Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

 The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3⁺PBL) and TIL (CD3⁺TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3⁺TIL and CD3⁺PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3⁺TIL produced significantly lower levels of IL-2 than CD3⁺PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3⁺TIL as compared to the CD3⁺PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3⁺TIL and CD3⁺PBL derived from patients with renal cell carcinomas. Received: 8 August 1995 / Accepted: 23 January 1996     

Effects of transforming growth factor β1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor α

The cytokine transforming growth factor β-1 (TGFβ1), was transfected into a TGFβ1-negative rat colon carcinoma. The growth of isografts of TGFβ1-expressing tumors was compared to that of vector control transfectants. The TGFβ1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFβ1-transfected tumor tissue had significantly greater infiltration of both CD4⁺ and CD8⁺ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFβ1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor α (TNFα) than did TIL from the vector control tumor. The TGFβ1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFβ1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFα on tumor proliferation in vitro. These results suggest that TGFβ1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth. Received: 7 July 1999 / Accepted: 12 August 1999     

Cellular immune responses in acute leukaemia patients with severe chemotherapy-induced leucopenia; characterization of the cytokine repertoire of clonogenic T cells

 T lymphocytes are important both for the host defence against infections and probably also as antileukaemic effector cells in patients with acute leukaemia. To investigate the T lymphocyte cytokine repertoire of clonogenic T lymphocytes, CD4⁺ and CD8⁺ T lymphocyte clones were prepared from acute leukaemia patients with chemotherapy-induced cytopenia (leucocytes <0.5×10⁹/l). A majority of both CD4⁺ and CD8⁺ clones secreted detectable interleukin-2 (IL-2), IL-10, IL-13, granulocyte/macrophage-colony-stimulating factor and interferon γ (IFNγ) in response to phytohaemagglutinin + accessory cells (Epstein-Barr-virus-transformed B cell line, 80-Gy-irradiated). The CD4⁺ clones showed significantly higher levels of IL-10 secretion than the CD8⁺ clones. Decreased levels of IL-2, IL-13 and IFNγ were observed when acute myelogenous leukaemia (AML) blasts were used instead of cells from the B cell line as accessory cells during phytohaemagglutinin activation, but the differences in IL-13 and IFNγ levels were reversed by addition of exogenous IL-2. On the basis of these results we conclude: (i) the remaining clonogenic T lymphocytes derived from acute leukaemia patients with therapy-induced leucopenia can respond to activation with a broad cytokine response, and T-cell-derived cytokines may then contribute to cytokine responses during complicating infections in these patients; (ii) although T cells can modulate AML blast functions and mediate antileukaemic effects, the leukaemia blasts will also modulate T cell functions and alter the cytokine profile of activated T lymphocytes. Received: 6 November 1997 / Accepted: 5 March 1998     

Differential alteration in peripheral T-regulatory and T-effector cells with change in P-glycoprotein expression in Childhood Nephrotic Syndrome: A longitudinal study

IntroductionChildhood Idiopathic Nephrotic Syndrome (INS) responds to glucocorticoid therapy, however, 60–80% of patients relapse and some of them become steroid non responsive. INS may occur because of T cell dysfunction, abnormal cytokines and podocytopathies which reverse on steroid treatment. The reason of relapses could be imbalances in T cells phenotypes and respective cytokines. Herein, we hypothesize that relapses in INS may occur due to imbalance in T-regulatory and T-effector cell with their respective cytokines and overexpression of P-gp on lymphocytes.MethodsThe frequency of peripheral blood CD4⁺CD25⁺FoxP3⁺ Treg, CD4⁺IFN-γ⁺ Th1 and CD4⁺IL-4⁺ Th2 lymphocytes and their respective cytokines and P-gp expression on peripheral blood lymphocytes (PBLs) were analyzed in INS patients at baseline (n = 26), during remission (n = 24) and at relapse (n = 15).ResultsCompared to baseline, the frequency of Tregs was significantly increased at remission and decreased during relapse. In contrast, the frequency of Th1 and Th2 lymphocytes was significantly decreased during remission and increased at the time of relapse. Similarly, expression of P-gp was significantly high at baseline and at the time of relapse as compared to remission. Levels of cytokines IL-10 and TGF-β in the supernatant of stimulated PBMCs was increased during remission and decreased during relapse. In contrast, levels of IFN-γ and IL-4 were decreased during remission and increased at the time of relapse.ConclusionsSteroid therapy in INS induces decreased P-gp expression on PBLs along with increased frequency and cytokine response of T-regulatory cells, and reduced frequency and respective cytokine response of Th1 and Th2 cells during remission. However, reversal in the frequency and respective cytokines of T-regs, Th1 and Th2, and P-gp expression on PBLs occurs during relapses on follow-up.     

Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes

BackgroundNatural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.AimThe aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4⁺ lymphocytes.MethodsPurified human CD4⁺ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf^®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.ResultsNeither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4⁺ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4⁺ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf^®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4⁺ lymphocytes.ConclusionFor the first time, the immunomodulatory capacity of CHF5633 on CD4⁺ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4⁺ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4⁺ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.     

Characterization of interleukin-2-initiated versus OKT3-initiated human tumor-infiltrating lymphocytes from glioblastoma multiforme: Growth characteristics,cytolytic activity,and cell phenotype

Summary Outgrowth of tumor-infiltrating lymphocytes (TIL) from the human primary brain tumor glioblastoma multiforme was achieved by OKT3 initiation (10 ng/ml), followed by sustained expansion by interleukin-2 (IL-2; 200 U/ml). Tumor-infiltrating lymphocyte (TIL) initiation by this process was performed in parallel with the standard IL-2-only method. Of ten tumors, seven yielded TIL in response to OKT3/IL-2, whereas only three of these seven grew after initiation with IL-2 alone. On the basis of cell doubling times, at least 60 doublings, resulting in (hypothetically) up to 10²³ TIL from as few as 2 × 10⁵ cells in tumor suspensions, could be achieved using OKT3/IL-2. OKT3-initiated TIL proliferated in culture for as long as 288 days, although senescence of some cultures occurred at as early as 73 days. Significant heterogeneity of lymphocytes infiltrating the fresh tumors and heterogeneity of resultant TIL phenotype and function were apparent, yet several common trends were noted. In all cases after OKT3 initiation, significant net growth was not apparent until approximately 14 days. In contrast, in the three samples that grew in response to IL-2 alone, log-phase growth was always observed earlier. During the early phase of the cultures, all TIL expressed some killing activity toward a broad spectrum of tumors, including the autologous tumor. No consistent preference of TIL for lysis of autologous tumor was observed. Glioblastoma multiforme TIL cultures contained a mixture of CD8⁺ and CD4⁺ cells, with few CD16⁺ or NKH-1⁺. Of the six TIL examined in detail for population phenotype in relationship to time in culture, four eventually became exclusively CD4⁺. Further analysis of these CD4⁺ TIL indicated that all were of the helper-inducer class, 4B4⁺ and 2H4^–. Concurrent with the decline in CD8⁺ cells, a decline in the cytolytic activity of these TIL cultures occurred. Furthermore, in two TIL that remained CD8⁺, a decline in the cytolytic activity also occurred. Therefore, loss of killing activity was not merely a reflection of the major cell phenotype changes. These results indicate that the OKT3/IL-2 process provides an alternative to IL-2 alone for TIL initiation and growth, as well as providing a novel system for further analysis of tumorderived lymphocyte and accessory cell functional potential.This work was supported by a grant from the Dunn Foundation, Houston, Texas     

Intracellular cytokine profiles and T cell activation in pulmonary sarcoidosis

In granulomatous inflammatory lung diseases such as sarcoidosis, the balance of cytokine production by activated T cells in the lungs may influence clinical disease outcome. To investigate the potential of T lymphocytes to produce cytokines and contribute to this process, T cells from bronchoalveolar lavage (BAL) and PB from 19 patients with active lung disease were stimulated, stained, and analysed by flow cytometry for intracellular production of cytokines and expression of the activation marker CD69. Higher proportions of BAL cells expressed CD69 compared with PB, in the absence of in vitro stimulation. The expression of IFN-γ was similar in unstimulated BAL and PB T cells, and there was no association between the expression of CD69 and IFN-γ. Following stimulation, there were increased numbers of IFN-γ⁺ T cells. A similar trend was found with IL-2⁺ T cells, but there were lower levels of IL-4⁺ T cells in BAL compared with PB, and similar levels of IL-10⁺ T cells. The presence of activated T lymphocytes in BAL samples from patients with sarcoidosis, with the potential to produce Th1 type 1 cytokines may contribute to the inflammatory processes in this granulomatous lung disease. The use of intracellular flow cytometry to investigate cytokine production by BAL T cells could help to indicate potential targets for future therapy.     

High expression of adhesion molecules/activation markers with little interleukin-2, interferon γ, and tumor necrosis factor β gene activation in fresh tumor-infiltrating lymphocytes from lung adenocarcinoma

Tumor-infiltrating lymphocytes (TIL) were derived from primary breast tumors, metastatic lymph nodes and malignant pleural effusions from 34 patients with breast cancer. TIL were cultured for approximately 30 days and studied for phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor stimulation. Tumor specimens were obtained from two different sites in 7 patients, resulting in 41 samples from which 38 TIL cultures were established. In addition to screening 38 bulk TIL cultures, TIL from 21 patients were separated into CD4⁺ and CD8⁺ subsets and extensively studied. Three CD4⁺ TIL were found specifically to secrete granulocyte macrophage-colony-stimulating factor and tumor necrosis factor when stimulated by autologous tumor and not by a large panel of stimulators (24–34) consisting of autologous normal cells, allogeneic breast or melanoma tumors and EBV-B cells. This cytokine release was found to be MHC-class-II-restricted, as it was inhibited by the anti-HLA-DR antibody L243. These 3 patients' EBV-B cells, when pulsed with tumor lysates, were unable to act as antigen-presenting cells and induce cytokine secretion by their respective CD4⁺ TIL. These findings demonstrate that MHC-class-II-restricted CD4⁺ T cells recognising tumor-associated antigens can be detected in some breast cancer patients.     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

Specific immune recognition of autologous tumor by lymphocytes infiltrating colon carcinomas: Analysis by cytokine secretion

Summary Tumor-infiltrating lymphocytes (TIL) were grown in the presence of interleukin-2 from 19 colon carcinoma specimens, including 1 primary lesion and 18 metastatic lesions. These cultures showed a median proliferation of 606-fold (range 13-fold to 28 000-fold) over 49 culture days (range 26–76 days). By phenotype, mature cultures were 69%–99% CD3⁺ (mean 93%) and contained mixed populations of CD4⁺ and CD8⁺ cells (CD4>CD8 in 10 of 19 cultures). Fresh cryopreserved colon tumors were not lysed by autologous TIL in short-term⁵¹Cr-release assays, and were poorly lysed by lymphokine-activated killer cells. Ten TIL cultures were assayed for cytokine secretion in response to autologous and allogeneic tumors during a 6- to 24-h coincubation. Culture supernatants were tested by ELISA for the presence of granulocyte/macrophage-colony-stimulating factor, interferon , and tumor necrosis factor . Of 10 TIL, 4 secreted at least two of these cytokines specifically in response to autologous and/or HLA-matched fresh allogeneic colon carcinomas, but not to melanomas or HLA-unmatched colon carcinomas. Cytokine secretion was mediated by both CD4⁺ and CD8⁺ TIL, and could be inhibited by mAb directed against the appropriate class of MHC antigen. These data provide evidence for specific, MHC-restricted immune recognition of human colon carcinomas by T lymphocytes.     

Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 22 human primary and metastatic liver tumors, and expanded in vitro in the presence of either interleukin-2 (IL-2, 100 U/ml) plus tumor necrosis factor (TNF, 1000 U/ml), IL-2 (1000 U/ml) plus IL-4 (1000 U/ml) or IL-2 (1000 U/ml) alone. TIL proliferated in culture in 20/22 cases. Among different cytokine combinations, TNF and IL-2 were most effective in promoting the outgrowth of CD3⁺ CD8⁺ T lymphocytes (mean ± SEM: 90%±5) in the cultures of TIL from primary liver tumors. Cytotoxicity against autologous tumor cells was demonstrated in all early cultures of TIL from primary liver cancers in the presence of IL-2 plus TNF. In contrast, cultures of TIL derived from colon cancer metastatic to liver had significantly lower levels of autotumor cytotoxicity and proportions of CD3⁺ CD8⁺ cells (40%±13) than those of TIL from primary liver tumors. The addition on day 0 of interferons ( or ) to TIL cultured in the presence of TNF and IL-2, significantly augmented cytotoxicity against autologous tumor. In contrast, incubation of TIL in the presence of IL-4 and IL-2 did not result in increased autotumor responses in the cultures of TIL from primary liver tumors. The expansion (-fold) of TIL (day 30) cultured in the presence of IL-2 alone compared to that in the presence of TNF and IL-2 was significantly greater for hepatocellular carcinoma (median, 280 vs 260) than for autologous peripheral blood lymphocytes (36 vs 27), cholangiocarcinoma (42 vs 51) or TIL from metastatic colon cancer (39 vs 30). Outgrowth of TIL in IL-2 plus TNF offers an opportunity for in vitro enrichment in cells with autotumor cytotoxicity in primary liver tumors. However, this cytokine combination was unable to promote and sustain growth of autotumor effectors from TIL in metastatic liver cancer.Supported by ACS grants IM27 077 and IM588 A (TLW) and Organ Transplant Program Project 1P01-CA-4744501 AZ     

Phenotypic and functional analysis of lymphocytes infiltrating paediatric tumours,with a characterization of the tumour phenotype

Summary Tumour-infiltrating lymphocytes (TIL) of paediatric tumours obtained from 37 lesions of different histo-type (12 osteosarcomas, 5 Wilms' tumours, 7 soft-tissue sarcomas, 5 neuroblastomas and 8 miscellaneous) were studied to establish their potential for therapy. Fresh isolated TIL were cultured for the first 2 weeks with low doses of interleukin-2 (IL-2) (20 Cetus U/ml) to select for tumour-specific lymphocytes potentially present in the neoplastic lesion, followed by culture with high doses of IL-2 (1000 Cetus U/ml) to achieve TIL expansion. TIL were grown with more than 10-fold expansion in only 9 cases (mean expansion: 58-fold, range 13.5–346). In 17 cases no viable cells were obtained. After 30 days of culture with IL-2 the proliferative ability of TIL declined sharply in the majority of cases and TIL became refractory to any further stimulus, including addition of IL-4, tumour necrosis factor (TNF) or interferon , and activation with OKT3 in solid phase. In 20 out of 37 cases TIL were available for phenotypic and functional analysis. TIL after long-term culture were predominantly CD3⁺ but 2 cases of osteosarcoma showed a predominance of CD3⁺TcR / cells. The CD4/CD8 ratio was more than 1 in 10 cases, without correlation with tumour histology, site of lesion or TIL growth. The number of CD16⁺ and CD25⁺ lymphocytes decreased progressively during culture, the latter concomitantly with a reduction of TIL growth rate. The lytic pattern of TIL against allogeneic and autologous tumour (Auto-Tu) cells was variable, but specific lysis of Auto-Tu was seen in only one case (Wilms' tumour) after culture with TNF and irradiated Auto-Tu cells. The immunohistochemical analysis of tumour lesions revealed a limited lymphocyte infiltrate, a low expression of histocompatibility leukocyte antigens (HLA) class I and of the adhesion molecules ICAM1, LFA3, and a significant production of transforming growth factor (TGF). These data indicate that TIL obtained from paediatric patients are difficult to expand at levels required for immunotherapy and lack a significant number of tumour-specific T lymphocytes. A low expression of immunomodulatory molecules on tumour cells or the production of suppressive factors may prevent activation and expansion of TIL in paediatric tumours.     

IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics

Background

Virus infections are the major cause of asthma exacerbations. CD8⁺ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8⁺ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8⁺ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8⁺ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.

Methods

Peripheral blood CD8⁺ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.

Results

Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8⁺ T cells from mild atopic asthmatics.

Conclusion

These data suggest that during a respiratory virus infection activated CD8⁺ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.     

Effect of cytokines (IL-2, IL-7, and IL-15) having common γ-chain of receptors on differentiation and maturation of CD4+/CD8+ T-cells in a CD45RA T-lymphocyte population in vitro

The effect of cytokines (IL-2, IL-7, and IL-15) having a common γ-chain of receptors on the maturation and differentiation of CD3⁺CD45RA⁺CD4⁺/CD8⁺ lymphocytes in homeostatic cultivation model in vitro was analyzed. It was found that the maximum IL-2 concentration in the helper CD45RA⁺-T-cell population mediates an increase in the number of CD45RA⁺CD4⁺ T lymphocytes with the phenotype of mature and immature terminally differentiated TEMRA T cells. IL-15 leads to the production of lymphocytes with CD27^–CD62L⁺ phenotype (presumably, TEMRA, in which the CD62L expression persists). In the CD45RA⁺CD8⁺ T lymphocyte populations, the studied cytokines (IL-2, IL-7, and IL-15) initiate the production of mature TEMRA (E) T lymphocytes and memory T cells with the CD45RA^?CD27⁺CD62L⁺ central phenotype (TCM).     

TcVac3 Induced Control of Trypanosoma cruzi Infection and Chronic Myocarditis in Mice

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8⁺T cell response with type-1 cytokine (IFN-γ⁺TNF-α>IL-4⁺IL-10) and cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ⁺CD8⁺T cells, a predominance of immunoregulatory IL-10⁺/CD4⁺T and IL10⁺/CD8⁺T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8⁺T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.     

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4⁺ and CD8⁺ TCRαβ⁺ T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin+IL-2+ B lymphocyte accessory cells). The CD4⁺ cells showed significantly higher IL-13 levels than the CD8⁺ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon γ levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells. Received: 24 April 1997 / Accepted: 24 July 1997     

Efficient expansion of tumor-infiltrating lymphocytes from solid tumors by stimulation with combined CD3 and CD28 monoclonal antibodies

Combined CD3 and CD28 monoclonal antibodies (mAb) may initiate efficient activation and expansion of tumor-infiltrating lymphocytes (TIL). In this study we compared phenotypical and functional characteristics of TIL from a group of 17 solid human tumors, stimulated either by high-dose recombinant interleukin 2 (rIL-2, 1000 IU/ml) or by a combination of anti-CD3 and anti-CD28 monoclonal antibodies in the presence of low-dose rIL-2 (10 IU/ml). Compared to activation with high-dose rIL-2, stimulation of TIL with CD3/CD28 mAb induced significantly stronger proliferation and yielded higher levels of cell recovery on day 14. Following the CD3/CD28 protocol, expansion of an almost pure population of CD3⁺ cells was obtained. Whereas CD4⁺ cells dominated in the first week of culturing, within 4 weeks the CD8⁺ population increased to over 90%. The specific capacity to kill autologous tumor cells was not increased as compared to the high-dose rIL-2 protocol, but all cultures showed high cytotoxic T cell activity as measured in a CD3-mAb-mediated redirected kill assay. These studies show that combined CD3 and CD28 mAb are superior to rIL-2 with respect to the initiation of expansion of CD8⁺ cytolytic TIL from solid tumors. Stimulation with specific tumor antigens at a later stage of culturing may further augment the expansion of tumor-specific cytolytic T cells.