bluuebunny/crossref_metadata_2025 huggingface dataset:
The V gamma locus of the human T cell receptor gamma gene. Repertoire polymorphism of the first variable gene segment subgroup.
Authors: Font M P , Chen Z , Bories J C , Duparc N , Loiseau P , Degos L , Cann H , Cohen D , Dausset J , Sigaux F
Published: June 2004

Southern blot analysis using a genomic probe of the human TCR-gamma chain first variable gene subgroup (V gamma I) was performed on DNA samples from both parents of 36 healthy Caucasian families. Two types of polymorphisms were found in these 72 unrelated DNA samples: three repertoire polymorphisms and two restriction fragment length polymorphisms (RFLP). In all cases, Mendelian inheritance of these polymorphisms was demonstrated. The most frequent repertoire polymorphism consists in the lack of the V gamma 4 and V gamma 5 segments. In 16% of chromosomes, the Eco RI and Taq I restriction fragments corresponding to V gamma 4 and V gamma 5 were lacking, with no additional bands. In these cases, a decrease of 10 kb was observed in the Bam HI fragment containing all V gamma I segments as compared with samples containing V gamma 4-V gamma 5 segments. To better understand this polymorphism, which takes place in a previously incompletely defined region, the central part of the V gamma I region, including the polymorphic V gamma 4-V gamma 5 segments, was cloned. This allowed us to localize precisely the V gamma 5 segment and thus complete the description of the V gamma I region. A striking homology of DNA and deduced amino acid sequences is present between V gamma 2 and V gamma 4 and between V gamma 3 and V gamma 5, much higher than that observed between V gamma 2 and V gamma 3 and between V gamma 4 and V gamma 5. The differences in nucleotide sequence occur mainly in the intron and three hypervariable regions. These results strongly suggest a gene duplication relationship between the segments V gamma 2-V gamma 3 and the segments V gamma 4-V gamma 5. The most frequent RFLP documented in this study is due to the combined absence of the Eco RI and the Taq I sites located in the noncoding region between V gamma 3 and V gamma 4. The haplotypic frequence of this RFLP is 6.9% of the general population. As the gamma/delta receptor may play an important role in immunological response, the biological relevance of the high degree of polymorphism occurring in the V gamma I region, as well as its possible association with some immune disturbances, should be further explored.
Keratinocyte growth regulation by the products of immune cells.
Authors: Hancock G E , Kaplan G , Cohn Z A
Published: June 2004

We describe a bioassay that allows the in vitro investigation of the stimulatory and suppressive factors derived from immune cells in short-term cultures of human keratinocytes. In agreement with other assays, epidermal growth factor is not mitogenic for human keratinocytes. Supernatant fluid from human PBMC stimulated with Con A, from allo-MLRs, as well as supernatants from nonstimulated PBMC, possess growth-promoting molecules. Our results show that both activated and nonactivated T cells release growth factors. Suppressive molecules are produced preferentially by monocyte cultures. Two T cell products, IFN-gamma and transforming growth factor beta are both inhibitory for keratinocyte proliferation. Two other T cell products, IL-3 and GM-CSF, stimulate keratinocyte proliferation at nanogram concentrations. These results suggest the existence of regulatory circuits between the T cells of a dermal inflammatory infiltrate and the overlying epidermal keratinocytes. This may determine the fine control of epidermal proliferation and turnover leading either to enhanced wound repair or skin pathology.
Bacterial cell wall-induced immunosuppression. Role of transforming growth factor beta.
Authors: Wahl S M , Hunt D A , Bansal G , McCartney-Francis N , Ellingsworth L , Allen J B
Published: June 2004

Group A streptococcal cell wall (SCW)-injected rats exhibit a profound immunosuppression that persists for months after the initial intraperitoneal injection of SCW. The goal of this study was to determine the mechanisms for the suppressed T lymphocyte proliferative responses in this experimental model of chronic inflammation. When spleen cell preparations were depleted of adherent cells, restoration of T cell proliferative responses to Con A and PHA occurred, implicating adherent macrophages in the regulation of immunosuppression. Furthermore, macrophages from SCW-treated animals, when cocultured with normal spleen cells in the presence of Con A or PHA, effectively inhibited the proliferative response. Supernatants from suppressed spleen cell cultures were found to inhibit normal T cell mitogenesis. Taken together, these results implicated a soluble macrophage-derived suppressor factor in the down regulation of T cell proliferation after exposure to SCW in vivo. Subsequent in vitro studies to identify this suppressor molecule(s) revealed the activity to be indistinguishable from the polypeptide transforming growth factor beta (TGF-beta). Furthermore, TGF-beta was identified by immunolocalization within the spleens of SCW-injected animals. The cells within the spleen that stained positively for TGF-beta were phagocytic cells that had ingested, and were presumably activated by, the SCW. These studies document that TGF-beta, previously shown to be a potent immunosuppressive agent in vitro, also effectively inhibits immune function in chronic inflammatory lesions in vivo.
Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastatic melanomas. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor.
Authors: Itoh K , Platsoucas C D , Balch C M
Published: June 2004

TIL from metastatic melanoma proliferated by greater than 1,000-fold (840-3,675, mean 1,543) after 6 wk in culture of mixtures of TIL and tumor cells with rIL-2 alone. Cytolysis was restricted to autologous tumor cells. CD8+ T cells were the predominant population of TIL before and after expansion, and were primarily responsible for autologous tumor-specific CTL activity. No other rIL-2-activated lymphocytes from peripheral blood, lymph nodes with melanoma metastasis, or TIL from sarcoma or renal cell carcinoma had autologous tumor-specific CTL activity. There were few or no CD16+ NK cells in TIL from metastatic melanoma before or after incubation with rIL-2, respectively. However, TIL from sarcoma or renal cell carcinoma contained a substantial proportion of CD3-CD16+ NK cells, which increased in number in culture with rIL-2. Purified CD16+ NK cells as well as CD3+CD16- T cells from rIL-2-activated TIL of renal cell carcinoma displayed MHC-nonrestricted cytotoxicity. At the clonal level as determined by limiting dilution, 8 of 10 clones from melanoma TIL displayed cytotoxicity restricted to autologous tumor cells, while all 13 clones from renal cancer TIL equally lysed autologous and allogeneic tumor cells. Anti-T cell receptor (TCR)-alpha/beta(WT31) mAb as well as anti-CD3 mAb inhibited autologous melanoma cell-specific CTL activity mediated by rIL-2-activated TIL at the effector phase. These two mAbs also inhibited rIL-2-dependent proliferation of these TIL when added to the culture. Pretreatment of fresh melanoma cells with mAb to MHC antigens followed by washing inhibited specific CTL activity. These results suggest that both TCR-alpha/beta on effector TIL and MHC antigens on fresh tumor cells are involved in the specific immune-recognition. After reaching maximum propagation, TIL from metastatic melanoma responded poorly to rIL-2 alone. However, stimulation with fresh autologous melanoma cells restored both CTL activity and proliferation in response to rIL-2. The latter is associated with IL-2 receptor (Tac antigen) expression on the surface. These results indicate that TIL from metastatic melanomas may have unique characteristics different from lymphocytes obtained from the other sources, and may contain precursor CTL sensitized in vivo to autologous tumor cells, and thus can be propagated in larger numbers with rIL-2 alone while retaining autologous tumor-specific CTL activity.
Neuraminidase-treated macrophages stimulate allogenic CD8+ T cells in the presence of exogenous interleukin 2.
Authors: Hirayama Y , Inaba K , Inaba M , Kato T , Kitaura M , Hosokawa T , Ikehara S , Muramatsu S
Published: June 2004

Prior work has shown that purified, resident, and inflammatory peritoneal macrophages are weak stimulators of the allogeneic MLR. We have identified conditions whereby thioglycollate-elicited macrophages become stimulatory, but primarily for the CD8+ T cell subset. The conditions were to treat the macrophages with neuraminidase and to supplement the MLR with rIL-2. These treatments together led to proliferative and cytotoxic responses by isolated CD8+ but not CD4+ T cells. Likewise when MHC-congenic strains were evaluated, an MLR was observed across isolated class I but not class II MHC barriers. Pretreatment of the macrophages with IFN-gamma further enhanced expression of class I MHC products and stimulatory activity, but did not seem essential. While these treatments did not render macrophages stimulatory for an MLR in purified CD4+ cells, blastogenesis of CD4+ cells was observed when the MLR involved bulk T cells. Small allogeneic B lymphocytes behaved similarly to macrophages, in the pretreatment with neuraminidase and supplementation with rIL-2 rendered B cells stimulatory for allogeneic, enriched, CD8+, but not CD4+, T cells. Spleen adherent cells, which are mixtures of macrophages and dendritic cells, stimulated both CD4+ and CD8+ T cells, and neither neuraminidase nor exogenous IL-2 was required. We think that these data suggest that most macrophages and small B cells lack three important functions of dendritic cells: a T cell-binding function that can be remedied by neuraminidase treatment, a T cell growth factor-inducing function that can be bypassed with exogenous IL-2, and an IL-2 responsiveness function that is required by CD4+ lymphocytes.
A novel activation pathway for mature thymocytes. Costimulation of CD2 (T,p50) and CD28 (T,p44) induces autocrine interleukin 2/interleukin 2 receptor-mediated cell proliferation.
Authors: Yang S Y , Denning S M , Mizuno S , Dupont B , Haynes B F
Published: June 2004

Prior studies have shown that thymocytes, unlike peripheral T cells, do not proliferate in response to mitogenic combinations of anti-CD2 mAbs. The present study demonstrated that stimulation by a mitogenic anti-CD2 combination (9-1 plus 9.6) with anti-CD28 induced vigorous thymocyte proliferation in the absence of exogenous IL-2. This thymocyte proliferation was IL-2 dependent as shown by the complete inhibition using anti-IL-2-R mAbs. Induction of IL-2-R transcripts was detected in thymocytes stimulated by the anti-CD2 antibody combination alone or the anti-CD2 combination plus anti-CD28 antibody. However, induction of IL-2 transcripts was observed only in thymocytes triggered jointly by the anti-CD2 combination plus anti-CD28 antibodies. The double-negative (CD4-8-) or CD1+ thymocytes isolated by sorting or by panning were unresponsive to CD2/CD28 triggering. The same mitogenic signal could induce vigorous proliferation of thymocytes with a mature phenotype, i.e., CD3+CD4+ or CD3+CD8+ thymocytes. Immunofluorescence studies demonstrated that the majority of CD3+ thymocytes were CD28+, and most of the CD28+ cells were located in the medullary compartment of thymus. These results indicated that the T cell lineage surface molecules CD28 and CD2 are involved in the regulation of expansion and further differentiation of mature thymocytes.
Acidic precursor revealed in human eosinophil granule major basic protein cDNA.
Authors: Barker R L , Gleich G J , Pease L R
Published: June 2004

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and various cell types, is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. A cDNA for human MBP was isolated from a gamma GT10 HL-60 cDNA library. The nucleotide sequence of the MBP cDNA indicates that MBP is translated as a 25.2-kD preproprotein. The 9.9-kD pro-portion of proMBP is rich in glutamic and aspartic acids and has a calculated pI of 3.9, while proMBP itself has a calculated pI of 6.2. We suggest that MBP is translated as a nontoxic precursor that protects the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the granule core toxic MBP, and we present results from the literature suggesting that other cationic toxins, which damage cell membranes, may also be processed from nontoxic precursors containing distinct anionic and cationic regions.
Prevention of experimental cerebral malaria by anticytokine antibodies. Interleukin 3 and granulocyte macrophage colony-stimulating factor are intermediates in increased tumor necrosis factor production and macrophage accumulation.
Authors: Grau G E , Kindler V , Piguet P F , Lambert P H , Vassalli P
Published: June 2004

IL-3 and granulocyte/macrophage colony stimulating factor (GM-CSF) are two cytokines released by activated T lymphocytes that stimulate the growth and differentiation of various hematopoietic cell lines, among which are macrophages. It has been shown that TNF/cachectin, another cytokine that is released mostly by activated macrophages, plays a central role in experimental cerebral malaria (CM), an acute and lethal neurological syndrome induced by Plasmodium berghei ANKA infection in CBA mice. Since CM requires functional CD4+ T lymphocytes to occur, we explored, by injecting rabbit antibodies to murine rIL-3 and/or GM-CSF, whether these cytokines are intermediates in the marked TNF release leading to CM. Treatment of infected mice with each antibody separately had no protective effect. In contrast, when both anti-rGM-CSF and anti-rIL-3 antibodies were injected together; (a) the occurrence of neurological syndrome was prevented in 90% of the cases; (b) the rise in serum TNF was prevented; and (c) macrophage accumulation in the spleen was significantly reduced. Murine CM appears to involve a cytokine cascade in which IL-3 and GM-CSF lead to the accumulation of TNF-releasing macrophages in vivo.
Properties of isolated red pulp macrophages from mouse spleen.
Authors: Nusrat A R , Wright S D , Aderem A A , Steinman R M , Cohn Z A
Published: June 2004

Dense monolayers of large, adherent macrophages were prepared from the red pulp of mouse spleen. These sinus-lining phagocytes resembled liver Kupffer cells in morphology, as well as expression of F4/80 and class II MHC antigens and receptors for IgG. C3-coated red cells attached at low levels to spleen macrophages, but attachment and endocytosis were enhanced on fibronectin-coated surfaces. The ionophore A23187 induced spleen macrophages to synthesize prostaglandin E2, but like Kupffer cells, spleen macrophages did not synthesize leukotrienes and made relatively small amounts of HETE and 12-hydroxyheptadecanoic acid. Resident spleen macrophages did not produce H2O2, but splenic inflammatory cells, induced by infection of animals with Listeria monocytogenes, actively released H2O2. We conclude that the functional properties of resident, sinusoidal-lining macrophages in liver and spleen are similar to one another but distinct from other pools of phagocytes.
B cell progenitors have different growth requirements before and after immunoglobulin heavy chain commitment.
Authors: Sauter H , Paige C J
Published: June 2004

Using the clonable pre-B cell assay, we have identified B cell progenitors that are not yet committed to the production of a particular H chain allele. These cells represent approximately 10-20% of clonable pre-B cells found in 15-d fetal liver. The clonable pre-B cell assay provides an environment adequate for the expansion and differentiation of these cells into mature, Ig-secreting, cells. Using the same methodology, we have also identified progenitors that are uncommitted to the production of a particular L chain isotype. Moreover, investigating the growth requirements for clonable pre-B cells has led to the discovery of selective growth conditions that distinguish cells before and after commitment to H chain allotype.
A new member of the immunoglobulin superfamily that has a cytoplasmic region homologous to the leukocyte common antigen.
Authors: Streuli M , Krueger N X , Hall L R , Schlossman S F , Saito H
Published: June 2004

A human gene (LAR) that hybridizes to mouse leukocyte common antigen cDNA under relaxed hybridization conditions was isolated. The LAR gene is expressed in a broad range of cells, including T lymphocytes, kidney, and prostate cells. The structure of the protein encoded by the LAR gene was deduced by determining the nucleotide sequences of a 7.7-kb LAR cDNA. The putative LAR protein is composed of a 1,234 amino acid extracellular region, a 24 amino acid transmembrane segment, and a 623 amino acid cytoplasmic region. The cytoplasmic region contains two homologous domains that have extensive sequence similarity to the cytoplasmic region of the leukocyte common antigens. The NH2-terminal region of the extracellular segment of the LAR protein contains three tandem Ig-like domains and nine non-Ig-like domains. Among the known Ig-like proteins, the LAR protein has the highest degree of similarity to neural-cell adhesion molecule. The non-Ig-like domains of the LAR protein are also similar to the non-Ig-like domains of neural-cell adhesion molecule.
Production of tumor necrosis factor/cachectin by human B cell lines and tonsillar B cells.
Authors: Sung S S , Jung L K , Walters J A , Chen W , Wang C Y , Fu S M
Published: June 2004

The production of TNF/cachectin by human B cell lines and tonsillar B cells was examined. Of the 15 B cell lines examined, 9 cell lines synthesize TNF mRNA constitutively. PMA stimulated most cell lines to accumulate increased amounts of TNF. SeD, 8866P, 32al, RPMI 1788, and four bone marrow-derived EBV-transformed cell lines accumulated high levels of TNF mRNA when stimulated by PMA. TNF production by these cell lines was examined. RPMI 1788 and WIH8 produced little TNF constitutively, but synthesized 5-7 ng/ml TNF when stimulated by PMA. A pre-B cell line, Nalm-6, did not synthesize any detectable amount of TNF mRNA, even with PMA stimulation. Tonsillar B cells could also be stimulated to produce TNF. PMA or Staphylococcus aureus Cowan I strain (SAC) alone stimulated some TNF mRNA accumulation, whereas B cell growth factor (BCGF) or anti-mu did not. This accumulation was synergistically elevated by the combinations of PMA and SAC, or PMA and anti-mu. BCGF increased PMA-, SAC-, PMA plus SAC-, or PMA plus anti-mu-induced TNF mRNA accumulations about twofold. The accumulation of TNF mRNA in tonsillar B cells stimulated by PMA plus SAC was between 32 and 48 h, the same peak interval as the accumulation of TNF and IL-2 mRNA in tonsillar T cells. This is in contrast to PMA or PMA plus A23187-stimulated RPMI 1788 cells in which TNF mRNA accumulation was maximal at 1-2 h. TNF activities found in tonsillar B cell supernatants correlated with the TNF mRNA levels in the cells. However, more TNF activity was found on the second-day than the third-day supernatants, indicating active TNF uptake by the B cells. Cyclosporin A (CsA) inhibited SAC and anti-mu responses in B cells in much the same way as the anti-CD3 responses in T cells. SAC-, PMA plus SAC-, and PMA plus anti-mu-stimulated, but not PMA-stimulated, increases in TNF mRNA accumulations in tonsillar B cells were inhibited by CsA. TNF production seems to increase in parallel with B cell proliferation, but the relationship of these two functions needs to be further examined.
The SJL/J T cell response to both spontaneous and transplantable syngeneic reticulum cell sarcoma is mediated predominantly by the V beta 17a+ T cell clonotype.
Authors: Katz J D , Ohnishi K , Lebow L T , Bonavida B
Published: June 2004

Previous studies have revealed that the reticulum cell sarcoma (RCS) of SJL/J (H-2s, IE-) mice express an "IE-like" stimulatory tumor-associated antigen, the expression of which is requisite for stimulating host T cells necessary for tumor growth. Herein, we present evidence that the predominant T cells raised in the syngeneic response to both spontaneous and transplantable RCS tumors are of the V beta 17a TCR clonotype. The V beta 17a+ clonotype of T cells has been shown to interact with IE allogeneic specificities. We demonstrate that all four characterized RCS-specific T cell hybridomas stained positively for the anti-V beta 17a mAb, KJ23a. Additionally, KJ23a, when added to cocultures of the T cell hybridomas and RCS tumors, inhibited the release of IL-2 by the hybridomas. Further, KJ23a was shown to markedly inhibit the proliferation of SJL/J T cells when cocultured with either spontaneous or transplantable RCS tumor cells. When analyzed by flow cytometry, the T cell blast population raised in response to both spontaneous and transplantable RCS were greater than 80% KJ23a+. These T cells were brightly stained by the anti-CD4 mAb, Gk1.5, and, therefore, represent class II-responsive T cells. In corroboration of the in vitro data, T cells derived from mesenteric lymph nodes of RCS tumor-bearing mice had likewise undergone a similar expansion of V beta 17a+, CD4+ T cells. Together, these results indicate that KJ23a+ T cells play an important and predominant role in the response of SJL/J mice to spontaneous RCS tumors and provide further suggestive evidence that the stimulatory antigen(s) on the RCS tumor is IE or an "IE-like" molecule. Significantly, the important role V beta 17a+ T cells play in the response to RCS suggests a potential therapeutic role for KJ23a mAb in the intervention and prevention of RCS tumors in SJL/J mice.
Genetic control of T cell responsiveness to the Friend murine leukemia virus envelope antigen. Identification of class II loci of the H-2 as immune response genes.
Authors: Miyazawa M , Nishio J , Chesebro B
Published: June 2004

T cells primed specifically for the envelope glycoprotein of Friend murine leukemia helper virus (F-MuLV) were prepared by immunizing mice with a recombinant vaccinia virus that expressed the entire env gene of F-MuLV. Significant proliferative responses of F-MuLV envelope-specific, H-2a/b T cells were observed when the T cells were stimulated with antigen-pulsed peritoneal exudate cells (PEC) having the b allele at the K, A beta, A alpha, and E beta loci of the H-2. On the other hand, PEC having only the kappa allele at these loci did not induce the envelope-specific T cell proliferation, even when the PEC had the b allele at the E alpha, S, or D loci. F-MuLV envelope-specific proliferation of H-2a/b T cells under the stimulation of antigen-pulsed, H-2a/b PEC was specifically blocked with anti-I-Ab and anti-I-Ek mAbs but not with anti-Kb, anti-Kk, or anti-I-Ak mAbs. Moreover, (B10.MBR x A/WySn)F1 mice that have the b allele only at the K locus but not in I-A subregion were nonresponders to the envelope glycoprotein, and the bm12 mutation at the A beta locus completely abolished the T cell responsiveness to this antigen. These results indicate that proliferative T cells recognize a limited number of epitopes on F-MuLV envelope protein in the context of I-Ab, hybrid I-Ak/b, and/or hybrid I-Ek/b class II MHC molecules but fail to recognize the same envelope protein in the context of I-Ak or I-Ek molecules. This influence of the H-2I region on T cell recognition of the envelope glycoprotein appeared to control in vivo induction of protective immunity against Friend virus complex after immunization with the vaccinia-F-MuLV env vaccine. Thus, these results provide, for the first time, direct evidence for Ir gene-controlled responder/nonresponder phenotypes influencing the immune response to a pathogenic virus of mice.
The natural history of encephalomyocarditis virus-induced myositis and myocarditis in mice. Viral persistence demonstrated by in situ hybridization.
Authors: Cronin M E , Love L A , Miller F W , McClintock P R , Plotz P H
Published: June 2004

Picornaviruses can initiate chronic inflammation that persists after the virus can no longer be cultured from inflamed tissues. In an attempt to understand this transition we have sought evidence for viral persistence by methods that detect viral genome independent of whether or not whole competent virus is present. In mice infected with a myotropic variant of encephalomyocarditis virus, EMC-221A, virus can be cultured in high yield at 1 wk and in low yield at 2 wk from skeletal muscle, heart, and brain; a small number of plaque-forming units could be cultured from brain at 4 wk. By contrast, in situ hybridization detected viral nucleic acid at least a week or two thereafter, often in single cells. In the skeletal muscle, inflammation disappeared by 3 wk, but in heart it remained for the full 12 wk of observation. In the brain, microglial nodules, sometimes with associated viral nucleic acid, were present for a long period. Application of this technique allows a more accurate assessment of the role of viral persistence in the pathogenesis of virus-initiated but apparently autoimmune inflammation.
In situ hybridization for interleukin 2 and interleukin 2 receptor mRNA in T cells activated in the presence or absence of cyclosporin A.
Authors: Granelli-Piperno A
Published: June 2004

RNA-RNA in situ hybridization was used to study the frequency of cells producing mRNA for IL-2 and IL-2-R in T lymphocytes stimulated by either of three mitogens: anti-CD3, anti-CD28, or PHA. Both CD4+ and CD8+ T cells expressed transcripts for IL-2 and the low-affinity IL-2-R when stimulated with these mitogens plus PMA. IL-2 transcripts peaked at 8-16 h, and IL-2-R at 24-40 h. Cyclosporin A (CSA) inhibited the synthesis of IL-2, but not IL-2-R mRNA, after stimulation by PHA or anti-CD3. However, higher concentrations of CSA were necessary to achieve 50-70% inhibition after stimulation with anti-CD28. At optimal points 12-22% of CD4+ and 5-13% of CD8+ cells expressed IL-2 mRNA, while 30-50% of cells of both subsets had IL-2-R mRNA. The IL-2 grain counts, which relate to the level of mRNA/cell, were higher in the CD4+ subset but could be increased several fold in the CD8+ subset in the presence of adherent accessory cells. The use of PMA as an accessory stimulus, in addition to adherent cells, greatly increased the frequency of lymphocytes with IL-2 mRNA and the amount of IL-2 activity in the culture medium, but the proliferative response was not significantly boosted. These observations indicate at the single cell level that many CD4+ or CD8+ lymphocytes can make IL-2 mRNA, and that the induction of IL-2 with several stimuli is reduced by CSA and enhanced by PMA.
Clonal analysis of functionally distinct human CD4+ T cell subsets.
Authors: Rotteveel F T , Kokkelink I , van Lier R A , Kuenen B , Meager A , Miedema F , Lucas C J
Published: June 2004

A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.
Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens.
Authors: Scott P , Natovitz P , Coffman R L , Pearce E , Sher A
Published: June 2004

BALB/c mice can be protected against a normally fatal Leishmania major infection by immunization with a partially purified, soluble subfraction of the parasite (fraction 9). In this study, we demonstrate that a T cell line established against fraction 9, designated line 9, transfers protection equivalent to that obtained by active immunization. In contrast, T cell lines (lines 1 and 9.2) responsive to a nonprotective soluble fraction (fraction 1) not only failed to protect BALB/c mice against L. major, but exacerbated the infection. Most importantly, in addition to differing in their antigen specificity, protective and exacerbative T cells lines could be distinguished on the basis of the lymphokines produced, a characteristic previously used to separate murine Th cells into two subsets, designated Th1 and Th2. We found that the protective cell line, line 9, displayed the Th1 property of secreting IL-2 and IFN-gamma, while the exacerbating lines secreted IL-4 and IL-5, a characteristic of Th2 cells. Our results demonstrate that Th1 and Th2 cells may have dramatically different effects on the outcome of an infection, and suggest that susceptibility and resistance in experimental leishmaniasis may depend upon a balance between the Th subsets induced.
Angioedema induced by a peptide derived from complement component C2.
Authors: Strang C J , Cholin S , Spragg J , Davis A E , Schneeberger E E , Donaldson V H , Rosen F S
Published: June 2004

Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin-cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus.
Essential role of the interleukin 2-interleukin 2 receptor pathway in thymocyte maturation in vivo.
Authors: Tentori L , Longo D L , Zuñiga-Pflucker J C , Wing C , Kruisbeek A M
Published: June 2004

The role of the IL-2-IL-2-R pathway in thymocyte differentiation in vivo is unknown. We have examined fetal thymocyte development in vivo, under conditions where all IL-2-R were saturated from day 13 of gestation with anti-IL-2-R mAbs that were previously shown to render mature T cells unable to respond to IL-2. This produced a dramatic change in the composition of developing T cells: thymocytes from day 1 neonatal mice born to anti-IL-2-R-treated mothers did not contain CD4+ or CD8+ single-positive cell populations. In addition, no generation of surface TCR beta chain-expressing T cells or antigen-reactive functional T cells occurred in treated mice. These data suggest that IL-2-IL-2-R interactions provide signals crucial to in vivo intrathymic development of mature T cells.
Presentation of the H-Y antigen on Langerhans' cell-negative corneal grafts downregulates the cytotoxic T cell response and converts responder strain mice into phenotypic nonresponders.
Authors: Peeler J S , Callanan D G , Luckenbach M W , Niederkorn J Y
Published: June 2004

We have used the murine cornea is an allograft model to investigate the relative roles of graft-derived IA+ APC (Langerhans' cells) and host-derived APC during the induction of CTL responses to H-Y. The natural exclusion of LC from the immunizing corneal graft led to a specific state of unresponsiveness to H-Y in responder strain mice, while inclusion of LC resulted in responsiveness. Failure to respond to H-Y could not be attributed to the absence of H-Y or IA antigen expression on the surface of LC-deficient grafts but instead, appeared to be due to active suppression of the T helper cell response during in vivo priming. Reprocessing of the H-Y antigen by host APC did not occur after immunization with H-Y presented on H-2-incompatible grafts unless presented initially by graft-derived LC. H-2 as well as some non-H-2 alloantigens were presented to the host without a requirement for donor-derived LC. Thus there appear to be differential requirements for the processing and presentation of alloantigens.
A human urine-derived interleukin 1 inhibitor. Homology with deoxyribonuclease I.
Authors: Rosenstreich D L , Tu J H , Kinkade P R , Maurer-Fogy I , Kahn J , Barton R W , Farina P R
Published: June 2004

We have previously reported that the urine of febrile humans contained large quantities of an inhibitor of IL-1-induced murine thymocyte proliferation that was a glycoprotein between 30 and 40 kD in size. In the present study this factor has been purified to homogeneity using a sequence of eight purification steps (ammonium sulfate precipitation, ion exchange chromatography, molecular sieve chromatography, hydrophobic affinity chromatography, hydroxylapatite chromatography, fast protein liquid chromatography, and two HPLC steps). SDS-PAGE analysis indicates that the purified material is a 38-kD molecule. Evidence based on a partial amino acid sequence analysis as well as enzyme studies indicates that this inhibitor is a type of human DNase I.
Augmented expression of a myeloid-specific protein tyrosine kinase gene (hck) after macrophage activation.
Authors: Ziegler S F , Wilson C B , Perlmutter R M
Published: June 2004

Protein tyrosine kinases are thought to participate in signal transduction pathways in a variety of cell types. Recent studies have identified a new src family protein tyrosine kinase (hck) that is preferentially expressed in myeloid cells. To examine the hypothesis that this kinase may regulate myeloid cell activity, antisera were generated that define the 59-kD product of the hck gene. Functional activation of human cultured macrophages with LPS augmented the expression of hck transcripts and of p59hck, but decreased the level of transcripts encoded by the closely related c-fgr protooncogene. Thus these two structurally similar src family kinases almost certainly subserve distinct functions. Reasoning from the known properties of the src family protein tyrosine kinases, it is likely that the products of these two protooncogenes assist in regulating the behavior of activated phagocytes.
Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes.
Authors: Kwon B S , Kestler D , Lee E , Wakulchik M , Young J D
Published: June 2004

Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2-terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35-kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.
The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.
Authors: Pawlowski N A , Kaplan G , Abraham E , Cohn Z A
Published: June 2004

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.
Characterization and specificity of antibodies to protein I of Neisseria gonorrhoeae produced by injection with various protein I-adjuvant preparations.
Authors: Wetzler L M , Blake M S , Gotschlich E C
Published: June 2004

A major goal of gonococcal research is the development of a gonorrheal vaccine. A vaccine candidate is the major outer membrane protein (PI) of the gonococcus, which has limited antigenic variability. Two main subtypes, PIA and PIB, and nine main serotypes have been described. To avoid raising anti-protein III (PIII)-blocking antibodies and limit potential lipooligosaccharide toxicity, PI was chromatographically isolated with minimal PIII contamination (less than 1%) from Pgh 3-2 (PIB), a serum-sensitive gonococcal strain and UU1 (PIA), a serum-resistant gonococcal strain. Alum was used as an adjuvant and the antibodies raised in rabbits did not agglutinate the organisms, were not opsonic, and bactericidal titers were not increased. To present PI in a form mimicking its in vivo disposition, it was inserted into liposomes. The resulting antisera did agglutinate the organism and contained opsonic and bactericidal activity greater than the preimmune sera or alum-generated sera. The PIB liposome antisera also had higher ELISA titers to a synthetic peptide equivalent to an exposed portion of PIB and a higher percentage of antibodies absorbed by whole organisms than the PIB alum antisera. We speculate that when PI is presented in liposomes, the antibodies raised are mainly to surface-exposed epitopes of the protein as opposed to when PI is presented absorbed to alum, where the antibodies are produced mainly to buried epitopes.
Rare peripheral T cells migrate to and persist in normal mouse thymus.
Authors: Michie S A , Kirkpatrick E A , Rouse R V
Published: June 2004

The traffic of T cells between the thymus and peripheral lymphoid organs is generally thought to be unidirectional. Using a technique of lymphocyte transfer between Thy-1 congenic mice, we demonstrate here the entry of rare peripheral lymph node T cells into the normal mouse thymus. At time points from 3 h to 24 wk after transfer, donor peripheral T cells were present in the host thymus, mainly as scattered single cells confined to the medulla. At 2 wk after transfer, donor T cells constituted 0.2% of the medullary thymocytes (compared with 11% of the peripheral lymph node T cells). As a population, these cells exhibited a stable mature immunophenotype (Ly-1hi, PNAlo, and mixed L3T4- and Lyt-2+). A minority of the donor T cells expressed high levels of the MEL-14 "homing receptor". The thymic medulla thus exhibits features of a peripheral lymphoid organ but differs in its low rate of turnover of recirculating T cells.
Identification of CD2-/CD3+ T cells in fetal human tissue.
Authors: Kabelitz D , Conradt P
Published: June 2004

From 1 to 23% of fetal human spleen or thymus cells (from the 20th to 24th week of gestation) were found to display a previously unrecognized CD2-/CD3+ phenotype. IL-2-dependent, long-term clones of CD2-/3+ T cells did not react with a panel of anti-CD2 mAbs and did not form rosettes with sheep erythrocytes. These results show that (a) significant numbers of CD2-/3+ T cells are present in fetal human spleen and/or thymus; and (b) in contrast to the widely accepted view, expression of CD2 is not a prerequisite for the expression of the CD3 molecular complex on human T cells.
The lysine residue in the membrane-spanning domain of the beta chain is necessary for cell surface expression of the T cell antigen receptor.
Authors: Morley B J , Chin K N , Newton M E , Weiss A
Published: June 2004

The TCR is a complex receptor composed of seven polypeptide chains consisting of a ligand-binding subunit, Ti, and a putative signal-transducing subunit, CD3. Phylogenetically conserved charged amino acid residues within the membrane-spanning domains present in all seven chains of the TCR have been proposed to be important in the association between Ti and CD3. Using a Ti beta chain-deficient mutant of the cell line Jurkat, site-directed mutagenesis and transfection of Ti beta chain cDNA was performed to assess the importance of the lysine residue at position 290 within the membrane-spanning domain of the Ti beta chain to expression of the TCR complex. These studies demonstrated that the lysine residue, and not simply conservation of either basic charge or secondary structure, is important at this position.
Monoreactive high affinity and polyreactive low affinity rheumatoid factors are produced by CD5+ B cells from patients with rheumatoid arthritis.
Authors: Burastero S E , Casali P , Wilder R L , Notkins A L
Published: June 2004

In patients with rheumatoid arthritis (RA), circulating CD5+ B lymphocytes, but not CD5- B lymphocytes, are increased in number and size, exist in an activated state, spontaneously proliferate, and secrete Ig that binds to the Fc fragment of IgG. By constructing continuous mAb-secreting cell lines from CD5+ B lymphocytes, the properties and dissociation constants (Kd) of these antibodies were determined. Two types of rheumatoid factors (RFs) with discrete reactivities were produced. The first type is polyreactive and binds with relatively low affinity (Kd, 10(-5) mol/liter) to the Fc fragment of IgG. These antibodies are similar to those produced by CD5+ B cells from healthy subjects. The second type of RF is monoreactive and binds with higher affinity (Kd, 10(-7) mol/liter) to the Fc fragment of IgG. These latter autoantibodies are produced by CD5+ B cells of RA patients, but not healthy subjects. Long-term longitudinal studies are needed to determine the role of these two types of RFs in the pathogenesis of RA.
A second chain of human CD8 is expressed on peripheral blood lymphocytes.
Authors: Shiue L , Gorman S D , Parnes J R
Published: June 2004

Human CD8 has been thought to consist of disulfide-linked homodimers and homomultimers of a single polypeptide chain homologous to mouse and rat CD8 alpha. In contrast, mouse and rat CD8 are composed of disulfide-linked heterodimers of alpha and beta chains. We have now isolated and sequenced cDNA clones encoding a human homologue of mouse and rat CD8 beta. One such clone was inserted into an expression vector and its encoded product was shown to be expressed on the cell surface after cotransfection into L cells with the human CD8 alpha gene. A second form of human CD8 beta cDNA encoding a protein with an altered cytoplasmic tail was similarly transfected, but its product could not be demonstrated on the cell surface. CD8 beta was further shown to be expressed on the surface of almost all CD8+ human peripheral blood T cells. These data provide the first evidence that human CD8 is a heterodimeric protein.