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J A Frelinger

Publications and source records attributed to J A Frelinger.

At least 55 records · Page 3Linked to original sources
PubMed1995

Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.

Amino Acid Sequence↗
PubMed1995

Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition.

During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide.

Amino Acid Sequence↗
PubMed1995

Alterations of a dominant epitope of lymphocytic choriomeningitis virus which affect class I binding and cytotoxic T cell recognition.

We have investigated mutation of a dominant cytotoxic T cell (CTL) epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Five NP peptide analogs with single substitutions at the predicted anchor residues (designated by the wild type amino acid, the position number and the new amino acid: P2A, P2R, M9L and M9K) and at a non-anchor position (S5N) were examined for binding to class I, H-2 Ld molecules. Each of the substitutions decreased or abolished the capacity of the NP peptide to increase cell surface Ld expression and to induce Ld stabilization in the cell lysates, indicating that these substitutions significantly affected peptide binding to Ld. We tested the peptide analogs for recognition by bulk primary CTL specific for LCMV, and for their ability to stimulate in vitro the CTL originally induced by wild type LCMV. Except for the M9L change, all mutations reduced CTL recognition by at least 100-fold, and the analogs failed to stimulate the CTL in vitro. The M9L peptide was recognized by the CTL and stimulated CTL in vitro almost as well as wild type; however, this peptide induced Ld stabilization in the cell lysates to a much lesser extent than wild type. Overall, this study demonstrates that mutations in the NP epitope affected peptide binding to the Ld molecule and CTL recognition.

Amino Acid Sequence↗
PubMed1995

Different MHC class I alleles compete for presentation of overlapping viral epitopes.

We previously identified an HLA-B8+ donor, NW, whose lymphoblastoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-B2702-restricted influenza-specific CTL response to an epitope in the nucleoprotein that overlaps the B8-restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the B8-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B2702 epitope. The other is that B8 and B2702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-B8.

Alleles↗
PubMed1995

Early, complete burn wound excision partially restores cytotoxic T lymphocyte function.

BACKGROUND: Cytotoxic lymphocytes (CTLs) are an important component of immune function, involved in antigen recognition and resistance to viral infection. Burn injury suppresses cell-mediated immunity, induces allograft tolerance, and increases the risk of viral infection, but the mechanisms are not well understood. This study analyzes the effect of burn size and burn wound excision on CTL activity. METHODS: Anesthetized CBA mice (n = 12) received a 0%, 20%, or 40% body surface area contact burn. Additional mice (n = 16) received a 40% burn that was totally, partially, or not excised 72 hours after burn. Excised areas were covered with normal, syngeneic skin. Two weeks later harvested splenocytes were cocultured with allogeneic stimulators. CTL activity was determined by a 51Cr release assay, in which CTL effectors were tested on allogeneic, radiolabeled targets. Dilution curves of CTL activity were compared by ANOVA: RESULTS: Both 20% and 40% burns significantly inhibited CTL activity (p < 0.05). Total but not partial excision of a 40% burn restored CTL activity (p < 0.01). Both total and partial wound excision also improved survival (p < 0.05). CONCLUSIONS: Burn injury inhibits CTL activity in a size-dependent manner, and total wound excision significantly improves both CTL function and survival after injury. This study suggests a mechanism for the immunosuppressive effects of burn injury and provides an immunologic rationale for early, complete burn wound excision.

Animals↗
PubMed1995

Natural killer cell activity in lymphocytic choriomeningitis virus-infected beta 2-microglobulin-deficient mice.

We have investigated the induction and role of natural killer (NK) activity in lymphocytic choriomeningitis virus (LCMV)-infected beta 2-microglobulin-deficient (beta 2m-) mice. We demonstrate that LCMV infection is more effective than polyinosinic:polycytidylic acid (poly I:C) at stimulating NK activity in beta 2m- mice. In addition, beta 2m- NK cells respond poorly to in vitro treatment with IL-12. The target specificity of the virally induced NK cells is similar to that previously reported for chemically induced beta 2m- NK cells. In both cases they can lyse YAC-1 tumor cells but are unable to kill beta 2m- or beta 2m+ T cell blasts. We have also found that the time course of induction of NK and cytotoxic T lymphocyte (CTL) activity by LCMV in beta 2m- mice is delayed compared with normal mice. Maximal NK and CTL activity is attained at day 8 and 10 post-infection respectively in beta 2m- mice compared with day 4 and 6-8 in B6 mice. Whereas normal mice die approximately 7 days following intracranial infection with LCMV, the course of disease in beta 2m- mice is protracted and characterized by a marked loss of body weight. We show that although the CD4+ CTL response in these mice is intimately involved in mediating weight loss, the virus-induced NK cells do not appear to play a role in the disease.

Animals↗
PubMed1994

Absence of MHC class II molecules reduces CNS demyelination, microglial/macrophage infiltration, and twitching in murine globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) is a severe genetic demyelinating disorder with an increased number of Ia (immune response antigen) positive brain microglia/macrophages. To assess the role of aberrant Ia expression in the central nervous system (CNS), twitcher mice, which represent the murine model for GLD, were mated with Ia- transgenic mice. Compared with the Ia+ controls, Ia- twitcher mice showed a profound reduction in the severity of demyelinating lesions correlated with significantly fewer microglia/macrophages. Most importantly, Ia- twitcher mice showed significantly reduced twitching compared with ia+ twitcher mice. In contrast with experimental allergic encephalomyelitis (EAE), there was no significant amount of inflammatory T cell infiltrates, implying that T cells may not play a predominant role in this disease. These findings may have broad therapeutic implications for Alzheimer's disease, Parkinson's disease, and Huntington's disease, which display enhanced Ia expression in the CNS without obvious T cell infiltrates.

Animals↗
PubMed1994

Cultured mouse keratinocyte allografts prime for accelerated second set rejection and enhanced cytotoxic lymphocyte response.

It has been reported that cultured keratinocyte (CK) allografts are not rejected in mice, unlike in other species. Several reports have suggested that mouse CK allografts are incapable of stimulating a primary alloresponse, including sensitization of recipients to alloantigens. In this study, we investigated the immunogenicity of mouse CK allografts in vivo by determining whether CK allografts primed for a second set rejection response. First, we grafted mice with either CK allografts, CK autografts, full-thickness (FT) allografts, or no graft at all. We then regrafted mice 4 weeks later with a tail skin allograft. Mice grafted with CK allografts rejected second allografts as rapidly and as vigorously as mice grafted with FT flank allografts. Next, we tested whether CK allograft primed recipients for enhanced CTL responses. We found that mice grafted with CK allografts generated a significantly enhanced CTL alloreactive response after in vitro stimulation. The response was similar to that of mice grafted with FT skin allografts. With evidence that CK allografts primed, we biopsied wounds immediately after CK allografting and, using Western immunoblotting, found that CK allografts had substantial expression of MHC class II antigens in vivo. We conclude from the results of our studies that mouse CK allografts unequivocally prime recipients to alloantigens in vivo and suggest that a possible mechanism for alloantigen priming may be CK allograft expression of MHC class II antigens.

Animals↗
PubMed1994

Analysis of mutant HLA-A2 molecules. Differential effects on peptide binding and CTL recognition.

Previous studies have identified several residues lining the groove of the HLA-A2.1 molecule that are critical for Ag presentation. However, it is not clear whether these residues are critical for binding of the peptide epitope per se or for determining the appropriate conformation of bound peptide. To distinguish between these possibilities, mutations at eight of these residues have been tested for their effects on the ability of the molecule to bind and present two known peptide epitopes--one derived from the influenza A matrix protein, the other from HIV pol. With only one exception, the mutations were found to affect the binding of the two peptides similarly. Most of the mutations resulted in intermediate deleterious effects on binding, with the B pocket mutant F9Y having the most dramatic negative effect on binding for both peptides. Two of the mutations significantly enhanced binding of both peptides and a peptide-specific effect on binding was seen with the substitution, Y99H, which enhanced binding of the matrix peptide yet diminished binding of the pol peptide. In contrast to the effects on binding, the effects of the mutations on presentation differed considerably for the two peptides. The most striking difference was seen with two alpha 2 alpha helix mutants that are fully recognized by pol peptide-specific CTL but not recognized by matrix peptide-specific CTL even though levels of binding were comparably diminished for the two peptides. These results suggest that some interactions, although not critical for binding per se, are critical for functional binding and the importance of these interactions differs among peptide epitopes.

Amino Acid Sequence↗
PubMed1994

Mutations inside but not outside the peptide binding cleft of the H-2 Ld molecule affect CTL recognition and binding of the nucleoprotein peptide from the lymphocytic choriomeningitis virus.

In order to investigate the role of residues inside and outside the peptide binding cleft of the Ld molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the Ld gene. We determined the effects of the mutations in the Ld molecule on the binding and recognition of an Ld-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV). Each of the mutations within the Ld peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface Ld expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the Ld residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding.

Amino Acid Sequence↗
PubMed1994

Significance of the six peptide-binding pockets of HLA-A2.1 in influenza A matrix peptide-specific cytotoxic T-lymphocyte reactivity.

To evaluate the roles of the six peptide-binding pockets of HLA-A2.1 in FMP-specific CTL recognition, we have constructed an extensive library of HMy2.C1R cell lines expressing mutant HLA-A2.1 molecules with different amino acid substitutions in each of the six pockets. These cell lines were tested for their ability to present synthetic FMP 58-66 to FMP-specific, HLA-A2.1-restricted human CTL lines. Six of 12 mutants with amino acid changes in pocket B significantly affect the FMP-specific CTL recognition, suggesting that pocket B plays a critical role in FMP-specific CTL recognition. Surprisingly, mutations in all other pockets, except for pocket F, also have significant effects on the CTL recognition. These results suggest that even the shallow pockets, which are likely to be less critical for peptide binding than the deep pockets, play a crucial role in FMP-specific CTL recognition.

Antigen Presentation↗
PubMed1994

Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*0201. Effect on maturation and peptide presentation.

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.

Amino Acid Sequence↗
PubMed1994

Differences in peptide presentation between B27 subtypes: the importance of the P1 side chain in maintaining high affinity peptide binding to B*2703.

Susceptibility to spondyloarthropathies is strongly associated with the MHC class I molecule HLA-B27, and is hypothesized to result from the presentation of arthritogenic peptides. Subtypes of B27 that differ structurally but are disease-associated ought to be capable of presenting such peptides, while nondisease-associated subtypes would not. We demonstrate that B*2703, the predominant West African B27 subtype that may not predispose to disease, is not recognized by most B*2705-alloreactive CTL, and does not efficiently present a known B*2705-restricted influenza A nucleoprotein (NP) peptide. We show inefficient presentation is due to a reduced binding affinity of B*2703 for the NP peptide. Furthermore, substituting Arg for the naturally occurring Ser at P1 of the NP peptide, restores high affinity binding and efficient presentation by B*2703. Our results suggest that B*2703 will bind and present efficiently only a subset of the peptides that bind to B*2705, in particular those with Arg or Lys at P1. The apparent lack of disease in individuals with B*2703 may be due to an inability to bind and present putative arthritogenic peptides.

Alleles↗
PubMed1993

Allele-specific B pocket transplant in class I major histocompatibility complex protein changes requirement for anchor residue at P2 of peptide.

To investigate the role of an anchoring pocket in allele-specific peptide presentation by a major histocompatibility complex class I molecule, we "transplanted" a B pocket from HLA-A*0201 into HLA-B*2705 by site-directed mutagenesis. The resulting protein, designated B27.A2B, binds a different set of endogenous peptides than B*2705 as evidenced by complete loss of allorecognition as well as restored expression in the antigen processing-defective mutant cell line T2. B27.A2B also fails to present an HLA-B27-restricted influenza virus peptide [nucleoprotein (383-391)] to cytotoxic T lymphocytes (CTLs). However, substitution of leucine, the predominant P2 anchor residue in A*0201-restricted peptides, for arginine, the P2 anchor in nucleoprotein-(383-391) and other B*2705-restricted peptides, restores recognition of B27.A2B by the same B*2705-restricted peptide-specific CTLs. These results demonstrate that a dominant polymorphic pocket in a class I molecule, through interaction with the anchor residue of an antigenic peptide, can distinguish among peptides differing by only a single amino acid and thus determine the allelic specificity of peptide presentation.

Alleles↗
PubMed1993

Roles of the six peptide-binding pockets of the HLA-A2 molecule in allorecognition by human cytotoxic T-cell clones.

To evaluate the contribution of the major histocompatibility complex class I pockets to the binding of self-peptides recognized by alloreactive cytotoxic T-lymphocyte (CTL) clones, we have constructed an extensive library of HLA-A2 mutants with different amino acid substitutions in each of the six pockets. When these mutants were tested in cytotoxicity assays with a panel of HLA-A2-specific alloreactive CTL clones, each CTL clone showed a unique pattern of reactivity, implying the different contributions of each pocket to binding individual peptides. We noted that the majority of the mutants in pocket B significantly affect recognition by the CTL clones. Unexpectedly, the mutations influencing allorecognition are found in all other pockets as well. Overall, this study demonstrates that each of the six peptide-binding pockets plays an important and distinct role in binding of self-peptides required for recognition of the HLA-A2 molecule by alloreactive CTLs.

Cell Death↗
PubMed1993

Transfer of lymphocytic choriomeningitis disease in beta 2-microglobulin-deficient mice by CD4+ T cells.

In this study we have investigated the role of CD4+, MHC class II-restricted cytotoxic T lymphocytes (CTLs) in the disease caused by lymphocytic choriomeningitis virus (LCMV) in beta 2-microglobulin deficient (beta 2m-) mice. Intracranial (i.c.) infection with LCMV resulted in death of six out of 11 beta 2m- mice. Mice that survived showed a marked loss in body weight. Death and loss of body weight could be prevented by immunosuppressing the mice with irradiation or cyclosporine prior to i.c. injection of LCMV. This treatment also prevented induction of virus-specific, MHC class II-restricted CTL following peripheral inoculation with LCMV. In vivo depletion of CD4+ cells with antibody also prevented death following i.c. injection whereas in vivo depletion of CD8+ cells had no effect. Disease could be transferred to recipient beta 2m- mice by adoptive transfer of beta 2m- derived immune spleen cells. Transfer of non-immune spleen cells did not result in illness. In vitro treatment of immune spleen cells with anti-CD4 antibody and complement eliminated class II-restricted CTL activity and also prevented mortality of recipients after adoptive transfer. Treatment with anti-CD8 antibody had no effect. We were unable to transfer LCM disease to beta 2m- recipients by adoptive transfer of immune spleen cells from C57BL/6 mice. These results suggest that, unlike normal mice, the pathology of LCM disease in beta 2m- mice is dependent upon virus-specific, CD4+CD8-, MHC class II-restricted T cells.

Animals↗