Biomedical subjects

J P Schneck

Publications and source records attributed to J P Schneck.

At least 19 recordsLinked to original sources

PubMed2001

Antigen-specific blockade of T cells in vivo using dimeric MHC peptide.

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.

Animals↗

PubMed2001

Lack of coreceptor allows survival of chronically stimulated double-negative alpha/beta T cells: implications for autoimmunity.

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.

Animals↗

PubMed2001

Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries.

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.

Antigens, Viral↗

PubMed2001

Antigen-induced T cell death is regulated by CD4 expression.

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.

Animals↗

PubMed2001

Increased TCR avidity after T cell activation: a mechanism for sensing low-density antigen.

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.

Animals↗

PubMed2000

Increased activated human T cell lymphotropic virus type I (HTLV-I) Tax11-19-specific memory and effector CD8+ cells in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with HTLV-I provirus load.

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.

Adult↗

PubMed2000

Enhanced antigen-specific antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex.

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.

Alanine↗

PubMed2000

Monitoring antigen-specific T cells using MHC-Ig dimers.

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.

Antigens↗

PubMed1999

An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines.

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 vaccinees immunized with a CP vector expressing portions 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 a novel technique for flow cytometric 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.

AIDS Vaccines↗

PubMed1999

Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses.

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.

Base Sequence↗

PubMed1999

MHC-based diagnostics and therapeutics - clinical applications for disease-linked genes.

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Animals↗

PubMed1999

Attrition of T cell memory: selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses.

Using a variety of techniques, including limiting dilution assays (LDA), intracellular IFNgamma 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 that accommodates memory T cell populations for multiple pathogens over the course of a lifetime.

Animals↗

PubMed1998

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.

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 cerebrospinal 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-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization 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.

Flow Cytometry↗

PubMed1997

Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

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 human histocompatibility 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.

Animals↗

PubMed1997

Differential effects of B cell receptor and B cell receptor-FcgammaRIIB1 engagement on docking of Csk to GTPase-activating protein (GAP)-associated p62.

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.

Binding Sites↗

PubMed1997

Peptide binding to MHC class I is determined by individual pockets in the binding groove.

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.

Animals↗

PubMed1996

Csk is constitutively associated with a 60-kDa tyrosine-phosphorylated protein in human T cells.

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.

Amino Acid Sequence↗

PubMed1996

Expression of human recombinant beta 2-microglobulin by Aspergillus nidulans and its activity.

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 HLA class I MHC complex.

Aspergillus nidulans↗