A purified protein from Salmonella typhimurium inhibits proliferation of murine splenic anti-CD3 antibody-activated T-lymphocytes

Abstract In a previous study, we observed that the purified substance Salmonella typhimurium -derived inhibitor of T-cell proliferation (STI) had an immunosuppressive effect, demonstrated as the suppression of mitogenic lectin-induced proliferation of murine spleen cells. In the present study, we confirmed the immunosuppressive effect of STI, which suppressed the proliferation of murine splenic T-lymphocytes activated with the anti-CD3 antibody (Ab) and phorbol 12-myristate-13 acetate (PMA) and this phenomenon was accompanied by augmentation of interferon-γ (IFN-γ) secretion and inhibition of interleukin-2 (IL-2) secretion. Furthermore, the augmentation of IFN-γ secretion caused IL-2 receptor α chain (IL-2R α) over expression on T-cells. However, the addition of an anti-IFN-γ Ab and recombinant IL-2 (rIL-2) did not reverse the suppressed T-cell proliferation, although the level of IL-2R α expression on T-cells recovered to around normal. Furthermore, Western blotting using an anti-phosphotyrosine Ab showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in T-cells was inhibited by incubation with STI for 48 h and this inhibition was not reversed by adding the anti-IFN-γ Ab and rIL-2. These results suggest that STI-induced suppression of T-cell proliferation involves a defect in IL-2R function and/or IL-2 signaling pathway in T-cells.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Chemical modification of macrophages enhances their response to human macrophage activation factor

Human monocyte-derived macrophages pretreated with the esterase inhibitors, antithrombin III and soybean trypsin inhibitor (STI) showed increased responsiveness to limiting concentrations of macrophage activation factor (MAF) as demonstrated by their enhanced cytotoxicity for tumor cells. Other proteins that are not esterase inhibitors did not enhance the effect of MAF on the macrophages. Enhancement of MAF activity was also obtained when macrophages were preincubated with the cell surface reactant diazotized sulfanilic acid (DSA). When MAF was preincubated with untreated, DSA-treated or STI-treated macrophages prior to testing on fresh macrophages, MAF preincubated with the untreated macrophages lost activity whereas the cytotoxicity induced by MAF preincubated with DSA-treated or STI-treated macrophages showed no decrease. These findings suggest that the enhanced responsiveness to MAF is the result of decreased destruction of MAF by macrophage-associated proteinases. Thus, the response of guinea pig and human macrophages to MAF appears to be regulated by similar mechanisms.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

Failure of CD4 T-cells to respond to liver-derived antigen and to provide help to CD8 T-cells

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.     

Definition and application of good manufacturing process-compliant production of CEA-specific chimeric antigen receptor expressing T-cells for phase I/II clinical trial

Adoptive cell therapy employing gene-modified T-cells expressing chimeric antigen receptors (CARs) has shown promising preclinical activity in a range of model systems and is now being tested in the clinical setting. The manufacture of CAR T-cells requires compliance with national and European regulations for the production of medicinal products. We established such a compliant process to produce T-cells armed with a first-generation CAR specific for carcinoembryonic antigen (CEA). CAR T-cells were successfully generated for 14 patients with advanced CEA⁺ malignancy. Of note, in the majority of patients, the defined procedure generated predominantly CD4⁺ CAR T-cells with the general T-cell population bearing an effector–memory phenotype and high in vitro effector function. Thus, improving the process to generate less-differentiated T-cells would be more desirable in the future for effective adoptive gene-modified T-cell therapy. However, these results confirm that CAR T-cells can be generated in a manner compliant with regulations governing medicinal products in the European Union.     

Comparison of influenza and SIV specific CD8 T cell responses in macaques

Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8(+) T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8(+) T-cells in 39 pigtail macaques expressing the common Mane-A*10(+) (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8(+) T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8(+) T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8(+) effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8(+) T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8(+) T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8(+) T-cell response, profile may assist in controlling HIV disease.     

Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus

Introduction  

Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by a disturbed T-cell balance skewed towards effector T-cells, in particular Th17-cells. The novel cytokine interleukin-21 (IL-21) is suggested to be crucial for triggering T-cell responses towards IL-17 producing cells. Thus, we aimed to investigate the ability of T-cells to produce IL-21 and IL-17 in SLE patients.     

Monitoring of peripheral blood cytotoxic T-cells under fluvastatin treatment in renal transplant recipients

Flow cytometric T-cell analysis is capable of adding valuable information for balancing immunosuppression in transplant recipients as it can take into account individual effects of immunosuppressive drugs on each patient as well as effects of other drugs which may modify the overall immunosuppression. Studies suggest that HMG-CoA-reductase-inhibitors (statins) reduce the frequency of organ rejection, although the precise mechanism of this effect is unknown. We therefore evaluated the effect of fluvastatin on size and activation of T-cell subpopulations and NK-cell activity in renal transplant recipients. At baseline, the population size of activated (HLA-DR+) T-cells was negatively correlated to serum HDL cholesterol suggesting an increased T-cell activation at low HDL levels. Fluvastatin treatment of a hypercholesterolemic group of patients for two months significantly decreased the LDL cholesterol. A longitudinal analysis revealed a relative increase in non-MHC restricted cytotoxic T-cells (CD3+/CD16+ or CD56+) over time which was significantly attenuated in fluvastatin treated patients but not in normocholesterolemic controls. Moreover, a relative decrease of activated MHC class I-restricted cytotoxic CD8+ T-cells was only observed upon fluvastatin treatment. NK-cell number and activity did not differ between groups. In summary, fluvastatin treatment of hypercholesterolemic renal transplant recipients is associated with a specific modulation of T-cells exerting cytotoxic effector functions.     

Hydrolysis of soybean trypsin inhibitor by the gamma subunit of 7SNGF and EGF-BP

Although soybean trypsin inhibitor (STI) does not inhibit the esterase activity of either epidermal growth factor binding protein (EGF BP) or the gamma subunit of 7SNGF, it does behave as a substrate for proteolysis. Cleavage of the active site peptide bond of STI does occur when incubated in the presence of either EGF-BP or the gamma subunit of 7SNGF. The hydrolysis id pH dependent with maximum proteolysis at pH 6.0-7.0. the newly formed C-terminal arginine residue in modified STI can be released by carboxypeptidase B digestion. Both enzymes are inhibited by low concentrations (2-4 microgram/ml) of the microbial protease inhibitors leupeptin and antipain. These inhibitors are specific for trypsin-like proteases. Since both enzymes can be found as part of high molecular weight complexes with growth factors these results confirm the hypothesis that they are involved during a postranslational modification event.     

Alterations in the phenotype of CMV-specific and total CD8+ T-cell populations in Wegener's granulomatosis

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.     

A method for the analysis of biological transduction phenomena

Biological transduction can be defined as the triggering of a cellular response by the binding of molecules of effector substances to specific cellular sites. An example of biological transduction, analyzed in this report, is the triggering of T-cell proliferation by the binding of T-cell growth factor (TCGF) to specific TCGF-binding sites on responsive T-cells. Sigmoidal or S-shaped curves often result when measurements of biological response are plotted as a function of concentration of effector substance. Such curves suggest that effector molecules must bind a critical number of cellular sites, and this critical number of bound complexes must undergo secondary events (cross-linking, association, internalization, second messenger release, etc.) in order to initiate the biological response. The method described here estimates the critical number of cellular sites (R) and the probability of these secondary events (PS/B) as follows: (1) The total number of cellular sites (N) is estimated from binding data, and the probabilities (PB) of effector molecules binding to a site are estimated from response data. (2) The response data are assumed to follow the summed binomial distribution function, which is equated to the incomplete beta function. (3) R and PS/B are estimated by applying nonlinear regression to the incomplete beta function. The T-cell data to which the method was applied gave N = 15,000, R = 5, and PS/B = 7.22 x 10(-4). These results show that the binding of very few TCGF molecules is required for activation of T-cells and that the probability of the secondary events leading to cell proliferation is much smaller than the probability of TCGF binding to T-cells. The method described can be used to analyze any biological transduction experiments where both binding and biological response data are available.     

T-cell inhibition of humoral responsiveness. I. Experimental evidence for restriction by the K- and/or D-end of the H-2 gene complex.

The inhibition (“suppression”) of an in vitro antibody response to SRBC by T-cells with specificity for histocompatibility antigens, is H-2 K/D-restricted whether or not the target determinant is H-2 I-region or non-H-2 encoded. By contrast, cytotoxic T-cells specific for histocompatibility antigens mediate a lysis of targets that is K/D-restricted in all cases except one: when the target determinant is H-2 I-region encoded, lysis is unrestricted. Further, it is shown that the target determinant and the restricting element must be on the same cell for effector function to be mediated. A spleen population immunized with histocompatibility antigens contains inhibitory and cytotoxic T-cells separable when I-region encoded determinants are the target. Thus, we conclude that the cytotoxic T-cell does not function as an inhibitory T-cell and vice versa. If the findings with this model experimental system may be extrapolated to the regulation of normal in vivo immune responsiveness then two K/D-restricted separable T-cell populations must mediate cytotoxicity and inhibition of humoral responsiveness. This finding, that with I-region encoded targets only, inhibitory T-cells are K/D-restricted whereas cytotoxic T-cells are not, raises questions about the mechanism of restrictive recognition which are dealt with in the context of the “two signal” model.     

Enhanced Inflammatory Potential of CD4+ T-Cells That Lack Proteasome Immunosubunit Expression,in a T-Cell Transfer-Based Colitis Model

Proteasomes play a fundamental role in intracellular protein degradation and therewith regulate a variety of cellular processes. Exposure of cells to (pro)inflammatory cytokines upregulates the expression of three inducible catalytic proteasome subunits, the immunosubunits, which incorporate into newly assembled proteasome complexes and alter the catalytic activity of the cellular proteasome population. Single gene-deficient mice lacking one of the three immunosubunits are resistant to dextran sulfate sodium (DSS)-induced colitis development and, likewise, inhibition of one single immunosubunit protects mice against the development of DSS-induced colitis. The observed diminished disease susceptibility has been attributed to altered cytokine production and CD4⁺ T-cell differentiation in the absence of immunosubunits. To further test whether the catalytic activity conferred by immunosubunits plays an essential role in CD4⁺ T-cell function and to distinguish between the role of immunosubunits in effector T-cells versus inflamed tissue, we used a T-cell transfer-induced colitis model. Naïve wt or immunosubunit-deficient CD4⁺ T-cells were adoptively transferred into RAG1^−/− and immunosubunit-deficient RAG1^−/− mice and colitis development was determined six weeks later. While immunosubunit expression in recipient mice had no effect on colitis development, transferred immunosubunit-deficient T- cells were more potent in inducing colitis and produced more proinflammatory IL17 than wt T-cells. Taken together, our data show that modifications in proteasome-mediated proteolysis in T-cells, conferred by lack of immunosubunit incorporation, do not attenuate but enhance CD4⁺ T-cell-induced inflammation.     

Troybodies and pepbodies

All antibodies (Abs) with effector function are produced in mammalian cells, whereas bacterial production is restricted to smaller targeting fragments (scFv and Fab) without effector functions. In this project, we isolated different peptides that bind one of several Ab effector molecules. We have developed bacterial expression vectors for direct cloning of these peptides as fusions to scFv and Fab, and have obtained targeting fragments that also have the ability to bind Ab effector molecules. Some of these fusions (pepbodies) may also initiate Ab effector functions. We have also genetically inserted T-cell epitopes into Abs with specificity for antigen-presenting cell (APC) surface molecules to target the Ab-T-cell epitope fusions (Troybodies) to APCs. The approach is to exchange loops in Ig constant domains with single copies of well-defined T-cell epitopes. We have shown that a number of such T-cell epitopes are loaded on to MHC class II on APCs and are presented to specific T-cells. An increase in T-cell activation of up to four orders of magnitude is achieved compared with synthetic peptide. Our current goal is to identify all the loops in all Ig constant domains that may be loaded with T-cell epitopes to produce a multi-vaccine.     

Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age

The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age.     

Mechanism and specificity of macrophage-mediated cytotoxity.

The cytotoxic effect of macrophages derived from alloimmunized mice (immune macrophages) was found to be immunologically specific. The immune macrophages killed only target macrophages carrying the alloantigens used for immunization in mixed macrophage cultures (MMC) under optimal conditions of contact between effector and target cells. T-sensitized lymphocytes, but not B cells, were capable of arming nonimmune macrophages and conferring upon them cytotoxic activity; the arming factor, which seemed to be a T mediator or T-cell receptor (membrane component) was removable by trypsin. Frequent rinsing or addition of hydrocortisone significantly decreased the cytotoxicity of the MMC. Pretreatment of peritoneal cells with anti-θ antisera and complement markedly decreased immune macrophage cytotoxic activity. It is suggested that the presence of a very small number of T-sensitized lymphocytes is required for strong cytotoxic activity to be manifested by the macrophages.