Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma: a cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance? A study of 136 cases

Patients with skin nodules characterized by the infiltrate of pleomorphic small/medium T lymphocytes are currently classified as "primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma" (SMPTCL) or as T-cell pseudolymphoma. The distinction is often arbitrary, and patients with similar clinicopathologic features have been included in both groups. We studied 136 patients (male:female = 1:1; median age: 53 years, age range: 3-90 years) with cutaneous lesions that could be classified as small-/medium-sized pleomorphic T-cell lymphoma according to current diagnostic criteria. All but 3 patients presented with solitary nodules located mostly on the head and neck area (75%). Histopathologic features were characterized by nonepidermotropic, nodular, or diffuse infiltrates of small- to medium-sized pleomorphic T lymphocytes. A monoclonal rearrangement of the T-cell receptor-gamma gene was found in 60% of tested cases. Follow-up data available for 45 patients revealed that 41 of them were alive without lymphoma after a median time of 63 months (range: 1-357 months), whereas 4 were alive with cutaneous disease (range: 2-16 months). The incongruity between the indolent clinical course and the worrying histopathologic and molecular features poses difficulties in classifying these cases unambiguously as benign or malignant, and it may be better to refer to them with a descriptive term such as "cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance," rather than forcing them into one or the other category. On the other hand, irrespective of the name given to these equivocal cutaneous lymphoid proliferations, published data support a nonaggressive therapeutic strategy, particularly for patients presenting with solitary lesions.

Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation

We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90(-/-) mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.

Characterization and optimization of antigen-specific T cell responses during ex vivo expansion of melanoma tumor-infiltrating lymphocytes

Treatment of metastatic melanoma with tumor reactive T cells (adoptive T cell therapy, ACT) is a promising approach associated with a high clinical response rate. However, further optimization of this treatment modality is required to increase the clinical response after this therapy. ACT in melanoma involves an initial phase (pre-REP) of tumor-infiltrating lymphocyte (TIL) expansion ex vivo from tumor isolates followed by a second phase, “rapid expansion protocol” (REP) generating the billions of cells used as the TIL infusion product. The main question addressed in this thesis was how the currently used REP affected the responsiveness of the CD8+ T cells to defined melanoma antigens. We hypothesized that the REP drives the TIL to further differentiate and become hyporesponsive to antigen restimulation, therefore, proper cytokine treatment or other ways to expand TIL is required to improve upon this outcome. We evaluated the response of CD8+ TIL to melanoma antigen restimulation using MART-1 peptide-pulsed mature DC in vitro. Post-REP TILs were mostly hypo-responsive with poor proliferation and higher apoptosis. Phenotypic analysis revealed that the expression of CD28 was significantly reduced in post-REP TILs. By sorting experiment and microarray analysis, we confirmed that the few CD28+ post-REP TILs had superior survival capacity and proliferated after restimulation. We then went on to investigate methods to maintain CD28 expression during the REP and improve TIL responsiveness. Firstly, IL-15 and IL-21 were found to synergize in maintaining TIL CD28 expression and antigenic responsiveness during REP. Secondly, we found IL-15 was superior as compared to IL-2 in supporting the long-term expansion of antigen-specific CD8+ TIL after restimulation. These results suggest that current expansion protocols used for adoptive T-cell therapy in melanoma yield largely hyporesponsive products containing CD8+ T cells unable to respond in vivo to re-stimulation with antigen. A modification of our current approaches by using IL-15+IL-21 as supporting cytokines in the REP, or/and administration of IL-15 instead of IL-2 after TIL infusion, may enhance the anti-tumor efficacy and long-term persistence of infused T cells in vivo.

Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs).

We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-gamma enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity.

Differentiative requirements for human CD8$\sp+$ suppressor lymphocytes

The aim of this research was to characterize the differentiative requirements of human CD8$\sp{+}$ suppressor lymphocytes. We investigated the role of monocytes in cellular interactions required for generation of T suppressor cells (Ts) in pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells (PBMC). We observed that the functional activity of CD8$\sp{+}$ T cells was dependent on the concentration of monocytes in the inductive cultures; at concentrations normally present in peripheral blood, PWM stimulation induced potent suppressor activity, whereas under conditions of moderate monocyte depletion the same phenotypic subset of CD8$\sp{+}$ cells enhanced responses. We also demonstrated that differentiation of CD8$\sp{+}$CD28$\sp{-}$ suppressor cells could be mediated by soluble products elaborated by monocytes and CD4$\sp{+}$ cells, identified as PGE$\sb2$ and IFN$\gamma$ respectively. These two signals were required sequentially to cause Ts induction. That is PGE$\sb2$ was required initially, followed by an IFN$\gamma$-dependent differentiative step. We also explored the possibility that PGE$\sb2$ caused modulation of the IFN$\gamma$ receptor number and/or affinity on CD8$\sp{+}$ cells, which might render these cells responsive to the differentiative effect of the IFN$\gamma$-signal. Using radiolabelled $\sp{125}$I-IFN$\gamma$, direct binding assays demonstrated that 10$\sp{-8}$M PGE$\sb2$ selectively increased the number of receptors on the CD8$\sp{+}$ cells. In contrast CD4$\sp{+}$ cells treated similarly exhibited no significant change in their number of IFN$\gamma$ receptors. These results, thus, suggest a relationship between PGE$\sb2$ induced expression of IFN$\gamma$ receptor and the initial requirement for PGE$\sb2$ in IFN$\gamma$-dependent differentiation of Ts cells. Together, our results suggest a crucial role for PGE$\sb2$ and IFN$\gamma$ in regulation of the immune response. Furthermore, such detailed definition of the differentiative requirements for CD8$\sp{+}$ suppressor cells should provide new insight into fundamental mechanisms of immunoregulation. ^

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

IMMUNOLOGICAL AND CYTOLOGICAL INVESTIGATION OF GAMMA INTERFERON PRODUCTION IN HUMAN T-LYMPHOCYTES INDUCED BY PMA AND PHA (IMMUNOGOLD, CELL CYCLE ANALYSIS)

The present study examined cellular mechanisms involved in the production and secretion of human (gamma)IFN. The hypothesis of this investigation was that (gamma)IFN is an export glycoprotein whose synthesis in human T lymphocytes is dependent on membrane stimulation, polypeptide synthesis in the rough endoplasmic reticulum, packaging in the Golgi complex, and release from the cell by exocytosis.^ The model system for this examination utilized T lymphocytes from normal donors and patients with chronic lymphocytic leukemia (CLL) induced in vitro with the tumor promoter, phorbol 12-myristate 13-acetate (PMA) and the lectin, phytohemagglutinin (PHA) to produce (gamma)IFN. This study reconfirmed the ability of PMA and PHA to synergistically induce (gamma)IFN production in normal T lymphocytes, as measured by viral inhibition assays and radio-immunoassays for (gamma)IFN. The leukemic T cells were demonstrated to produce (gamma)IFN in response to treatment with PHA. PMA treatment also induced (gamma)IFN production in the leukemic T cells, which was much greater than that observed in similarly treated normal T cells. In these same cells, however, combined treatment of the agents was shown to be ineffective at inducing (gamma)IFN production beyond the levels stimulated by the individual agents. In addition, the present study reiterated the synergistic effect of PMA/PHA on the stimulation of growth kinetics in normal T cells. The cell cycle of the leukemic T cells was also responsive to treatment with the agents, particularly with PMA treatment. A number of morphological alterations were attributed to PMA treatment including the acquisition of an elongated configuration, nuclear folds, and large cytoplasmic vacuoles. Many of the effects were observed to be reversible with dilution of the agents, and reversion to this state occurred more rapidly in the leukemic T cells. Most importantly, utilization of a thin section immuno-colloidal gold labelling technique for electron microscopy provided, for the first time, direct evidence of the cellular mechanism of (gamma)IFN production and secretion. The results of this latter study support the idea that (gamma)IFN is produced in the rough endoplasmic reticulum, transferred to the Golgi complex for accumulation and packaging, and released from the T cells by exocytosis. ^

Tumor infiltrating lymphocytes in ovarian cancer.

Several improvements in ovarian cancer treatment have been achieved in recent years, both in surgery and in combination chemotherapy with targeting. However, ovarian tumors remain the women's cancers with highest mortality rates. In this scenario, a pivotal role has been endorsed to the immunological environment and to the immunological mechanisms involved in ovarian cancer behavior. Recent evidence suggests a loss of the critical balance between immune-activating and immune-suppressing mechanisms when oncogenesis and cancer progression occur. Ovarian cancer generates a mechanism to escape the immune system by producing a highly suppressive environment. Immune-activated tumor infiltrating lymphocytes (TILs) in ovarian tumor tissue testify that the immune system is the trigger in this neoplasm. The TIL mileau has been demonstrated to be associated with better prognosis, more chemosensitivity, and more cases of optimal residual tumor achieved during primary cytoreduction. Nowadays, scientists are focusing attention on new immunologically effective tumor biomarkers in order to optimize selection of patients for recruitment in clinical trials and to identify relationships of these biomarkers with responses to immunotherapeutics. Assessing this point of view, TILs might be considered as a potent predictive immunotherapy biomarker.

High intratumoral levels of FoxP3+ lymphocytes are associated with better prognosis in primary resected esophageal adenocarcinomas.

Background: Tumor infiltrating T-lymphocytes (TILs) have been shown to play an important prognostic role in many carcinomas. The identification of prognostic relevant morphological or molecular factors is a major area of interest in the diagnostic process and for the treatment of highly aggressive esophageal adenocarcinoma. Studies about the impact of TILs in this tumor have not shown completely congruent results yet. We present a comprehensive study about the clinical and pathological impact of TIL in esophageal adenocarcinomas. Methods: A next generation tissue microarray (TMA) of 117 primary resected esophageal adenocarcinomas was analyzed for CD3+, CD8+ and FoxP3+ TIL using immunohistochemistry. The TMA contained three cores of the tumor center and the tumor periphery per each case. Slides were scanned with a high-resolution scanner (ScanScope CS; Aperio) and an image analysis software (Aperio Image Scope) was used to determine the TIL counts. The results were correlated with clinicopathological parameters. Results: CD3+, CD8+ and FoxP3+ TIL counts showed a significant correlation among each other (p<0.001 each, range: 0.27-0.77). TIL counts were categorized as high and low levels, according to the median. Tumors with high FoxP3+ intratumoral lymphocyte counts were more frequently of lower pT category (p<0.001) and without lymph node metastasis (p=0.04). High levels of FoxP3+ lymphocytes in the tumor center and the periphery were also associated with better prognosis (p<0.001 and p=0.041, respectively) in univariate analysis. A similar prognostic impact was seen for high levels of CD3+ and CD8+ TIL in the tumor center, but not in the periphery (p=0.047 and p=0.011, respectively). In multivariate analysis high central FoxP3+TIL levels were an independent prognostic factor (HR=0.4; p=0.023) which was similar to a combination score of CD3+/CD8+/FoxP3+ TIL (HR=0.54; p=0.027) or CD8+/Foxp3+ TIL (HR=0.052; p=0.020) and superior to pT- and pN category (p>0.05 each). Conclusion: This study demonstrates a significant beneficial prognostic impact of high TIL counts in the tumor center of esophageal adenocarcinomas, in particular with regards to the subpopulation of FoxP3+ and CD8+ T-regulatory cells. The determination of intratumoral lymphocytic counts and application of TIL scores can improve prognostic accuracy of pathologic reports of these tumors and may be helpful for better risk stratification of esophageal adenocarcinoma patients.

Constitutive IL-2 mRNA expression in lymphocytes, infected with the intracellular parasite Theileria parva.

Theileria parva-infected lymphoblastoid cell lines of T or B cell origin were examined for IL-2 mRNA expression. T. parva-infected T cell lines could be of the CD4-CD8-, CD4+CD8-, CD4-CD8+, or CD4+CD8+ phenotype and express alpha beta or gamma delta TCR. By Northern blot analysis and amplification by the polymerase chain reaction, IL-2 mRNA could be detected in all T. parva-infected cell lines tested. IL-2 mRNA expression was also shown to be dependent on the continuous presence of the parasite in the host cell cytoplasm, because elimination of the parasite by treatment of T. parva-infected cell cultures with the theilericidal drug BW720c resulted in the disappearance of detectable IL-2 mRNA. The effect of anti-IL-2 antibodies on the proliferation of T. parva-infected cells was also tested. Inhibition experiments suggest that although IL-2 mRNA can be detected in all cell lines tested, not all T. parva-infected cell lines are dependent on IL-2 for their proliferation. Our data provide the first example for the constitutive expression of IL-2 mRNA in T and B cells caused by infection with an intracellular parasite.