro and in vivo. In this study, we identified four novel functional splice variants of ULBPs including ULBP4-I, ULBP4-II, ULBP4-III and RAET1G3 in HepG2 liver carcinoma cells, WISH human amnion cells, Hep-2 larynx carcinoma cells and K562 leukemia cells, respectively, by reverse transcription-PCR and T vector cloning strategy. Analysis of alignments of amino acid sequences of the splice variants illustrated that there were important modifications between splice variants and their individual parental ULBP. All ULBP4 splice variants (ULBP4-I, ULBP4-II and ULBP4-III) were type 1 membrane-spanning molecules and had the ability to bind with human NKG2D receptor in vitro. Ectopic expressions of ULBP4 and ULBP4 splice variants resulted in the enhanced cytotoxic sensitivity of target cells against NK cells, which could be blocked by anti-NKG2D mAb. Moreover, co-culture-free soluble forms of ULBP4 splice variants (their alpha1 + alpha2 ectodomains) and RAET1G3 (soluble splice variant of RAET1G2) with NK cells down-regulated the cell surface expression of NKG2D. Finally, immobilized in a plate-bound form of RAET1G3 stimulated NK cells to secrete IFN-gamma. Taken together, all the identified functional splice variants will help to advance our knowledge regarding the overall functions of ULBP gene family.

Clinical-scale single-step CD4+ and CD8+ cell depletion for donor innate lymphocyte infusion (DILI)

The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection to reduce the risk of GVHD holds significant promise for application in adoptive immunotherapy. Current clinical-scale approaches for T-cell depletion (e.g., CD34(+) selection, CD3(+) depletion), usually deplete gammadelta T cells, which may be advantageous in mediating graft-versus-tumor (GVT) effects and augmenting the innate immune response against infections. Here, we present a new method for depletion of T cells with potential GVHD reactivity by using a single-step immunomagnetic protocol, which efficiently depletes CD4(+) and CD8(+) alphabeta T cells under good manufacturing practice (GMP) conditions. Depletion from unstimulated leukapheresis products (n=6) containing up to 2.0 x 10(10) cells showed high efficiency (mean log depletion of CD4(+) cells: 4.12, CD8(+) cells: 3.77). In addition, immunomagnetic CD4/CD8 depletion resulted in passive enrichment of innate lymphocytes (mean recovery of natural killer (NK) cells: 38%, gammadelta T cells: 50%). We demonstrated that gammadelta/NK cells preserved their proliferative and cytotoxic capacity and conclude that simultaneous large-scale depletion of CD4(+)/CD8(+) T cells is feasible and can be performed under GMP conditions with high-depletion efficacy for alphabeta T cells and recovery of functionally intact innate effector lymphocytes for potential use in adoptive immunotherapy studies.

Changes in thymus size, cellularity and relation between thymocyte subpopulations in young adult rats induced by Somatostatin-14

The role of somatostatin on inhibition of both normal and tumor cell cycle, secretion of endocrine and exocrine cells, as well as induction apoptosis is well documented. However, its effect on T cell development and thymic structure is not fully clarified. In order to investigate the influence of somatostatin in vivo on the thymus structure and T cell development, the young adult Albino Oxford male rats were intracerebroventriculary treated with somatostatin-14. We examined the thymus compartments and its cellularity, through assessment of morphometric parameters by stereological method, and the relation between thymocytes subpopulations, over expression of CD4, CD8 and T-cell receptor (TCR) alpha beta by flow cytometry. Additionally, we also determined the body and thymus weight of the rats, during the first three months of life, to define the time of SRIH-14 application. A decrease of relative thymus weight from the fourth weeks of postnatal life, and an unchanged relative thymus weight obtained in treated group indicates that SRIH-14 in young adult rats inhibits growth of whole organism, not only thymus. The changes in the absolute number and numerical density of cortical thymocytes indicate that SRIH-14 alters the true lymphoid tissue. SRIH-14 changes relation between thymocyte subsets, increase number of CD4(-)CD8(-)TCR alpha beta(-) and CD4(-)CD8(+)TCR alpha beta(hi) thymocyte subsets as well as the CD4(-)CD8(-)TCR alpha beta(low/hi) thymocytes, while decrease number of CD4(+)CD8(+) TCR alpha beta(-/low/hi) thymocyte subsets. These results indicate that somatostatin-14 is not involved in the control of the physiologic involution of the thymus, although induces thymic weight loss through the reduction of true lymphoid tissue. In addition, changes in frequency of thymocyte subpopulations, especially immature cells, indicate that SRIH-14 modulates thymocytes development and maturation.

Concomitant increase of IL-10 and pro-inflammatory cytokines in intraepithelial lymphocyte subsets in celiac disease

Celiac disease (CD) is a small intestinal enteropathy caused by permanent intolerance to wheat gluten. Active disease is characterized by a prominent cytokine response of intraepithelial lymphocytes (IELs) to gluten-containing diet with concomitant increase in expression of pro-inflammatory IFN-gamma and down-regulatory IL-10 without increase in tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). The aim was to understand the local immune reaction by determining which intraepithelial T cell subsets produce the different cytokines. The three major IEL-subsets gammadeltaIELs, CD4(+)alphabetaIELs and CD8(+)alphabetaIELs, as well as CD94(+)CD8(+)alphabetaIELs, selectively expanded in active CD, were retrieved from small intestinal biopsies of children with active CD and controls and analyzed quantitatively for cytokine mRNA expression. In active CD, CD8(+)alphabetaIELs showed a significant increase in expression levels of both IFN-gamma and IL-10. CD8(+)alphabetaIELs were also the IEL subset with highest expression level per cell of both cytokines and constituted the cellular source for almost all IFN-gamma and most IL-10. Expression levels of both cytokines were higher in CD94(-)CD8(+)alphabetaIELs than CD94(+)CD8(+)alphabetaIELs. TNF-alpha levels were only increased in CD4(+)alphabetaIELs, which also showed the highest expression level per cell and constituted the major source of this cytokine. Interestingly, IL-10 was increased also in CD4(+)alphabetaIELs. Cytokine levels were low in gammadeltaIELs. 'Classical' CD94(-)CD8(+)alphabeta T cells within the epithelium are responsible for the excessive production of IFN-gamma, believed to drive the formation of intestinal lesions in active CD. Production of IL-10 may be a common feature of IELs producing pro-inflammatory cytokines, thereby attempting to limit inflammation in an autocrine fashion.

In Vivo Expression Pattern of MICA and MICB and Its Relevance to Auto-Immunity and Cancer

Non-conventional MHC class I MIC molecules interact not with the TCR, but with NKG2D, a C-type lectin activatory receptor present on most NK, γδ and CD8+ αβ T cells. While this interaction is critical in triggering/calibrating the cytotoxic activity of these cells, the actual extent of its in vivo involvement, in man, in infection, cancer or autoimmunity, needs further assessment. The latter has gained momentum along with the reported expansion of peripheral CD4+CD28−NKG2D+ T cells in rheumatoid arthritis (RA). We first initiated to extend this report to a larger cohort of not only RA patients, but also those affected by systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). In RA and SS, this initial observation was further tested in target tissues: the joint and the salivary glands, respectively. In conclusion and despite occasional and indiscriminate expansion of the previously incriminated T cell subpopulation, no correlation could be observed between the CD4+CD28−NKG2D+ and auto-immunity. Moreover, in situ, the presence of NKG2D matched that of CD8+, but not that of CD4+ T cells. In parallel, a total body tissue scan of both MICA and MICB transcription clearly shows that despite original presumptions, and with the exception of the central nervous system, both genes are widely transcribed and therefore possibly translated and membrane-bound. Extending this analysis to a number of human tumors did not reveal a coherent pattern of expression vs. normal tissues. Collectively these data question previous assumptions, correlating a tissue-specific expression/induction of MIC in relevance to auto-immune or tumor processes.

Mycobacterial heat shock protein‐induced blood T lymphocytes subsets and cytokine pattern: Comparison of sarcoidosis with tuberculosis and healthy controls

Sarcoidosis (SA) is a disorder of unknown aetiology. Mycobacterium tuberculosis heat shock proteins (Mtb-hsp) have been considered as causative agents of SA. The role of Mtb-hsp in the immune response in SA has not been investigated. Mtb-hsp-stimulated T-cell subsets and Th1/Th2 cytokine patterns in the supernatant from peripheral blood mononuclear cell cultures from 22 SA patients, 20 tuberculosis (TB) patients and 20 healthy volunteers were compared using flow cytometry. In unstimulated cultures, a significantly higher percentage of CD8(+)alphabeta(+)T-cells were present in SA versus controls. Similarly there was a significantly increased IL-6 and decreased IL-4 level in SA and significantly lower INF-gamma, IL-2, IL-4, IL-10 production in TB versus controls. After Mtb-hsp stimulation, there was a significantly increased TNF-alpha, IL-6, IL-10 and decreased INF-gamma, IL-2, IL-4 production in SA and significantly increased TNF-alpha, IL-6 concentrations in TB versus controls. CD8(+)gammadelta(+)IL-4(+)T-cells were detected significantly less often in Mtb-hsp-induced cultures in SA versus controls. Comparing SA versus TB, CD4(+)gammadelta(+)TCR-cells were significantly increased in Mtb-hsp-induced cultures in TB versus controls and SA. Before stimulation, significantly increased IL-6, IL-10 and decreased IL-4 level in SA versus TB was revealed, whereas Mtb-hsp stimulation caused significantly increased IL-10 and decreased IL-4 concentrations in SA. After Mtb-hsp stimulation, increased levels of pro-inflammatory cytokines, TNF-alpha and IL-6 were found in sera from SA and TB patients in comparison with healthy controls; SA patients demonstrated the lowest levels of IL-4 and the highest levels of IL-10.

Characterizing tumor-promoting T cells in chemically induced cutaneous carcinogenesis.

There is a longstanding but poorly understood epidemiologic link between inflammation and cancer. Consistent with this, we previously showed that alphabeta T cell deficiency can increase resistance to chemical carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene and promoted by phorbol 12-myristate 13-acetate. This provoked the hypothesis that alphabeta T cell deficiency removed T regulatory cells that limit the anti-tumor response or removed a specific tumor-promoting (T-pro) T cell population. Here we provide evidence for the latter, identifying a novel CD8(+) subset that is a candidate for T-pro cells. We demonstrate that CD8 cell-deficient mice show substantially less tumor incidence and progression to carcinoma, whereas susceptibility is restored by CD8(+) cell reconstitution. To characterize the putative T-pro cells, tumor-infiltrating lymphocytes were isolated from normal and CD4(-/-) mice, revealing an activated population of T cell receptor alphabeta(+)CD8(+)CD44(+)CD62L(-) cells expressing the inflammatory mediators IFNgamma, TNFalpha, and cyclooxygenase-2, but deficient in perforin, relative to recirculating cells of equivalent phenotype. This novel population of CD8(+) T cells has intriguing similarities with other lymphocytes that have been associated with tissue growth and invasiveness and has implications for inflammation-associated carcinogenesis, models of cancer immunosurveillance, and immunotherapeutic strategies.