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1. Webb, T, Bieler, J, Schneck, JP, Oelke, M. Ex vivo induction and expansion of Natural Killer T cells be CD1d1-Ig coated artificial antigen presenting cells. J Immunol Methods 2009. May 14

   Natural killer T (NKT) cells play a pivotal role in maintaining immune homostasis. They recognize lipid antigen in the context of CD1d molecules and subsequently produce cytokines that activate cells of both the innate and adaptive immune responses. Many studies examining patients with autoimmune disease or cancer have shown that there is a reduction in both NKT cell number and function. Due to the complexities of manipulating NKT cells in vivo, ex vivo expanded effector NKT cells would be an excellent therapeutic modality. To date, immunotherapy utilizing the NKT/CD1d system has been dependent on the use of autologous DC in the presence or absence of a synthetic glycolipid, alpha-galactocylceramide. Here we report a novel technique that facilitates the growth and analysis of NKT cells through the use of CD1d-expressing aAPC. CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical (Valpha14(-)) NKT cells. Importantly, CD1d-Ig aAPC can expand NKT cells from cancer patients. Thus, CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies

2. Ndhlovu, Z, Angenendt M, Heckel D, Schneck, JP, Griffin DE and Oelke M. Development of artificial antigen- presenting cell (aAPC)- based assay for the detection of low frequency virus- specific CD8+4 Tcells in whole blood with application to measles virus. Clin Vaccine Immunol. 2009. Jun 3.

   Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8(+) T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8(+) T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-gamma) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8(+) T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8(+) T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8(+) T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-gamma mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8(+) T cells in vaccine trials. The technology should be transferable to analysis of CD8(+) T-cell responses to other antigens.

3. Webb T, Giuntoli R, Rogers O, Schneck J.P. Oelke M. Ascites Specific Inhibition of CD1d-Mediated Activation of NKT cells. Clinical Cancer Research. 2008. Dec 1.

   Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer-associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells. EXPERIMENTAL DESIGN: To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-immunoglobulin-based artificial antigen presenting cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classic CD8+ T cells. RESULTS: Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the ability of the cells to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of alpha-galactosylceramide-loaded CD1d-immunoglobulin-based aAPC to activate NKT cells. In addition, our data show that treatment with ascites does not inhibit HLA-A2-mediated activation of classic CD8+ T cells. CONCLUSIONS: Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.

4. Durai M., Krueger C., Ye Z., Cheng L., Mackensen A., Oelke M., Schneck J.P. In vivo Functional efficacy of tumor-specific T cells expanded using HLA-lg based artificial Antigen Presenting Cells (aAPC). Cancer Immunology and Immunotherapy. 2008. Dec 1.

   Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.

5. Tao S, Li Y , Zhou J , Qian J, Schnaar R, Zhang Y , Goldstein I , Zhu H, Schneck J.P. Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers Glycobiology. 2008. Oct 18 .

   Glycosylation is among the most complex posttranslational modifications with an extremely high level of diversity that has made it refractory to high-throughput analyses. Despite its resistance to high-throughput techniques, glycosylation is important in many critical cellular processes that necessitate a productive approach to their analysis. To facilitate studies in glycosylation, we developed a high-throughput lectin microarray for defining mammalian cell surface glycan signatures. Using the lectin microarray we established a binary analysis of cell binding and hierarchical organization of 24 mammalian cell lines. The array was also used to document changes in cell surface glycosylation during cell development and differentiation of primary murine immune system cells. To establish the biological and clinical importance of glycan signatures, the lectin microarray was applied in two systems. First, we analyzed the cell surface glycan signatures and were able to predict mannose-dependent tropism using a model pathogen. Second, we used the glycan signatures to identify novel lectin biomarkers for cancer stem-like cells in a murine model. Thus, lectin microarrays are an effective tool for analyzing diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, and cell surface biomarker identification.

6. Shaikh SR, Mitchell D, Carroll E, Li M, Schneck J.P., and Edidin M. Differential effects of a saturated and a monounsaturated fatty acid on MHC> class I antigen presentation. Scand. J. Immunol. 2008 Jul;68(1):30-42.

   Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids.

7. Nagaraj, S., Gupta, K., Pisarev,V., Kinarsky, L., Sherman, S., Loveleen, K., Herber, D., Schneck, J.P., and Gabrilovich, D. Altered recognition of antigen is a mechanism of CD8+T cell tolerance in cancer. Nature Medicine 13, 2007: 828-835. Download PDF

   Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide–major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.

8. Mohamood , A.S., Guler, M.L., Xiao, Z., Zheng, D., Hess, A., Wang, Y., Yagita, H., Schneck, J.P., Hamad, A.R. Protection from autoimmune diabetes and T-Cell lymphoproliferation induced by FasL mutation are differentially regulated and can be uncoupled pharmacologically. Am J Pathol. 2007 Jul; 171(1):97-106. Download PDF

9. Fahmy,T., Schneck ,J.P., Saltzman, W. A nanoscopic multivalent antigen-presenting carrier for sensitive detection and drug delivery to T Cells. Nanomedicine .2007Mar;3(1)75-85.

10. Deviren, G., Gupta, K., Paulaitis, M.E., Schneck, J.P. Detection of Antigen–Specific T cells on p/MHC Microarrays. J. Mol. Recognit. 2007Jan-Feb:20(1):32-8; Aug 11 Pub.Online 9 Nov 2006. Download PDF

    The development of high-throughput protein microarrays for rapidly determining antigen-specific T-cell receptor repertoires of diverse T-cell populations can enable comprehensive, broad-based analyses of T-cell responses. Promising applications include medical diagnostics, vaccine development, treatment of autoimmune diseases and detection of potential agents of bioterrorism. In this study, we examined the feasibility of using peptide/major histocompatibility complex (p/MHC) microarrays to selectively capture and enumerate antigen-specific T cells. Results are presented for p/MHC microarrays consisting of a dimeric MHC-immunoglobulin complex, K&b-Ig, loaded with either a cognate or non-cognate peptide for binding CD8R T cells.We quantified the sensitivity of these Kb-Ig microarrays by measuring a lower detection limit of 0.05% antigen-specific CD8R T cells mixed with splenocytes from C57BL/6J mouse. A fivefold increase in this lower detection limit (0.01%) was achieved using a secondary capture anti-Ig antibody to coat the microarray surface. This higher sensitivity is comparable to that obtained using standard state-of-the-art fluorescence activated cell sorting (FACS) instruments. We also found that contacting the T-cell suspension with the Kb-Ig microarrays under mild shear flow conditions produced more uniform distributions of captured T cells on the individual spots and better spot-to-spot reproducibility across the entire microarray. Copyright © 2006 John Wiley & Sons, Ltd.

11. Mohamood, A.S., Trujillo, C.J., Zheng, D., Jie, C., Martinez, M.F., Schneck, J.P., Hamad, A.R.A. Gld mutation of Fas ligand increases the frequency and up-Regulates Cell survival genes in CD25+CD4+ TR cells. Int. Immunol. 2006 Jun 12; [Epub ahead of print]. Download PDF

12. Boin F. Wigley FM, Schneck J.P.,Oelke M., Rosen A. Evaluation of topoisomerase-1- specific CD8+ T-cell response in systematic sclerosis. Ann NY Acad Sci. 2005; Dec;1062:137-45.

13. Oelke, M., Krueger, C., Giuntoli, R.L., II, Schneck, J.P. Artificial antigen-presenting cells: artificial solutions for real diseases. Trends in Molecular Medicine 2005; 11:412-420. Download PDF

14. Karabekian, Z., Simon, D., Lytton, Silver, P.B., Sergeev, Y., Schneck, J.P., and Caspi, R.R. Antigen/MHC class II/Ig dimmers for study of uveitogenic T cells: IRBP p161-180 presented by both IA and IE molecules. Investigative Ophthalmology & Visual Scienc, 2005; 46:3769-3776. Download PDF

15. Oelke, M., Krueger, C., and Schneck, J. P. Technological advances in adoptive immunotherapy. Drugs of Today 2005; 41:13-21. Download PDF

    Adoptive immunotherapy is an attractive and elegant strategy for treating a variety of life-threatening diseases. Several approaches have been developed to generate antigen-specific CD4+ and CD8+ T cells for adoptive T-cell therapy in cancer and infectious diseases. Currently, many approaches are based on either the use of autologous peptide pulsed dendritic cells as antigen-presenting cells or nonspecific expansion of T cells. Unfortunately, current approaches lack the ability to serve as reproducible and economically viable methods. Several groups are developing new artificial approaches to overcome problems associated with dendritic cells and the nonspecific expansion of Tcell clones in order to make adoptive immunotherapy more feasible and effective. Thus, by increasing the availability of adoptive immunotherapy, we will be able to better determine the efficacy of the approaches in the treatment of a variety of diseases.

16. Technological advances in adoptive immunotherapy. Mathias Oelke, Christine Krueger and Jonathan P. Schneck. Drugs of Today, 41:13-21, 2005. Download PDF

    Adoptive immunotherapy is an attractive and elegant strategy for treating a variety of life-threatening diseases. Several approaches have been developed to generate antigen-specific CD4+ and CD8+ T cells for adoptive T-cell therapy in cancer and infectious diseases. Currently, many approaches are based on either the use of autologous peptide pulsed dendritic cells as antigen-presenting cells or nonspecific expansion of T cells. Unfortunately, current approaches lack the ability to serve as reproducible and economically viable methods. Several groups are developing new artificial approaches to overcome problems associated with dendritic cells and the nonspecific expansion of T-cell clones in order to make adoptive immunotherapy more feasible and effective. Thus, by increasing the availability of adoptive immunotherapy, we will be able to better determine the efficacy of the approaches in the treatment of a variety of diseases. In this review, we focus on technological advances that will facilitate adoptive immunotherapy. Specifically, we summarize current strategies which are either based on artificial antigen-presenting cells or on T-cell receptor gene transfer.

17. Effect of human papillomavirus-16 infection on CD8+ T-cell recognition of a wild-type sequence p53264-272 peptide in patients with squamous cell carcinoma of the head and neck. Sirianni N, Ha PK, Oelke M, Califano J, Gooding W, Westra W, Whiteside TL, Koch WM, Schneck JP, DeLeo A, Ferris RL. Clin Cancer Res. 10:6929-37, 2004.

    PURPOSE: Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, currently considered primarily for patients whose tumors overexpress p53. Circumstances exist, however, where increased p53 degradation may result in appreciable presentation of p53-derived peptides, despite low p53 expression. Squamous cell carcinoma of the head and neck is associated with oncogenic human papillomavirus (HPV) subtypes, which inactivate p53 through proteasomal degradation. The criterion of p53 overexpression would exclude these individuals from wt p53-based immunotherapy. EXPERIMENTAL DESIGN: We tested the correlation of HPV infection with enhanced antigenicity of the p53 protein and postulated that removal of HPV-16(+) tumors with enhanced p53(264)-(272) peptide presentation might lead to a drop in T cells specific for this peptide in vivo. Circulating frequencies of T cells specific for the HLA A*0201:p53(264)-(272) complex were measured ex vivo using dimeric HLA:peptide complexes in 15 head and neck cancer patients before and 6 months after tumor excision. RESULTS: CD8+ T-cell recognition of HLA A*0201 restricted wt p53(264)-(272) peptide presented by HPV-16(-) squamous cell carcinoma of the head and neck lines was enhanced by HPV-16 E6 expression, sometimes exceeding that of a naturally transformed, HPV-16(+) wt p53 expressing squamous cell carcinoma of the head and neck cell line. In patients with HPV-16(-) tumors, the frequency of wt p53(264-272)-specific T cells remained largely unchanged after tumor removal. However, a significant decline in frequency of anti-p53(264-272) T cells was observed postoperatively in HPV-16(+) patients (P < 0.005).

    CONCLUSIONS: Recognition of HPV-associated squamous cell carcinoma of the head and neck appears associated with levels of wt p53-specific T cells and inversely with p53 expression. p53 peptides may be useful tumor antigens for squamous cell carcinoma of the head and neck immunotherapy in addition to viral gene products.

18. Immunotherapy with enhanced self immune cells. Mathias Oelke and Jonathan Schneck. Discovery Medicine, 4:203-207, 2004.

19. Quality and quantity: New strategies to improve immunotherapy of cancer. Krueger C, Schneck JP and Oelke M. Trends in Molecular Medicine 10:205-208, 2004. Download PDF

    Adoptive immunotherapy is a promising approach for the treatment of infectious diseases and cancer. Several lines of research are currently focusing on the development of different technologies to facilitate the induction and expansion of antigen-specific T cells. Here, we discuss two current articles that affect the field of adoptive immunotherapy. One article describes the engineering of artificial antigen-presenting cells, which promise to replace the cumbersome dendritic-cell approach for the in vitro generation of large numbers of antigen-specific T cells. The second development is a description of a new technique for the detection of functionally active antigen-specific T cells, which will enhance the ability to control the quality of the T cells to be used in adoptive immunotherapy. Together, these exciting findings will advance the field of immunotherapy.

20. HLA-Ig based artificial Antigen-presenting cells: Setting the terms of engagement. Mathias Oelke and Jonathan P. Schneck. Clinical Immunology 10:243-251. 2004. Download PDF

    Recent advances in molecular medicine have shown that soluble MHC-multimers can be valuable tools for both the stimulation of as well as the analysis of antigen-specific T cells in vitro. In this review, we describe the use of dimeric major histocompatibility complexes, HLA-Ig, to visualize antigen-specific T cells as well as their potential to stimulate immune responses as part of an artificial Antigen Presenting Cell (aAPC). The use of HLA-Ig based aAPC represents an exciting new approach to generate antigen-specific CTL for adoptive immunotherapy that helps to overcome many of the obstacles associated with limitations in current approaches to adoptive immunotherapy.

21. B220+ DN T cells suppress polyclonal T cell activation by a Fas-independent mechanism that involves inhibition of IL-2 production. Hamad AR, Mohamood AS, Trujillo CJ, Huang C-T, Yuan E and JP Schneck. J. Immunol. 171:2421-2426, 2003. Download PDF

    Fas-mediated apoptosis is a key mechanism for elimination of autoreactive T cells, yet loss of function mutations in the Fas signaling pathway does not result in overt T cell-mediated autoimmunity. Furthermore, mice and humans with homozygous Fas(lpr) or Fas ligand(gld) mutations develop significant numbers of B220+ CD4- CD8- double-negative (DN) alphabeta T cells (hereafter referred to as B220+ DN T cells) of poorly understood function. In this study, we show that B220+ DN T cells, whether generated in vitro or isolated from mutant mice, can suppress the ability of activated T cells to proliferate or produce IL-2, IL-10, and IFN-gamma. B220+ DN T cells that were isolated from either lpr or gld mice were able to suppress proliferation of autologous and syngeneic CD4 T cells, showing that suppression is Fas independent. Furthermore, restoration of Fas/Fas ligand interaction did not enhance suppression. The mechanism of suppression involves inhibition of IL-2 production and its high affinity IL-2R alpha-chain (CD25). Suppression also requires cell/cell contact and TCR activation of B220+ DN T cells, but not soluble cytokines. These findings suggest that B220+ DN T cells may be involved in controlling autoreactive T cells in the absence of Fas-mediated peripheral tolerance.

22. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig coated artificial Antigen Presenting Cells. Oelke M, Maus MV, Didiano D, June CH, Mackensen A, and Schneck JP. Nature Medicine, 9:619-624, 2003. Download PDF

    Adoptive immunotherapy holds promise as a treatment for cancer and infectious diseases, but its development has been impeded by the lack of reproducible methods for generating therapeutic numbers of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs). As a result, there are only limited reports of expansion of antigen-specific CTLs to the levels required for clinical therapy. To address this issue, artificial antigen-presenting cells (aAPCs) were made by coupling a soluble human leukocyte antigen-immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody to beads. HLA-Ig-based aAPCs were used to induce and expand CTLs specific for cytomegalovirus (CMV) or melanoma. aAPC-induced cultures showed robust antigen-specific CTL expansion over successive rounds of stimulation, resulting in the generation of clinically relevant antigen-specific CTLs that recognized endogenous antigen-major histocompatibility complex complexes presented on melanoma cells. These studies show the value of HLA-Ig-based aAPCs for reproducible expansion of disease-specific CTLs for clinical approaches to adoptive immunotherapy.

23. Peptide-b2-microglobulin-MHC fusion molecules bind antigen-specific T cells and can be used for multivalent MHC-Ig complexes. Greten TF, Korangy F, Neumann G, Wedemeyer H, Schlote K, Heller A, Scheffer S, Pardoll DM, Garbe AI, Schneck JP, Manns MP. J Immunol Methods, 271:125-135, 2002. Download PDF

    Recombinant soluble MHC molecules are widely used for visualization, activation and inhibition of antigen-specific immune responses. Using a genetic approach, we have generated two novel peptide-beta2-microglobulin-MHC constructs. We have linked the MHC molecule with the peptide of interest, without limiting the recognition by the cognate TCR. This molecule can also be joined with the IgG heavy chain resulting in a dimeric MHC-Ig fusion protein. These molecules bind antigen-specific T cells with high specificity and sensitivity, therefore, providing a valuable tool for detection as well as enrichment of antigen-specific T cells.

24. Probing T cell membrane organization using dimeric MHC-Ig complexes. Fahmy T, Bieler J, Schneck J. J Immunol Methods. 2002 Oct 1;268(1):93. Download PDF

    In this report, we review a novel method for probing the membrane organization of T cells using dimeric major histocompatibility complexes (MHC), MHC-Ig. MHC-Ig complexes are useful reagents for quantitative analysis of binding data since their valency is controlled. These complexes can be easily labeled and loaded with a variety of peptides. A binding assay using these dimers and quantitative analysis of the MHC-Ig dimer-T cell binding curves is described in detail. Using this approach, we show that the organization of TCR on activated T cells is different from TCR organization on nai;ve T cells. The implications of these findings are discussed with regards to current models of T cell recognition. This analysis offers insight into how T cell controls their biological range of responsiveness. Specifically, these findings reveal the biophysical basis of the ability of activated T cells to recognize low amounts of antigen independent of costimulation.

25. T cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens. Brehm MA, Pinto AK, Daniels KA, Schneck JP, Welsh RM, Selin LK. Nat Immunol. 2002 Jul;3(7):627-34.

    We show here that T cell cross-reactivity between heterologous viruses influences the immunodominance of virus-specific CD8(+) T cells by two mechanisms. First, T cells specific for cross-reactive epitopes dominate acute responses to viral infections; second, within the memory pool, T cells specific for cross-reactive epitopes are maintained while those specific for non-cross-reactive epitopes are selectively lost. These findings suggest an immunological paradigm in which viral infections shape the available T cell repertoire, causing alterations in the hierarchies of both the primary and memory CD8(+) T cell responses elicited by subsequent viral infections. Thus, immunodominance is a function of the host's previous exposure to unrelated pathogens, and this may have an impact on protective immunity and immunopathology.

26. Development and use of multimeric major histocompatibility complex molecules. Greten TF, Schneck JP. Clin Diagn Lab Immunol 2002 Mar;9(2):216-20, 2002. Download PDF

27. Antigen-induced T cell death is regulated by CD4 expression. Hamad AR, Schneck JP. Int Rev Immunol 2001 Oct;20(5):535-46. 2001

    Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.

28. Antigen-specific blockade of t cells in vivo using dimeric mhc peptide. O'Herrin SM, Slansky JE, Tang Q, Markiewicz MA, Gajewski TF, Pardoll DM, Schneck JP, Bluestone JA. J Immunol 167(5):2555-2560, 2001. Download PDF

    Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.

29. Lack of coreceptor allows survival of chronically stimulated double-negative alpha/beta T cells: implications for autoimmunity. Hamad AR, Srikrishnan A, Mirmonsef P, Broeren CP, June CH, Pardoll D, Schneck JP. J Exp Med 193(10):1113-1121, 2001. Download PDF

    Lymphoproliferative diseases are characterized by massive accumulation of CD4(-)CD8( -)B220(+) (double-negative [DN]) T cells in peripheral organs. Although evidence indicates these cells are derived from mature autoreactive alpha/beta T cells, the significance of coreceptor downregulation is not known. In this study, we examined the role CD4 coreceptor plays in the survival of repeatedly stimulated T cells. CD4(+/+) and CD4(-/-) T cells from AND T cell receptor (TCR) transgenic mice exhibited similar phenotypes after antigenic stimulation, but the CD4(-/-) T cells survived in much larger numbers than the CD4(+/+) cells upon primary and secondary major histocompatibility complex (MHC)/peptide stimulation. Enhanced survival of CD4(-/-) T cells was due to decreased apoptosis rather than enhanced proliferation. Similarly, circumvention of the CD4/MHC interaction by using a surrogate TCR ligand that does not engage CD4 led to significant enhancement of CD4(+/+) cells than when stimulated with MHC/peptide. Finally, we generated DN B220(+) T cells using an in vitro model system and showed they were more tolerant to chronic stimulation than CD4(+/+) cells. Together, these results indicate that coreceptor engagement controls expansion of normal T cells. In the absence of coreceptor, T cells survive chronic stimulation and express B220 as seen in autoimmune lymphoproliferative diseases.

30. Activation induced death of T cell regulated by CD4 coreceptor. AR Hamad and J. Schneck. Intern Rev Immunol 20:1-12, 2001.

    Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/ FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.

31. Increased TcR avidity after T cell activation: A mechanism for sensing low density antigen. Fahmy, T., Bieler, J.G., Edidin, M., and Schneck, J.P. Immunity 14, 135-143, 2001, Cover Article. Download PDF

    While activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery.

32. Enhanced Immunogenicity of CTL Epitope Analogues Derived from Positional Scanning Synthetic Combinatorial Libraries. La Rosa, C., Krishnan, R., Papp, J., Gardner, A., Markel, S., Mead, J Schneck, J.P., Houghten, R., Pinilla, C., and Diamond, D.J. Blood. 97:1776-1786, 2001. Download PDF

    The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.

33. Increased activated HTLV-I Tax11-19-specific CD8+ cells in patients with HTLV-I-associated myelopathy/Tropical spastic paraparesis: Correlation with HTLV-I proviral load. Nagai, M., Ryuji, R., Greten, T.F., Schneck, J.P., Leist, L.P. and Jacobson, S. J. Infectious Diseases 183:197-205, 2001.Download PDF

    To discern the T cell subtype associated with T cell differentiation, the expression of CD45RA and CD27 was measured from total CD8(high) cells and from human T cell lymphotropic virus type I (HTLV-I) Tax11-19 peptide-specific CD8(+) cells in peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phenotypically defined memory and/or effector cells (CD45RA(-)CD27(+), CD45RA(+)CD27(-), and CD45RA(-)CD27(-)) were increased in HAM/TSP CD8(+) cells, compared with those of HTLV-I-seronegative healthy control subjects. The percentage of human leukocyte antigen (HLA)-DR-positive cells was also increased in CD8(+) cells of HAM/TSP, compared with those in HLA-DR(+)CD8(+) cells of healthy control subjects. HTLV-I provirus load correlated with the frequency of Tax11-19-specific CD8(+) cells. The high frequency of memory and/or effector type HTLV-I Tax11-19-specific CD8(+) cells suggests that continuous restimulation driven by HTLV-I antigens in vivo may be associated with the pathogenesis of HAM/TSP.

34. Enhanced Antigen-Specific Antitumor Immunity with Altered Peptide Ligands that Stabilize the MHC-Peptide-TCR Complex. Slansky, J.E., Rattis, F.M., Boyd, L.F., Fahmy, T., Jaffee, E.M., Schneck, J.P., Margulies, D.H., and Pardoll, D.M. Immunity 13:529-538, 2000. Download PDF

    T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I-restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.

35. Monitoring Antigen-Specific T Cells Using MHC-Ig Dimers. Schneck, J.P., Immunol. Invest. 29:163-169, 2000.

    To summarize, two novel approaches are currently being examined that allow for identification of antigen-specific T cells. A biochemical approach to generating soluble multivalent MHC complexes has been to generate tetrameric MHC complexes linked to avidin. We have also generated a general approach for producing soluble divalent versions of class I and class II MHC molecules, using Ig as a molecular scaffold. The experimental system described here outlines a general approach of using multivalent high affinity ligands to study cell-cell interactions, driven by multivalent ligand-receptor interactions. Our work indicates that divalent chimeric molecules are high-avidity analogs of proteins useful in probing and selectively regulating cellular responses.

36. Monitoring Antigen-Specific T Cells Using MHC-Ig Dimers. Schneck, J.P., Slansky, J.E., O'Herrin, M.O., and Greten, T.F. Current Protocols of Immunological Techniques Supplement 35:17.0.1-17.0.2 & 17.2.1-17.2.17.

37. Attrition of T cell memory: selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses. Selin, L.K., Lin, M.Y., Kraemer, K.A., Pardoll, D.M., Schneck, J.P. Varga, S.M., Santolucito, P., Pinto, A.K., and Welsh, R.M. Immunity. 11:733-42, 1999. PDF Version

    Using a variety of techniques, including limiting dilution assays (LDA), intracellular IFN-g assays, and Db-IgG1 MHC dimer staining to measure viral peptide-specific T cell number and function, we show here that heterologous virus infections quantitatively delete and qualitatively alter the memory pool of T cells specific to a previously encountered virus. We also show that a prior history of a virus infection can alter the hierarchy of the immunodominant peptide response to a second virus and that virus infections selectively reactivate memory T cells with distinct specificities to earlier viruses. These results are consistent with a model for the immune system which accommodates memory T cell populations for multiple pathogens over the course of a lifetime.

38. Lateral Diffusion of GFP-Tagged H2Ld Molecules and of GFP-TAP 1 Reports on the Assembly and Retention of these Molecules in the Endoplasmic Reticulum. Marguet, D., Spiliotis, E.T., Pentcheva, T., Lebowitz, M., Schneck, J., and Edidin, M. Immunity, 11:231-240, 1999.PDF Version

    Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.

39. The Major Histocompatibility Complex Leads the Way for Disease-Linked into the Clinic. Howard, M., Spack, E.G., Choudhury, K., Greten, T.F., and Schneck, J.P. Immunol. Today, 20:161-165, 1999.

40. Soluble, High-Affinity Dimers of T-Cell Receptors and Class II Major Histocompatibility: Biochemical Probes for Analysis and Modulation of Immune Responses. Lebowitz, M. S., O'Herrin, S.M., Hamad, A.-R., Fahmy, T., Marguet, D., Barnes, N. B., Pardoll, D., Bieler, J. G., and Schneck, J. P. Cellular Immunology, 192:175-184, 1999. PDF Version

    T cell receptors (TCR) and major histocompatibility complex (MHC) molecules are integral membrane proteins that have central roles in cell-mediated immune recognition. Therefore, soluble analogs of these molecules would be useful for analyzing and possibly modulating antigen-specific immune responses. However, due to the intrinsic low affinity and inherent solubility problems, it has been difficult to produce soluble high affinity analogs of TCR and class II MHC molecules. This report describes a general approach which solves this intrinsic low affinity by constructing soluble divalent analogs using IgG as a molecular scaffold. The divalent nature of the complexes increases the avidity of the chimeric molecules for cognate ligands. The generality of this approach was studied by making soluble divalent analogs of two different classes of proteins, a TCR (2C TCR2Ig) and a class II MHC (MCCI-Ek2Ig) molecule. Direct flow cytometry assays demonstrate that the divalent 2C TCR2Ig chimera retained the specificity of the native 2C TCR, while displaying increased avidity for cognate peptide/MHC ligands, resulting in a high affinity probe capable of detecting interactions that heretofore have only been detected using surface plasmon resonance. TCR2IgG was also used in immunofluorescence studies to show ER localization of intracellular peptide-MHC complexes after peptide feeding. MCCI-Ek2Ig chimeras were able to both stain and activate an MCC-specific T cell hybridoma. Construction and expression of these two diverse heterodimers demonstrate the generality of this approach. Furthermore, the increased avidity of these soluble divalent proteins makes these chimeric molecules potentially useful in clinical settings for probing and modulating in vivo cellular responses.

41. An Algorithm for Evaluating Human Cytotoxic T Lymphocyte Responses to Candidate AIDS Vaccines. Carruth, L.M., Greten, T.F., Murray, C.E., Castro, M.G., Crone, S. N., Pavlat, W., Schneck, J., and Siliciano, R. F. AIDS Res. and Human Retro. 15:1021-1034, 1999. PDF Version

    Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccines immunized with a CP vector expressing portins of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by novel technique for flow cytometry visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.

42. Direct Analysis of Viral-Specific CD8+ T Cells with Soluble HLA-A2/Tax11-19 Tetramer Complexes in Patients with Human T Cell Lymphotropic Virus-Associated Myelopathy. Bieganowska K, Hollsberg P, Buckle GJ, Lim DG, Greten TF, Schneck J, Altman JD, Jacobson S, Ledis SL, Hanchard B, Chin J, Morgan O, Roth PA, Hafler DA. J Immunol 162:1765-1771, 1999. PDF Version

    Human T cell lymphotropic virus-I (HTLV-1)-associated myelopathy is a slowly progressive neurologic disease characterized by inflammatory infiltrates in the central nervous system accompanied by clonal expansion of HTLV-I-reactive CD8+ T-cells. In patients carrying the HLA-A2 allele, the immune response is primarily directed to the Tax11-19 peptide. The frequency, activation state, and TCR usage of HLA-A2/Tax11-19 binding T cells in patients with HTLV-I-associated myelopathy was determined using MHC class I tetramers loaded with the Tax11-19 peptide. Circulating Tax11-19-reactive T cells were found at very high frequencies, approaching 1:10 circulating CD8+ T cells. T cells binding HLA-A2/Tax11-19 consisted of heterogeneous populations expressing different chemokine receptors and the IL-2R beta-chain but not the IL-2R alpha-chain. Additionally, Tax11-19-reactive CD8+ T cells used one predominant TCR Vbeta-chain for the recognition of the HLA-A2/Tax11-19 complex. These data provide direct evidence for high frequencies of circulating Tax11-19-reactive CD8+ T cells in patients with HTLV-I-associated myelopathy.

43. Potent T Cell Activation with Dimeric Peptide-MHC Class II Ligand: The Role of CD4 Coreceptor. Hamad, A.R.A., O'Herrin, S., Lebowitz, M., Srikrishnan, A., Bieler, J., Schneck, J., and Pardoll, D. JEM 188:1633-1640, 1998. PDF Version

    The interaction of the T cell receptor (TCR) with its cognate peptide-major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a primary event during T cell activation. Here we used a dimeric IEk-MCC molecule to study ist capacity to activate antigen-specific T cells and to directly analyze the role of CD4 in physically stabilizing the TCR-MHC interaction. Dimeric IEk-MCC stably binds to specific T cells. In addition, immobilized dimeric IEk-MCC can induce TCR downregulation and activate antigen-specific T cells mor efficiently than anti-CD3. The potency of the dimeric IEk-MCC is significantly enhanced in the presence of CD4. However, CD4 does not play any significant role in stabilizing peptide-MHC-TCR interactions as it fails to enhance binding of IEk-MCC to specific T cells or influence peptide-MHC-TCR dissociation rate or TCR downregulation. Moreover, these results indicate that dimerization of peptide-MHC class II using an IgG molecular scaffold significantly increases its binding avidity leading to an enhancement of its stimulatory capacity while maintaining the physiological properties of cognate peptide-MHC complex. These peptide-MHC-IgG chimeras may, therefore, provide a novel approach to modulate antigen-specific T cell responses both in vitro and in vivo.

44. Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19 specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Greten, T. F., Slansky, J. E., Kubota, R., Soldan, S. S., Jaffee, E. M., Leist, T. P., Pardoll, D. M., Jacobson, S., and Schneck, J. P. PNAS 95:7568-7573, 1998. PDF Version

    Human T lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8+ lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrocpinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specifc CD8+ T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-a and g-interferon depending on the severity of the disease. Thus, visulaization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8+ T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

45. Analysis of the expression of peptide-major histocompatability complexes using high affinity soluble divalent T cell receptors. O'Herrin, S. M., Lebowitz, M. S., Bieler, J. G. , Al-Ramadi, B., Utz, U., Bothwell, A., and Schneck, J. J. Exp. Med. 186 (8);1333-1345, 1997. PDF Version

    Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by humanhistocompatibility leukocyte antigens-A2. Further, using 2C TCR- Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.

46. Differential Effects of B Cell Receptor and B Cell Receptor-FcgRIIB1 Engagement on Docking of Csk to GAP-associated p62. Vuica, M., Desiderio, S. and Schneck, J. P. J. Exp. Med. 186 (2):259-267, 1997. PDF Version

    The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcgamma receptors of the IIB1 type (FcgammaRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein-associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab')2 resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcgammaRIIB1-deficient cell line IIA1.6 and recovered when FcgammaRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.

47. Peptide binding to MHC class I is determined by individual pockets in the binding groove. Johansen, T. E., McCullough, K., Catipovic, B., Su, X.-M., Amzel, M. and Schneck, J. Scand. J. Immunol. 46:137-146, 1997.

    H-2Kb and HLA-A2 are MHC4 class I molecules with a similar overall structure. Important differences between these two class I molecules reside in the structure of the individual pockets in the antigenic-peptide-binding groove. H-2Kb, which has a deep C pocket, binds specifically peptides with a tyrosine or a phenylalanine at position 5. In contrast, HLA-A2 has a shallow C pocket, which cannot accommodate large side chains at position 5. Site-directed mutagenesis was used to generate a chimera between the murine H-2Kb and the human HLA-A2 [H-2Kb/HLA-A2(C')]. The structure of this chimera is similar to H-2Kb except for the region around the deep C pocket, where residues at positions 9, 97 and 99 were substituted with those bulkier residues from HLA-A2. Peptide binding between this chimera and H-2Kb-binding peptides [VSV (52-59), OVA (257-264), and MCMV pp89 (168-176)], revealed that the deep C pocket of H-2Kb was crucial for high-affinity binding. While a peptide, VSV (52-59), was found to bind with severalfold lower 'affinity' to H-2Kb/HLA-A2(C') than to the wild-type H-2Kb, a VSV analogue with the tyrosine in position 5 (Tyr5) substituted with an alanine was found to bind with a similar 'affinity' to both MHC class I molecules. Computer-aided modelling of the H-2Kb/HLA-A2(C') complex indicates that the VSV (52-59) peptide probably binds to the chimeric MHC molecule with the peptide side chain of anchor residue Tyr5 pointing away from the groove. These results confirm a role of the individual pockets in determining peptide-binding affinity and specificity and suggest that this may be accomplished by changes in side-chain orientation.

48. Expression of human recombinant b2 microglobulin by Aspergillus nidulans and its activity. O'Herrin, S. M., Kulkarni, S., Kenealy, W. R., Fechner Jr., J. H., Sollinger, H., Schneck, J. P., and Burlingham, W. J. Human Immunol. 50: 63-72, 1996.

    The light chain of HLA class I protein (beta 2m) has been expressed in Aspergillus nidulans. The cDNA of beta 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in beta 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of beta 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 177 micrograms/liter of f beta 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human beta 2m produced in fungi (f beta 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified f beta 2m from culture supernatants appeared biochemically similar to beta 2m obtained from human urine (u beta 2m) as seen in immunoblot analysis. Functionally, f beta 2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELISA for detecting properly folded HLA class I heavy chain and in assays showing cell-surface beta 2m exchange into the mouse class I MHC H-2Kd. In these experiments the biological activity of f beta 2m was indistinguishable from u beta 2m. The successful expression of biologically active beta 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLAclass I MHC complex.

49. Csk associates with the TCR d and e chains via its SH2 domain: A mechanism for turning off TCR signalling. Catipovic, B., Schneck, J. P., Brummet, M. E., Marsh, D. G., and Rafnar, T.. Journal Biol.Chem. 271(16) 9698-9703, 1996.

    Csk associates with the TCR d and e chains via its SH2 domain. Rafnar, T., Schneck, J. P., Brummet, M. E., Marsh, D.,G., and Catipovic, B. Ann NY Acad. Sci. 766:206-8, 1995 Sept. 7. PDF Version

    The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase-activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP.

50. Abnormal B-cell function in HTLV-I-tax transgenic mice. Peebles, S., Maliszewski, C. R., Sato, T. A., Hyde, J., Maroulakou, I. G., Huziker, R., Schneck, J., and Green, J. R. Oncogene 10 (6): 1045-51, 1995 Mar. 16.

    Transgenic mice that carry the HTLV-I Tax gene develop an exocrinopathy with some similarities to Sjoegren's syndrome. Our experiments reveal that these mice have lymphadenopathy and splenomegaly composed primarily of B lymphocytes, as well as abnormal levels of secreted immunoglobulins. To gain insight into whether the lymphadenopathy manifested by these transgenic mice was the result of induction of cytokines by Tax, we utilized cell lines from these mice to study in vitro B-cell responses. Conditioned media (CM) derived from the cell lines caused B-cells to proliferate when a second signal, surface Ig cross-linking, was provided. The CM also caused a marked enhancement of IgM secretion by spleen cells or by purified B-cells treated with supplemental cytokines. The B-cell proliferative response and enhanced IgM secretion have not been attributed to a known cytokine. These results suggest that the CM from the cell lines contain a factor(s) involved in novel pathways of B-cell growth and differentiation that may participate in the pathologic development of autoimmune disease.

51. Analysis of the structure of empty and peptide-loaded MHC molecules at the cell surface. Catipovic, B.C., Talluri, G., Oh, J., Wei, T., Su, X.-M., Johansen, T E., Edidin, M., and Schneck, J. P. J. Exp. Med. 180:1753-1761, 1994.

    We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.

52. A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations. Dal Porto, J., Johansen, T.E., Catipovic, B.C., Parfitt, D.-J.,Tuveson, D., Gether, U., Kozlowski, S., Fearon, D., and Schneck, J. PNAS 90:6671-6675, 1993.

    Genetically engineered or chemically purified soluble monovalent major histocompatibility complex (MHC) molecules, which have previously been used to study T cells, have not blocked cytotoxic T-cell responses. Here we describe a genetically engineered divalent class I MHC molecule which inhibits lysis of target cells by alloreactive cytotoxic T cells. This protein, H-2Kb/IgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, H-2Kb, and an immunoglobulin heavy chain polypeptide. The chimeric protein has serological and biochemical characteristics of both the MHC and IgG polypeptides. Nanomolar concentrations of H-2Kb/IgG inhibited lysis of H-2Kb-expressing target cells not only by alloreactive H-2Kb-specific T-cell clones but also by alloreactive H-2Kb-specific primary T-cell cultures. A direct binding assay showed high-affinity binding between the H-2Kb/IgG molecule and an H-2Kb-specific alloreactive T-cell clone. Unlabeled H-2Kb/IgG displaced 125I-labeled H-2Kb/IgG from T cells with an IC50 of 1.2 nM.

53. Role of conserved regions of class I MHC molecules in the activation of CD8+ CTL by peptide and purified cell-free class I molecules. Takeshita, T., Kozlowski, S., England, R.D., Brower, R., Schneck, J., Takahashi, H., DeLisi, C., Margulies, D.H. and Berzofsky, J.A. Intern. Immunol. 5:1129-1138, 1993.

    To analyze the molecular interactions involved in CD8+ cytotoxic T lymphocyte (CTL) recognition quantitatively, we developed a cell-free antigen presenting system. Genetically engineered soluble H-2Dd molecules coated on plastic microtiter plates could present HIV envelope peptide to an antigen-specific CTL clone, inducing it to produce IFN-gamma in the absence of accessory cells and their accessory or co-stimulatory molecules. The peptide-MHC complexes were functionally stable for over 24 h. The magnitude of T cell activation was dependent on the concentrations of both class I MHC molecule and the peptide, but was more sensitive to the concentration of the MHC molecule than to that of peptide. This result suggests that one MHC molecule can play more than one role in activating the CTL. One such role is the interaction between CD8 and a conserved region of class I MHC, as suggested by the finding that holding the total MHC concentration constant with an irrelevant class I MHC molecule (H-2Kb engineered to have the same alpha 3 domain as H-2Dd) made the T cell response less sensitive to the change in concentration of the relevant MHC molecule (H-2Dd). The irrelevant class I MHC molecule (H-2Kb), unable to present this peptide by itself, augmented the T cell response at lower concentrations of peptide.These results suggest that the conserved alpha 3 domain of the class I MHC heavy chain as well as polymorphic regions play an important role in T cell activation and that T cell interaction with MHC molecules not presenting peptide can still augment the response.

54. Major histocompatibility complex conformational epitopes are peptide specific. Catipovic, B., Dal Porto, J., Mage, M., Johansen, T.E., and Schneck, J.P. JEM 176, 1611-1618, 1992.

    Serologically distinct forms of H-2Kb are stabilized by loading cells expressing "empty" class I major histocompatibility complex (MHC) molecules with different H-2Kb binding peptides. The H-2Kb epitope recognized by monoclonal antibody (mAb) 28.8.6 was stabilized by ovalbumin (OVA) (257-264) and murine cytomegalovirus (MCMV) pp89 (168-176) peptides, but not by vesicular stomatic virus nucleoprotein (VSV NP) (52-59) and influenza NP (Y345-360) peptides. The H-2Kb epitope recognized by mAb 34.4.20 was stabilized by VSV NP (52-59) peptide but not by OVA (257-264), MCMV pp89 (168-176), or influenza NP (Y345-360) peptides. Immunoprecipitation of H-2Kb molecules from normal cells showed that 28.8.6 and 34.4.20 epitopes were only present on a subset of all conformationally reactive H-2Kb molecules. Using alanine-substituted derivatives of the VSV peptide, the 28.8.6 epitope was completely stabilized by substitution of the first residue and partially stabilized by substitution of the third or the fifth residues in the peptides. These results indicate that distinct conformational MHC epitopes are dependent on the specific peptide that occupies the antigenic peptide binding groove on individual MHC molecules. The changes in MHC epitopes observed may also be important in understanding the diversity of T cell receptors used in an immune response and the influence of peptides on development of the T cell repertoire.

55. CD8 expression alters the fine specificity of an alloreactive MHC class I-specific T hybridoma. Blok, R., Margulies, D.H., Pease, L., Ribaudo, R.K., Schneck, J., and McCluskey, J. Inter. Immunol. 4;455-66, 1992.

    The influence of CD8 on the fine specificity of MHC class I-restricted T cell allorecognition was evaluated by comparing the reactivity of CD8- and CD8-transfected forms of an allospecific, H-2Kb-restricted T hybridoma. The CD8- T hybridoma responded to cells expressing H-2Kb, H-2Kbm6, and the individual H-2Kb----bm10 back mutations 165V----M, 173K----E, and 174N----L. Under the same conditions the CD8- T hybridoma responded poorly or not at all to cells expressing H-2Kbm10, H-2Kbm8, the individual H-2Kb----bm10 back mutants163T----A and 167W----S, and the individual H-2Kb----bm8 back mutations 22Y----F and 24E----S. In contrast, T hybridoma cells expressing high levels of CD8 reacted strongly with antigen presenting cells (APC) expressing H-2Kb and H-2Kbm6 molecules, as well as APC expressing H-2Kbm10 (weakly), H-2Kbm8, and all five individual H-2Kb----bm10 and the two H-2Kb----bm8 back mutants 22Y----F and 24E----S. The mutations which distinguish the T cell recognition of both H-2Kbm10 and H-2Kbm8 from H-2Kb are predicted to control the interaction of these class I molecules with antigenic peptides in the binding site, implying an important role for peptide antigen in T cell allorecognition. Nonetheless, CD8 expression by the H-2Kb-restricted T cells conferred novel or enhanced alloreactivity with cells expressing H-2Kbm10, H-2Kbm8, and each of the individual H-2Kb----bm10 and H-2Kb----bm8 back mutants. These findings reflect an important role for CD8 in influencing the fine specificity of MHC class I recognition by T cells and may indicate a limited structural role for peptide antigen in defining the ligand recognized by these alloreactive T cells.

56. Immunochemical analysis of a recombinant, genetically engineered, secreted HLA-A2/Q10b fusion protein. DeVito, L.D., Mason, B., Schneck, J., Margulies, D.H., Sollinger, H.W., and Burlingham, W.J. Human Immunol. 32:125-133, 1991.

    We engineered a fusion gene which encodes the alpha 1 and alpha 2 domains of HLA-A2 with the alpha 3 and truncated transmembrane domains of the murine class I-like protein Q10b, and transferred it into mouse L cells along with the gene for human beta 2-microglobulin (beta 2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human beta 2m light chain. Native detergent-solubilized HLA-A2 and secreted rA2/Q10b proteins were found to be similar by: (a) the binding to mouse monoclonal anti-HLA antibodies in an ELISA; (b) the blocking of lysis of HLA-A2+ cells by human anti-HLA-A2,-B17, anti-HLA-A2,9,28, and anti-HLA-A2,28 cross-reactive group (CREG) antisera in a complement-dependent cytotoxicity assay; and (c) the ability when coupled to Sepharose to selectively purify HLA-A2,9,28 and HLA-A2,28 CREG-specific antibodies. Mouse L cells expressing rA2/Q10b produced as much as 2.5 micrograms protein per 10(6) cells/day, or 50- to 100-fold more antigen on a per cell basis than the level of HLA-A2 expressed by B-lymphoblastoid cell line or spleen cells. Thus rA2/Q10b represents a viable alternative to detergent-solubilized HLA-A2 for purification of anti-HLA-A2 antibodies and analysis of anti-HLA-A2 immune responses.

57. Multivalent requirement for the stimulation of alloreactive T cells: Studies with engineered soluble MHC class I proteins in vitro and in vivo. Margulies, D.H., Boyd, L.F., Kozlowski, S., Kjer-Nielson, L., Lopez, R., McCluskey, J., Schneck, J., and Hunziker, R. In: Transgenic Mice and Mutants in MHC Research. David, C. and Egorov, I. (eds.) Springer-Verlag, Berlin, pp. 39-45, 1990.

58. A peptide derived from the alpha helical region of class I MHC blocks CTL engagement of the class I MHC molecule. Munitz, T.I., Schneck, J., Coligan, J.E., Maloy, W.L., Henrich, J.P., Sharrow, S.O., Margulies, D.H., and Singer, A. Cold Spring Harbor Symp. Quant. Biol. 54:557-561, 1989.

59. Inhibition of allorecognition by an H-2Kb-derived peptide is evidence for a T-cell binding region on a major histocompatibility complex molecule. Schneck, J., Munitz, T., Coligan, J.E., Maloy, W.L., Margulies, D.H., and Singer, A. Proc. Natl. Acad. Sci. U.S.A. 86:8516-8520, 1989.

    The class I and class II major histocompatibility complex (MHC) antigens are polymorphic cell-surface glycoproteins that present antigenic peptides to T lymphocytes in the generation of immune responses. While much is known about the recognition and processing of antigens, the nature of T-cell recognition sites on MHC molecules is poorly understood. Both structural and functional studies have suggested that the two major alpha-helical regions of the class I MHC molecule not only define the site for binding of antigenic peptide but also provide potential sites for interaction of the MHC molecule with the T-cell receptor. A peptide derived from one of these regions on the H-2Kb molecule, peptide Kb163-174, was previously shown to specifically inhibit the stimulation of an alloreactive T-cell hybridoma. To further investigate the role of this region in the recognition of H-2Kb, the effects of peptide Kb163-174 on allospecific T-cell lines and clones were studied. When peptide Kb163-174 was cocultured with either an H-2Kbm10 anti-H-2Kb cytotoxic T-lymphocyte (CTL) clone or a CTL line, this peptide inhibited lysis of H-2Kb targets. Pretreatment experiments showed that the blockade was due to interaction of the peptide with the effector T cells. Surprisingly, peptide Kb163-174 also inhibited lysis of H-2Kb targets by H-2Kbm1-, H-2Kbm3-, H-2Kbm6, and H-2Kbm8-anti-H-2Kb CTLs. These CTLs, which identify multiple antigenic sites on H-2Kb in the alpha 1 and alpha 2 domains, are not directed against amino acid residues163-174 of H-2Kb. In addition, peptide Kb163-174 specifically blocked lysis of only H-2Kb and not H-2Ld targets by a single bulk CTL culture that was alloreactive on both H-2Kb and H-2Ld. These results indicate that peptide Kb163-174 interferes with T-cell receptor engagement of a contact site on the H-2Kb molecule. Thus, amino acid residues 163-174 define a site used by many alloreactive T cells to engage the H-2Kb molecule.

60. Inhibition of an allospecific T cell hybridoma by soluble class I proteins and peptides: Estimation of the affinity of a T cell receptor for class I MHC molecules.Schneck, J., Maloy, W.L., Coligan, J.E., and Margulies, D.H. Cell. 55:47-53, 1989.

    To investigate the molecular basis of the interaction between the T cell receptor and the MHC class I antigen in an allogeneic response, a soluble counterpart of the murine class I molecule, H-2Kb, was genetically engineered. Cells secreting this soluble molecule, H-2Kb/Q10b, inhibited stimulation of an H-2Kb-reactive T cell hybridoma by cells transfected with H-2Kbm10, a weak stimulus, but not by H-2Kb- or H-2Kbm6-transfected cells. Soluble purified H-2Kb/Q10b protein also blocked T cell stimulation. In addition, a peptide from the wild-type H-2Kb molecule spanning the region of the bm10 mutation specifically inhibited activation of the T cell hybridoma by H-2Kbm10 cells, thus suggesting that amino acid residues 163-174 of H-2Kb define a region important for T cell receptor binding. An estimate for the Kd of the T cell receptor for soluble H-2Kb/Q10b was 10(-7) M, while the Kd for soluble peptide 163-174 was 10(-4) M.

61. Interaction between interferon and cells of the immune system. Bloom, B.R., Schneck, J., Rager-Zisman, B., Quan, P.C., Neighbour, A., Minato, N., Reid, L., and Rosen, O.M. In: Chemistry and Biology of Interferons: Relationship to Therapeutics - UCLA Symposia on Molecular and Cellular Biology, Vol. XXV. (Merrigan, T.C., and Friedman, R.M., editors). Academic Press, New York. p. 198, 1982.

62. Genetic analysis of the role of cAMP in mediating effects of interferon. Schneck, J., Rager-Zisman, B., Rosen, O.M., and Bloom, B.R. Proc. Natl. Acad. Sci. U.S.A. 79:1879-1883, 1982.

    The effects of interferon (IFN) on Fc receptor-mediated phagocytosis, intracellular cAMP levels, antiviral activity, and growth inhibition were analyzed in a cloned macrophage-like cell line, J774.2, and variants derived from it. Purified IFN increased Fc receptor-mediated phagocytosis in J774.2 cells, and in cAMP-responsive nonphagocytic variants but was without effect in cAMP-unresponsive nonphagocytic variants, in adenylate cyclase-deficient variants, and in cAMP-dependent protein kinase-deficient variants. Under conditions in which IFN augmented phagocytosis, it increased intracellular levels of cAMP. Parental cells were highly sensitive to IFN-mediated growth inhibition. In contrast, cAMP-dependent protein kinase-deficient variants were only 1/100th as sensitive to growth inhibition by IFN. All cell lines tested, both responsive and unresponsive to cAMP, were equally protected by IFN against infection with vesicular stomatitis virus, demonstrating that the antiviral state was independent of cAMP. These results indicate that, in transformed macrophages, stimulation of phagocytosis and inhibition of growth by IFN are mediated through intracellular cAMP, whereas the antiviral state induced by IFN is independent of cAMP.

63. Trifluperazine inhibits phagocytosis in a macrophage-like cultured cell line. Horwitz, S.B., Chia, G.H., Harracksingh, C., Orlow, S., Pifko-Hirst, S., Schneck, J., Sorba, L., Speaker, M., Wilk, E.W., and Rosen, O.M. J. Cell. Biol. 91:798-802, 1981.

    Trifluoperazine, a drug that binds to Ca2+-calmodulin and inhibits its interaction with other proteins, was found to inhibit growth and phagocytosis in a macrophagelike cell line, J774.16. Both effects were reversible and occurred at the same concentrations of drug (25--50 microM) that inhibited the activation of cyclic nucleotide phosphodiesterase by calmodulin in vitro. Fc-mediated phagocytosis was also depressed by W-7, a sulfonamide derivative that inhibits the activity of Ca2+-calmodulin. In contrast, taxol, a drug that stabilizes cellular microtubules, had no effect on Fc-mediated phagocytosis although it inhibited cell growth at nanomolar concentrations. The inhibitory effects of trifluoperazine and W-7 on phagocytosis suggest that calmodulin may be involved in this complex cellular function.

64. Modulation of Fc-receptor expression and Fc-mediated phagocytosis in variants of a macrophage-like cell line. Schneck, J., Rosen, O., Diamond, B., and Bloom, B.R. J. Immunol. 126:745-749, 1981.

65. Genetic and functional studies of continuous macrophage-like cell. Bloom, B.R., Diamond, B., Newman, W., Schneck, J., Piscatello, J., Damiani, G., Rosen, N., Muschel, R., and Rosen, O. In: Mononuclear Phagocytes Functional Aspects, Part II. (van Furt, R., editor). Martinus Nijhoff Publishers, pp. 941-967, 1980.

66. Properties of protein kinase and adenylate cyclase-deficient variants of a macrophage-like cell line. Rosen, N., Piscatello, J., Schneck, J., Muschel, R.J., Bloom, B.R., and Rosen, O. J. Cell. Physiol. 98:125-136, 1979.

    Stable variants of the macrophage-like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth-inhibitory effect of cholera toxin and 8-bromo-adenosine 3':5' cyclic monophosphoric acid (8 Br-cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP-mediated enhancement of phagocytosis and cyclic AMP-mediated inhibition of plasminogen activator secretion and growtn. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br-cAMP-mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br-cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br-cAMP or cholera toxin; they were also insensitive to cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2--inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc-mediated phagocytosis--are mediated by a cyclic AMP-dependent portein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.

67. Inhibition of plasminogen activator secretion by cyclic AMP in a macrophage-like cell line. Rosen, N., Schneck, J., Bloom, B.R., and Rosen, O. J. Cyclic Nucleotide Res. 5:345-358, 1978.

    The continuous cell line, J774.2, exhibits many macrophage-like functions such as latex and Fc-mediated phagocytosis, antibody mediated phagocytosis, antibody mediated cytotoxicity, chemotaxis, and lysozyme secretion. Cyclic AMP stimulates Fc-mediated phagocytosis and inhibits the growth of J774.2. To further evaluate the relationship between cyclic AMP and the specialized functions exhibited by these cells. Variants deficient in phagocytosis, adenylate cyclase and cyclic AMP-dependent protein kinase were derived. We have now shown that J774.2 also secretes plasminogen activator and that this secretion is rapidly and specifically inhibited by 8-bromoadenosine 3':5'-cyclic monophosphoric acid (8 Br--cAMP) or cholera toxin under conditions where lysozyme secretion is unaltered. Utilizing protein kinase-deficient variants, the ability of cyclic AMP to inhibit plasminogen activator secretion was shown to be mediated by a cyclic AMP-dependent protein kinase. We conclude that cyclic AMP has diametrically opposing effects on two macrophage-like functions: Fc-mediated phagocytosis and plasminogen activator secretion.

68. Genetic approaches to the mechanisms of macrophage functions. Bloom, B.R., Diamond, B., Muschel, R., Rosen, N., Schneck, J., Damiani, G., Rosen, O., and Scharff, M. Fed. Proc. 37:2765-2771, 1978.

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