bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐reactive memory T cells for Epstein‐Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex‐bound peptide by T cells and its role in alloreactivity
Authors: Burrows Scott R. , Silins Sharon L. , Khanna Rajiv , Burrows Jacqueline M. , Rischmueller Maureen , McCluskey James , Moss Denis J.
Published: March 2007

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein‐Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV‐exposed individuals, and each distinct cross‐reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross‐reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft‐versus‐host disease. We have also explored the molecular basis for T cell cross‐reactivity with alloantigens using the HLA B35 allo‐reactive CTL clonotype. To elucidate the structural features of peptides that may be cross‐recognized by these T cells, mono‐substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)‐bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self‐MHC‐restricted T cells to alloantigens.
Maternal immunoglobulins have no effect on the rate of maturation of the B cell compartment of the offspring
Authors: Delassus Sylvie , Darche Sylvie , Kourilsky Philippe , Cumano Ana
Published: March 2007

To elucidate a general role of maternal immunoglobulins (Ig) on the kinetics of B cell development of the offspring, we studied non‐genetic influences of maternal Ig on the developing immune system of B cell‐competent mice. These animals were the offsprings of either B cell‐deprived μMT or of normal C57BL/6 females. In these mice, we have compared the kinetics of Ig production, the numbers of B cell progenitors, the expression of surface markers specific of the B lineage and the progression of Ig variable gene expression. We show that the absence of maternal Ig has no detectable effect on the kinetics of IgM and IgG production by the offspring's immune system. The number of B cell precursors, the kinetics of generation of B cells and their pattern of surface markers expression is identical in both types of mice. The acquisition of diversity in the B cell repertoire and the changes in the ratios of variable gene family expression are also indistinguishable. We conclude that maternally derived Ig has no influence on the rate of development and maturation of the B cell compartment of the offspring.
Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice
Authors: Neurath Markus F. , Fuss Ivan , Pasparakis Manolis , Alexopoulou Lena , Haralambous Sylva , Meyer zum Büschenfelde Karl‐Hermann , Strober Warren , Kollias George
Published: March 2007

Antibodies to tumor necrosis factor (TNF)‐α have been recently proposed as effective treatment for patients with Crohn's disease. Here, we analyze the functional role of TNF‐α in a mouse model of chronic intestinal inflammation induced by the hapten reagent 2,4,6,‐trinitrobenzene sulfonic acid (TNBS) that mimics some characteristics of Crohn's disease in humans. Macrophage‐enriched lamina propria (LP) mononuclear cells from mice with TNBS‐induced colitis produced 10–30‐fold higher levels of TNF‐α mRNA and protein than cells from control mice. When mice with chronic colitis were treated by intraperitoneal injection of antibodies to TNF‐α, an improvement of both the clinical and histopathologic signs of disease was found. Isolated macrophage‐enriched LP cells from anti‐TNF‐α‐treated mice produced strikingly less pro‐inflammatory cytokines such as interleukin (IL)‐1 and IL‐6 in cell culture. The predominant role of TNF‐α in the mouse TNBS‐induced colitis model was further underlined by the finding that striking colonic inflammation and lethal pancolitis was induced in TNF‐α‐transgenic mice upon TNBS treatment. Conversely, no significant TNBS‐induced colitis could be induced in mice in which the TNF‐α gene had been inactivated by homologous recombination. Complementation of TNF‐α function in TNF−/− mice by the expression of a mouse TNF‐α transgene was sufficient to reverse this effect. Taken together, the data provide direct evidence for a predominant role of TNF‐α in a mouse model of chronic intestinal inflammation and encourage further clinical trials with antibodies to TNF‐α for the treatment of patients with Crohn's disease.
CD5− CD8αβ intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen‐specific activation: CD5− and CD5+ CD8αβ IEL do not represent separate T cell lineages
Authors: Müller Stefan , Jungo Myriam , Aichele Peter , Mueller Christoph
Published: March 2007

We followed αβ T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I‐restricted αβ TCR‐transgenic (tg) mice. The proportion of tg αβ TCR+ CD8αβ IEL is reduced compared with CD8+ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8αβ IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR+ CD5− CD8αβ IEL are as frequent as in specific pathogen‐free (SPF) mice, whereas tg TCR+ CD5int or, even more pronounced, tg TCR+ CD5hi CD8αβ IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen‐specific stimulation of CD5− CD8αβ IEL in vitro, CD5 surface expression is up‐regulated on a large fraction of cells within 48 h. Up‐regulation of CD5 surface expression is further enhanced by the presence of the anti‐αIEL monoclonal antibody 2E7. This clearly demonstrates that CD5−, and CD5+ CD8αβ IEL cannot be considered as separate T cell lineages.
Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope
Authors: Kaneko Takashi , Moriyama Takashi , Udaka Keiko , Hiroishi Kazumasa , Kita Hiroto , Okamoto Hiroaki , Yagita Hideo , Okumura Ko , Imawari Michio
Published: March 2007

An epitope that acted as a weak agonist in the cytotoxicity assay was identified as part of the capsid protein of a hepatitis C virus (HCV) variant. In a low concentration, the variant epitope also had a weak antagonistic effect. When a minute amount of this variant epitope was added to the culture for induction, it selectively attenuated the expansion of major cytotoxic T cell populations and drastically reduced the cytotoxic responses against the wild‐type epitope. Thus, antagonism to induction suppressed immune responses against both the wild type and the variant, thereby helping the persistence of not only variant itself but also the wild‐type HCV. Because this variant was a weak agonist, most cytotoxic T cells induced with the wild‐type epitope were cross‐reactive with the variant and susceptible to the antagonism to induction. Only the T cells which were not cross‐reactive with the variant and not susceptible to the antagonism survived the antagonism in induction. This implied that the specificity of the remaining immune response, if any, was directed exclusively to the wild‐type epitope after the emergence of the variant. For viruses like HCV, being heterogeneous itself may contribute significantly toward persistent infection through antagonism to induction.
Organ‐selective regulation of vascular adhesion protein‐1 expression in man
Authors: Arvilommi Anna‐Maija , Salmi Marko , Jalkanen Sirpa
Published: March 2007

Vascular adhesion protein‐1 (VAP‐1) is an endothelial molecule which mediates lymphocyte binding to endothelium in peripheral lymph nodes and at certain sites of inflammation. The expression of VAP‐1 in vivo is strongly up‐regulated in inflamed tissues, such as gut and skin. The purpose of this work was to examine the factors responsible for this induction of VAP‐1. Since the expression of VAP‐1 could not be induced in cultured endothelial cells with a large panel of mediators, we used an organ culture technique for the investigation of the regulation of VAP‐1 expression in a more physiological micromilieu. Indeed, we found that the expression of endothelial VAP‐1 could be up‐regulated in human tonsillar tissue with interleukin (IL)‐1, IL‐4, tumor necrosis factor (TNF‐α), interferon (IFN)‐γ and lipopolysaccharide, whereas histamine, thrombin, dibutyryl cAMP, N‐formyl‐Met‐Leu‐Phe (fMLP) and phorbol 12‐myristate 13‐acetate (PMA) had no effect. The induced VAP‐1 protein was similar in molecular weight to the non‐induced VAP‐1, suggesting that VAP‐1 synthesized de novo carries appropriate carbohydrate moieties. In contrast to tonsil organ culture, similar inductions performed with human appendix showed no up‐regulation of VAP‐1 expression, indicating that the regulation of VAP‐1 expression exhibits organ‐selective characteristics. Furthermore, in these tissues the smooth muscle cells, which constitutively express VAP‐1, could not be stimulated to alter their level of expression of this molecule. In conclusion, the expression of VAP‐1 can be markedly up‐regulated with several mediators in tonsil but not in appendix organ culture, whereas cultured endothelial cells cannot be induced to express VAP‐1. These results indicate that the expression of VAP‐1 is regulated in a tissue‐ and cell type‐selective manner, and a correct micromilieu is required for the up‐regulation to occur.
G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated activated/memory phenotype CD4+ cells of older lupus mice
Authors: Sabzevari Helen , Propp Stephanie , Kono Dwight H. , Theofilopoulos Argyrios N.
Published: March 2007

A general characteristic of lupus‐prone mice (and humans) is the expedited accumulation of large numbers of presumably self‐reactive activated/memory phenotype T cells. The mechanism by which these cells escape apoptosis has not been defined. We used activated/memory phenotype CD4+ cells from male BXSB mice with early‐life severe lupus‐like disease to investigate cell cycle status and apoptosis susceptibility, and to determine the role of corresponding genes in survival of these cells. In vitro acridine orange staining indicated that most of the rapidly accumulating memory phenotype CD4+ T cells of 4‐month‐old male BXSB mice are G1 arrested. Long‐term bromodeoxyuridine in vivo labeling also showed that with advanced age, there was a shift of the CD4+ CD44hi male cells from predominantly cycling to predominantly noncycling. Moreover, the CD4+ CD44hi cells of older males were refractory to anti‐CD3‐induced proliferation and apoptosis. Using a multiprobe RNase protection assay encompassing riboprobe panels for cell cycle and apoptosis‐related genes, we found that these cells exhibited high expression of certain members of the Ink4 (p18Ink4C) and Cip/Kip (p21Cip1) families of cyclin kinase inhibitors as well as of the apoptosis‐inhibiting Bcl‐xL gene. Western blot analysis confirmed increased levels of Bcl‐xL and p21Cip1, and also identified increases in another cyclin kinase inhibitor, p27Kip1. We propose that in autoimmunity, self‐reactive CD4+ cells are subjected to successive rounds of activation/division that eventually lead to a build‐up in cyclin‐dependent kinase inhibitors. Once high levels of such inhibitors are reached, they cause refractoriness to further activation, impaired cell cycle entry and resistance to apoptosis, a situation akin to replicative senescence.
Affinity of the interaction between Fcgamma receptor type III (FcγRIII) and monomeric human IgG subclasses. Role of FcγRIII glycosylation
Authors: Galon Jérǒme , Robertson Michael W. , Galinha Annie , Maziéres Noëlle , Spagnoli Roberto , Fridman Wolf‐Herman , Sautès Catherine
Published: March 2007

Binding of the Fc region of IgG antibodies to low affinity Fcγ receptors (FcγR) triggers important effector functions in the immune system. The type IIIb FcγR (FcγRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand‐receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of FcγRIIIb (sFcγRIIIb or sCD16) corresponding to the 188 N‐terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non‐glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcoreTM instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen‐binding fragments (F(ab′)2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (KA) of 1.3 ± 0.6 × 106 M−1 and 2.6 ± 0.4 × 105 M−1, respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 ± 0.2 × 106 M−1), whereas that for human IgG3 was twofold higher (4.2 ± 0.4 × 105 M−1). The specificity of the non‐glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 ≫ IgG4 ⋙ IgG2. Thus, the extracellular polypeptide of FcγRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.
Tumor necrosis factor blockade in actively induced experimental autoimmune encephalomyelitis prevents clinical disease despite activated T cell infiltration to the central nervous system
Authors: Körner Heinrich , Lemckert Frances A. , Chaudhri Geeta , Etteldorf Susanne , Sedgwick Jonathon D.
Published: March 2007

Recently, we demonstrated that experimental autoimmune encephalomyelitis (EAE) in the rat, passively transferred using myelin basic protein (MBP)‐reactive encephalitogenic CD4+ T cells, was preventable by administration of a p55‐tumor necrosis factor‐IgG fusion protein (TNFR‐IgG). This was despite quantitatively and qualitatively normal movement of these MBP‐specific T cells to the central nervous system (CNS). To extend these findings, the effect of TNFR‐IgG on EAE actively induced by injection of MBP in complete Freund's adjuvant was examined. This form of EAE in the rat typically involves an acute, self‐limiting neurological deficit, substantial CNS inflammation, but minimal demyelination. Here we show that administration of TNFR‐IgG prior to onset of disease signs completely prevented the neurological deficit or markedly reduced its severity. This blockade of clinical disease was dissociated from weight loss which occurred at the same tempo and magnitude as in control rats exhibiting neurological signs of disease such as paralysis. The timing of TNF blockade was critical as established clinical disease was relatively refractory to TNFR‐IgG treatment. Activated CD4+ T cells expressing normal or elevated levels of VLA4, major histocompatibility complex class II, MRC OX40 and CD25 were isolated from or immunohistochemically localized in the CNS of clinically healthy rats treated before disease onset. There was a reduction of the amount of other inflammatory leukocytes in the CNS of these treated animals but, more importantly, the activation state of inflammatory leukocytes, as well as that of microglia isolated from treated animals, was reduced. Thus, TNFR‐IgG, when administered before disease onset, appears to act by inhibiting an effector function of activated T cells and possibly other inflammatory leukocytes necessary to bring about the neurological deficit. However, while TNF is a critically important cytokine for the early events leading to initiation of EAE, it is not a necessary factor in the acute neurological deficit characteristic of this form of EAE, once disease onset has occurred.
The fate of activated T cells migrating through the body: rescue from apoptosis in the tissue of origin
Authors: Bode Ulrike , Wonigeit Kurt , Pabst Reinhard , Westermann Jürgen
Published: March 2007

After activation within a lymphoid tissue, T lymphocytes enter the blood, where they circulate and then re‐enter many organs. However, they predominantly end up in the tissue of origin, a phenomenon so far thought to be caused by organ‐specific homing. We analyzed the fate of T cells from different sources stimulated via the T cell receptor and CD28 and then injected intravenously into rats. Our results showed that preferential proliferation and reduced apoptosis, rather than preferential immigration, were responsible for the accumulation of activated T cells in the tissue of origin, explaining how immune responses can spread from site to site but still be restricted to certain regions. Manipulating the life span of such cells might be a promising approach to influencing immune responses.
Qa‐1 interaction and T cell recognition of the Qa‐1 determinant modifier peptide
Authors: Cotterill Lisa A. , Stauss Hans J. , Millrain Margaret M. , Pappin Darryl J. C. , Rahman Dinah , Canas Benito , Chandler Phillip , Stackpoole Arthur , Simpson Elizabeth , Robinson Peter J. , Dyson P. Julian
Published: March 2007

The peptide‐binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa‐1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa‐1b and the α3 domain of H‐2Db. This allowed the use of a monoclonal antibody directed against H‐2Db whilst retaining the peptide‐binding groove of Qa‐1b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2‐microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2‐microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa‐1b ‐presented peptide Qdm (AMAPRTLLL). A whole‐cell binding assay was used to determine which residues of Qdm were important for binding to Qa‐1b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa‐1b/Db molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H‐2Dk, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non‐classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.
Massive mammary gland infection and pregnancy‐dependent mammary tumor development in mice infected neonatally with mouse mammary tumor virus (SW) but not in mice infected as adults, despite a dramatic local response
Authors: Papiernik Martine , Waché Anne‐Claude , Pontoux Christiane , Nabarra Bernadette
Published: March 2007

Mouse mammary tumor virus (MMTV) (SW) caused a high incidence (65%) of pregnancy‐dependent adenocarcinomas in BALB/c(SW) mice infected as new‐borns by suckling their mothers' milk. These tumors were type B adenocarcinomas which developed early, at about 1 year of age. Uninfected breeding females and those infected at an age of 8 weeks by injection of virus had the same low incidence of malignant tumors (13%), and the tumors developed later (at approx. 23–24 months). The low incidence of tumors in adult‐infected mice was correlated with partial infection of the mammary glands, and delayed transmission of MMTV(SW) to the offspring. Although the virus was rapidly disseminated in both types of infection, the responses of neonatally infected and adult‐infected mice to MMTV(SW) infection and viral superantigen (vSAG) presentation were different. Activation by and presentation of the vSAG was impaired in mice infected neonatally, and tolerance induction by clonal deletion was delayed. Local activation was dramatic in mice infected as adults and clonal deletion followed rapidly. Although interaction between B and T cells is needed for completion of the virus life cycle and viral amplification, the strong local immune response to MMTV(SW) in adult‐infected mice limits mammary gland infection, and protects them against mammary tumor development.
Peptide‐induced T cell receptor down‐regulation on naive T cells predicts agonist/partial agonist properties and strictly correlates with T cell activation
Authors: Bachmann Martin F. , Oxenius Annette , Speiser Daniel E. , Mariathasan Sanjeev , Hengartner Hans , Zinkernagel Rolf M. , Ohashi Pamela S.
Published: March 2007

Recent experiments defining T cell agonists, partial agonists and antagonists have suggested that the T cell can discriminate between subtle differences in interactions leading to T cell activation. To further understand the complexities of T cell activation, we have analyzed the requirements for the induction of a variety of effector functions using naive T cells and a variety of altered peptide ligands. Using a strong agonist peptide, massive T cell receptor (TCR) down‐regulation correlated with a wide range of effector functions that were all induced above the same threshold peptide concentration. Interestingly, the kinetics of TCR down‐regulation correlated with the concentration of the peptide, whereas the maximal degree of TCR down‐regulation correlated with the induction of all monitored effector functions. A selected group of altered peptide ligands was also examined that were able to render target cells susceptible for lysis by effector cytotoxic T lymphocytes. The extent of TCR down‐regulation induced by these peptides corresponded to the induction of a subset of effector functions. These studies have shown that the extent of TCR down‐regulation defines the strength of TCR‐mediated “signal 1” which correlates with the spectrum of effector functions activated within the T cell. Thus, activation of different T cell functions requires the triggering of distinct numbers of TCR. The different parameters that influence TCR down‐regulation define important distinctions between our results and previously reported findings with T cell clones and may outline decisive parameters for the consequences of T cell activation in vivo.
T cell repertoire in patients with multiple myeloma and monoclonal gammopathy of undetermined significance: Clonal CD8+ T cell expansions are found preferentially in patients with a low tumor burden
Authors: Halapi Eva , Werner Åsa , Wahlström Jan , Österborg Anders , Jeddi‐Tehrani Mahmood , Yi Qing , Janson Carl Harald , Wigzell Hans , Grunewald Johan , Mellstedt Håkan
Published: March 2007

The T cell receptor (TCR) variable (V) gene repertoire was analyzed in patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 17), multiple myeloma (MM) stage I (n = 16), MM stages II/III (n = 31) and agematched controls (n = 27) by immunofluorescence and flow cytometry using a panel of mouse monoclonal antibodies (mAb) (n = 10) against TCR Vα and Vβ gene products. T cell expansion was defined as a value ⩾ thrice the normal median value for each respective TCR V mAb. Fifty‐three percent of all patients displayed CD8+ expansion(s) as compared to 30% of age‐matched controls (p = 0.001). Within the CD4 subset, 18% of the patients displayed T cell expansion(s) in comparison to 11% of the controls (not significant). Interestingly, the CD8+ expansion(s) were more frequently noted in patients with a low tumor burden (MGUS/MMI) (73%) as compared to those with advanced disease (MM II/III) (32% and control donors (30%) (p < 0.01). Likewise, multiple CD8+ expansions (two or more) were more common in MGUS/MM I patients than in MM II/III and controls (p = 0.01). The T cell expansions were stable over time in patients with a stable disease. A high degree of clonality of the expansions was detected by TCR CDR3 fragment length analysis, determination of Jβ gene usage and nucleotide sequencing. The frequent finding of oligoclonal CD8+ T cell expansions in patients with a low tumor mass, but not in patients with advanced disease justifies further work in order to identify the relevance of expanded CD8+ T cells. In one patient with T cell reactivity against the autologous myeloma idiotype, two expansions within the CD8 population (Vβ3 and Vβ5.2 respectively) displayed no reactivity against the idiotype. Instead, idiotype recognition was confined to a CD8 non‐expanded Vβ22+ T cell population, with a highly restricted TCR usage (CDR3 fragment length analysis).
Nephritogenic T cells use granzyme C as a cytotoxic mediator
Authors: Bailey Naila Celesta , Kelly Carolyn J.
Published: March 2007

Cytoplasmic granules of cytotoxic T lymphocytes contain several proteins that may be involved in cell‐mediated cytotoxicity. We have previously described nephritogenic T cell clones that are cytotoxic to cultured renal proximal tubular epithelial cells (MCT). One of these clones, M52.34.1, expresses perforin, a cytotoxic mediator. We investigated the expression of other granule‐associated proteases of M52.34.1. Granzymes A and B have been extensively studied in T cell‐mediated cytotoxicity, and associated with tissue destruction in models of transplantation. However, the activity of other granzymes has not been as extensively investigated. We focused our studies on granzyme C. Northern blots showed very high levels of granzymes B and C mRNA expression in M52.34.1 cells 3 days following T cell activation. There was no expression of granzyme A mRNA. An antisense oligonucleotide made from the 5′‐upstream region of the murine granzyme C exon 1 inhibited granzyme C mRNA expression in M52.34.1 when added at a concentration of 50 μM to the culture medium for 2 days. There was no inhibition of granzyme C mRNA expression with the sense oligonucleotide. The granzyme C antisense oligonucleotide inhibited M52.34.1 cytotoxicity to MCT at effector: target ratios of 20:1 and 40:1. M52.34.1 cells mediate inflammatory interstitial nephritis following adoptive transfer. If T cells were resuspended in 200 μM of the antisense oligonucleotide prior to subcapsular transfer, the recipient kidneys showed markedly diminished tubular cell destruction, suggesting that granzyme C can also be an important mediator of cytotoxicity in vivo.
Antigen‐specific CD8+ T cell subset distribution in lymph nodes draining the site of herpes simplex virus infection
Authors: Cose Stephen C. , Jones Claeroen M. , Wallace Morgan E. , Heath William R. , Carbone Francis R.
Published: March 2007

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis. Recently, studies using more advanced molecular approaches have suggested that such functionally derived precursor frequencies considerably underestimate the proportion of T cells specific for the antigen under investigation. We have defined T cell receptor sequences characteristic of CTL populations directed to a dominant determinant of the herpes simplex virus (HSV) glycoprotein B (gB). In this investigation, we used this receptor signature as a probe to directly monitor changes occurring within lymph nodes draining the sites of active infection with HSV. We found that although lymph node CD8+ T cell numbers increase as a consequence of HSV infection, the majority of these cells are small resting cells that are not enriched for gB‐specific receptors. In contrast, a significant proportion of activated T cells are highly enriched for CTL bearing gB‐specific receptors. Our results are therefore consistent with a nonspecific migration of CTL precursors into the lymph nodes draining the site of infection, followed by the activation and proliferation of the antigen‐specific subset that normally makes up a small proportion of the naive T cell repertoire.
Phenotypic and functional analysis of CD4+ NKRP1A+ human T lymphocytes. Direct evidence that the NKRP1A molecule is involved in transendothelial migration
Authors: Poggi Alessandro , Costa Paola , Zocchi Maria Raffaella , Moretta Lorenzo
Published: March 2007

In this report, we show that among human CD4+ T lymphocytes 5–20% express the C‐type lectin molecule NKRP1A. This lymphocyte subset displays a slightly more limited T cell receptor Vβ repertoire than the CD4+ NKRP1A− counterpart. CD4+ NKRP1A+ T lymphocytes are characterized by a high expression of β1 and β2 integrins, thus representing a T lymphocyte subset that can possibly adhere and migrate through vascular endothelium. Indeed, resting CD4+ NKRP1A+ lymphocytes, differently from the CD4+ NKRP1A− subset, migrated across endothelial cell monolayers in a Transwell chamber system. Pre‐treatment of CD4+ NKRP1A+ T lymphocytes with an anti‐NKRP1A monoclonal antibody (mAb) strongly reduced transendothelial migration, suggesting the involvement of the NKRP1A molecule in the transmigration process. Furthermore, cells of the NKRP1A− Jurkat CD4+ T cell line stably transfected with NKRP1A cDNA migrated more rapidly and efficiently than either untransfected or mock‐transfected Jurkat cells. Finally, mAb‐mediated cross‐linking of NKRP1A molecules in CD4+ T lymphocytes induced the up‐regulation of the lymphocyte function‐associated antigen 1 Mg2+‐binding site as well as β1 and β2 integrin chains. Altogether, these findings suggest that the NKRP1A molecule is involved in transendothelial migration of resting CD4+ T lymphocytes.
Glucocorticoids inhibit human antigen‐specific and enhance total IgE and IgG4 production due to differential effects on T and B cells in vitro
Authors: Akdis Cezmi A. , Blesken Thorsten , Akdis Mübeccel , Alkan Sefik S. , Heusser Christoph H. , Blaser Kurt
Published: March 2007

Although anti‐inflammatory properties of glucocorticoids (GC) are well documented, their activity in allergic diseases is still controversial. Recently, it has been reported that GC can increase, both in vivo and in vitro, the polyclonal production of total IgE. In this study we investigated the effects of GC on the antigen (Ag)‐specific IgE response in a human in vitro system with peripheral blood mononuclear cells or B cells of bee venom‐sensitized individuals that allows the production of bee venom phospholipase A2 (PLA)‐specific IgE and IgG4 antibodies (Ab). PLA‐specific Ab were induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti‐CD2 and soluble CD40 ligand (sCD40L) in the presence of interleukin (IL)‐4. Indeed, dexamethasone and prednisolone enhanced the formation of total IgE and IgG4 in PBMC, while the production of PLA‐specific IgE and IgG4 Ab was selectively inhibited in a dose‐dependent manner. The suppressive effect of GC was mediated during Ag‐specific stimulation and T cell‐B cell interaction. This was due to GC suppressing specific T cell proliferation and cytokine production, whereas neither allergen‐specific nor total IgE and IgG4 production by sCD40L/IL‐4‐stimulated pure B cells was affected. In contrast to GC, cyclosporine A inhibited both total and PLA‐specific IgE and IgG4 secretion in peripheral blood mononuclear cells and B cell cultures. Further experiments showed that increase in nonspecific total isotype response resulted from inhibition of IL‐4 uptake by cells other than B cells and sufficient availability of IL‐4 to B cells for isotype switch and synthesis. Furthermore, demonstration of opposite regulatory effects of GC on specific and total isotype formation in vitro, including the inhibition of allergy‐relevant Ag‐specific IgE response, may contribute to a better understanding of apparently controversial observations, and explain why most allergic patients benefit from GC therapy.
Ligation of the T cell co‐stimulatory receptor CD28 activates the serine‐threonine protein kinase protein kinase B
Authors: Parry Richard V. , Reif Karin , Smith Graham , Sansom David M. , Hemmings Brian A. , Ward Stephen G.
Published: March 2007

The intracellular signaling pathways activated upon ligation of the co‐stimulatory receptor CD28 remain relatively ill‐defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3‐kinase. The downstream effector targets of the CD28‐activated PI 3‐kinase‐dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3‐kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3‐kinase and the known protective effects of both CD28 and PI 3‐kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti‐CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood‐derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.
Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen
Authors: Fidock David A. , Pasquetto Valérie , Gras Hélène , Badell Edgar , Eling Wijnand , Ballou W. Ripley , Belghiti Jacques , Tartar André , Druilhe Pierre
Published: March 2007

Antibody(Ab)‐mediated inhibition of sporozoite invasion of hepatocytes is a mechanism that has been clearly demonstrated to act upon Plasmodium falciparum pre‐erythrocytic stages in humans. Consequently we have analyzed the Ab response to a recently identified P. falciparum sporozoite surface protein, STARP, in malaria‐exposed individuals and tested the inhibitory effect of these Ab upon hepatocyte invasion in vitro. STARP‐specific IgG were detected in 90 and 61 % of sera from regions where individuals were exposed to 100 and 1–5 infectious bites per year, respectively. These IgG were predominantly of the cytophilic IgG1 or IgG3 type. STARP and the major sporozoite surface protein, CS, elicited equivalent IgG levels in adults. When affinity purified from either African immune sera or the serum of an individual experimentally protected by irradiated sporozoite immunization, STARP‐specific Ab prevented up to 90% of sporozoites from invading human hepatocytes. The dose‐dependent and reproducible inhibition was more pronounced than that observed with human CS‐specific Ab affinity purified under identical conditions. Substantial reduction of sporozoite invasion was also observed with Ab induced by artificial immunization with recombinant STARP protein and reactive with the native protein. Taken together with recent findings of human cytotoxic T lymphocytes specific for this antigen, these results promote the interest of studying the efficacy of STARP as a target for immune effector mechanisms operating upon preerythrocytic stages.
Zinc inhibits interleukin‐1‐dependent T cell stimulation
Authors: Wellinghausen Nele , Martin Michael , Rink Lothar
Published: March 2007

Zinc is a trace element which is essential for immune functions. It directly induces monokine secretion by monocytes; however, effects of zinc on T cells appear contradictory. Apart from enhanced lymphocyte proliferation in peripheral blood mononuclear cells (PBMC), inhibitory properties of high zinc dosages have also been described. In this study, PBMC failed to produce lymphokines like interferon (IFN)‐γ after stimulation with zinc in a serum‐ and LPS‐free cell culture system, whereas monokine secretion [interleukin (IL)‐1β] occurred. Zinc‐uptake studies with the zinc‐specific fluorescent probe zinquin revealed that zinc is taken up by PBMC within a few minutes, reaching nearly equal levels in PBMC, isolated monocytes, and T cells. However, if zinc was depleted 1 h after monocyte induction, zinc‐free pre‐cultured T cells were stimulated to secrete IFN‐γ by zinc‐induced monokines. Furthermore, the necessity for a cell‐cell interaction between monocytes and T cells for IFN‐γ induction was elucidated. Zinc ions inhibited the proliferation of the IL‐1‐dependent T cell line D 10N in a dose‐dependent manner, suggesting a direct inhibitory effect of zinc. By immunoprecipitation we revealed a specific inhibition of IL‐1 receptor‐associated protein kinase (IRAK) by zinc ions. Therefore, in contrast to an indirect stimulation of T cells due to zinc‐induced monokines, higher concentrations of zinc directly inhibit T cell functions by means of specific inhibition of IRAK and subsequent signaling events such as NFxB activation. The divergent effects of zinc on different cell populations, depending on the zinc concentration, could explain contradictory results of zinc stimulation. Furthermore, our data suggest new strategies of specific zinc‐mediated immune modulation.
A murine transmembrane tumor necrosis factor (TNF) transgene induces arthritis by cooperative p55/p75 TNF receptor signaling
Authors: Alexopoulou Lena , Pasparakis Manolis , Kollias George
Published: March 2007

The arthritogenic activities of tumor necrosis factor (TNF) and its p55TNF‐receptor (R) have been well documented in experimental animal models of arthritis, and in transgenic mice expressing wild‐type or mutant transmembrane human TNF proteins in their joints. In this study we show that chronic inflammatory arthritis also develops in transgenic mice made to overexpress a mutant transmembrane from of the murine TNF protein (muTNFΔ1–12) which is known to utilize efficiently both the p55 and the p75TNFR. Cross‐breeding of the transgene into a TNF knockout background did not alter development of disease. Analysis of TNF bioactivity in sera from lipopolysaccharide‐stimulated mice orex vivomacrophage cultures demonstrated that the muTNFΔ1–12protein accumulates on the cell surface and is not processed to bioactive soluble TNF, indicating that transmembrane TNF is by itself sufficient to mediate pathogenesis of arthritis. Furthermore, using TNFR knockout mice, it is shown that development of transmembrane TNF‐mediated arthritis requires the presence of the p55TNFR but is significantly delayed in the absence of the p75TNFR, suggesting a positive cooperation between the two TNFR in the arthritogenic process. These results indicate that blocking the activities of both soluble and transmembrane TNF may be required to effectively neutralize the pathogenic potential of this cytokine in arthritis.
Antigen‐specific CD8+ T cells inhibit IgE responses and interleukin‐4 production by CD4+ T cells
Authors: Holmes Beverly J. , Macary Paul A. , Noble Alistair , Kemeny D. Michael
Published: March 2007

There is a growing body of evidence which suggests that CD8+ T cells play an important part in regulating the IgE response to non‐replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4+ T cell responses to ovalbumin (OVA) by CD8+ T cell depletion in vivo. Following intraperitoneal immunization with alum‐precipitated OVA, OVA‐specific T cell responses were detected in the spleen and depletion of CD8+ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8+ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8+ T cells on days 12–18 greatly enhanced the IgE response, which rose to 26 μ/ml following a second injection of anti‐CD8 on day 35 and remained in excess of 1 μ/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8‐depleted on day 12 with purified CD8+ T cells from animals immunized on day 12 completely inhib ited the IgE response. This effect was antigen specific; CD8+ T cells from OVA‐primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8+ T cell depletion decreased interferon (IFN)‐γ production but enhanced interleukin (IL)‐4 production by OVA‐stimulated splenic CD4+ T cells. Furthermore, CD8+ T cell depletion and addition of anti‐IFN‐γ antibody enhanced IgE production in vitro in an IL‐4‐supplemented mixed lymphocyte reaction. These data clearly show that antigen‐specific CD8+ T cells inhibit IgE in the immune response to non‐replicating antigens. The data indicate two possible mechanisms: first, CD8+ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA‐specific IL‐4 production but enhance IFN‐γ production by CD4+ T cells.
Secreted proteophosphoglycan of Leishmania mexicana amastigotes activates complement by triggering the mannan binding lectin pathway
Authors: Peters Christopher , Kawakami Masaya , Kaul Marcus , Ilg Thomas , Overath Peter , Aebischer Toni
Published: March 2007

Cutaneous lesions induced by infection of mice with the protozoan parasite, Leishmania mexicana, contain abundant amounts of a high molecular mass proteophosphoglycan (PPG), which is secreted by the amastigote stage residing in phagolysosomes of macrophages and can then be released into the tissue upon rupture of the infected cells. Amastigote PPG forms sausage‐shaped but soluble particles and belongs to a novel class of serine‐rich proteins that are extensively O‐glycosylated by phosphooligosaccharides capped by mannooligosaccharides. The purified molecule is shown here to efficiently activate complement (C) and deplete hemolytic activity of normal serum and may prevent the opsonization of L. mexicana amastigotes. Complement activation is Ca2+ dependent but does not depend on antibodies or the complement component C1. PPG binds to serum mannan binding protein (MBP), thus activating the MBP‐associated serine protease, P100. Subsequently, the C cascade is triggered through C4 leading to covalent modification probably of carbohydrate hydroxyls of PPG by C3 fragments. Thus, PPG is able to activate C via the mannan binding lectin pathway which is unusual for secreted, soluble products of microbial origin. The proteophosphoglycan‐induced complement activation is postulated to contribute to the lesion development and pathology caused by the parasite.
Complement C3b fragment covalently linked to tetanus toxin increases lysosomal sodium dodecyl sulfate‐stable HLA‐DR dimer production
Authors: Serra Vincent A. , Cretin François , Pépin Elsa , Gabert Françoise M. , Marche Patrice N.
Published: March 2007

Processing and presentation of covalently linked C3b‐tetanus toxin (TT) complexes, as compared to unlinked C3b + TT, lead to increased T cell proliferation. The aim of this study was to analyze the effect of coupling C3b to TT on the efficiency of TT peptide loading on HLA‐DR1 molecules. In the Epstein‐Barr virus‐transformed B cell line HOM 2, we detected a significant increase of sodium dodecyl sulfate (SDS)‐stable major histocompatibility complex (MHC) class II molecules after exposure to C3b‐TT as compared to unlinked C3b and TT. The ratio of compact form/unbound form (C/U ratio) obtained with C3b‐TT as antigen (Ag) is about twice that obtained with uncomplexed TT + C3b as Ag. Similar results were obtained using HLA‐DR1‐transfected fibroblasts that do not express C3b complement receptors, indicating that the SDS‐stable HLA‐DR1 increase did not result simply from C3b opsonization but rather from a direct effect of C3b‐TT linkage on peptide generation. Exposure of HOM 2 cells to C3b‐TT resulted in an increase in concentration of SDS‐stable HLA‐DR molecules in lysosomes but not in endosomes. Thus, C3b attachment to Ag induces a redistribution of peptide/MHC complex which results in a higher efficiency of Ag presentation by MHC class II molecules.
Strong mucosal adjuvanticity of cholera toxin within lipid particles of a new multiple emulsion delivery system for oral immunization
Authors: Tomasi Maurizio , Dertzbaugh Mark T. , Hearn Thomas , Hunter Robert L. , Elson Charles O.
Published: March 2007

Cholera toxin (CT) is an effective mucosal adjuvant but causes significant intestinal secretion which limits its usefulness. In the present study we developed a new multiple emulsion (ME) delivery system into which antigen and CT could be incorporated and asked whether CT would retain its mucosal adjuvanticity when sequestered within emulsion particles. ME were selectively taken up into Peyer's patches, and those containing antigen plus CT generated intestinal secretory IgA and serum IgG antibody responses in mice comparable quantitatively and qualitatively to those occurring after oral immunization with soluble antigen plus CT. The ME particles containing CT did not cause intestinal secretion. The adjuvanticity of CT within ME was due to the CT present in the inner aqueous phase of the ME and was lost if CT binding was blocked by pre‐incubation with GM1 ganglioside. Proteins incorporated in ME were protected from external acid, protease, and bile. We conclude that CT sequestered in ME, although unable to bind to the epithelium and thus stimulate intestinal secretion, still retains its mucosal adjuvanticity. Thus, the ability of CT to bind to enterocytes is not obligatory for its mucosal adjuvanticity.