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J L Hurwitz

Publications and source records attributed to J L Hurwitz.

At least 73 records · Page 4Linked to original sources

Concomitant T-cell receptor alpha and delta gene rearrangements in individual T-cell precursors.

A debate has recently surfaced concerning the degree of precommitment attained by alpha beta and gamma delta T-cell precursors prior to T-cell receptor (TCR) gene rearrangement. It has been suggested that precursors may be precommitted to rearrange either alpha or delta genes, but not both, thus giving rise to alpha beta- and gamma delta-producing T cells, respectively. Alternatively, the precursors may be flexible with regard to potential TCR gene rearrangements. To address this controversy, the gene rearrangements among a group of T-cell hybridomas from fetal, newborn, and early postnatal mouse thymi were examined. Six probes spanning the delta and alpha loci were used in Southern blot analyses to characterize the rearrangements which occurred on homologous chromosomes in each cell. Although homologous chromosomes often rearranged in synchrony within the alpha locus, a number of hybridomas were found which had retained a delta rearrangement on one chromosome and an alpha rearrangement on the second. Results show that a precommitment by T cells to rearrange delta or alpha genes in a mutually exclusive manner is not an absolute feature of mouse thymocyte development.

Animals↗

Occurrence of atrial fibrillation in patients with paroxysmal supraventricular tachycardia due to atrioventricular nodal reentry.

The occurrence of atrial fibrillation in patients with paroxysmal supraventricular tachycardia (PSVT) has been well documented when PSVT is secondary to atrioventricular reentry, but not when PSVT is secondary to atrioventricular nodal reentry (AVNRT). Seventeen patients with AVNRT were followed using transtelephonic electrocardiogram monitoring to document symptomatic tachycardias. The median length of telephone monitor surveillance was 357 days. Fifteen of 17 patients transmitted electrocardiograms that showed PSVT. Three of 17 patients (18%) transmitted electrocardiograms that showed atrial fibrillation. A transition from PSVT into atrial fibrillation was not recorded, but all three did have PSVT recorded on other days of follow-up. We report the occurrence of atrial fibrillation in patients with AVNRT and that its incidence is higher than expected for the general population.

Atrial Fibrillation↗

Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture.

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.

Aging↗

Progression of rearrangements at T cell receptor beta and gamma gene loci during athymic differentiation of bone marrow cells in vitro.

A previously described in vitro system that supports T cell differentiation from bone marrow (BM) precursors was analyzed for T cell receptor gene rearrangement and expression. Cultured populations from days (d.) 0, 3, 6, and 12-13 were fused with BW5147. Only hybridomas from d.6 and d.12-13 bore BM-derived rearrangements. The rearrangement complexity was higher on d.12 than d.6 in that patterns consistent with D beta 1-J beta 1 and D beta 2-germ-line configurations decreased while V-D-J beta 2 and V gamma 2-J gamma 1 joining became dominant. Northern blots of d.13 BM cultures revealed gamma, alpha, and beta (1.0 and 1.3 kb) transcripts. Parallels between patterns in BM cultures and thymus ontogeny were recognized.

Animals↗

A programmed functional and phenotypic development of bone marrow-derived cytotoxic cell precursors in vitro.

An analysis of function and marker expression during cytotoxic cell differentiation in vitro from T cell-depleted bone marrow precursors is described. Cytotoxic activity is not detectable during the first five days of culture, but rises abruptly soon after. Antibody plus complement depletion studies show that cytotoxic cells derive from Thy-1-negative precursors and undergo a continual increase in Thy-1 and Lyt-2 marker expression as the culture progresses. A reciprocal decrease in asialo-GM1 antigen expression on effector cells is seen. The J11d-negative precursor cells acquire J11d (an antigen known to mark cortical thymocytes) at an intermediate stage of culture, but revert to the J11d-negative phenotype prior to functional acquisition. At least some effectors are T cell receptor positive as illustrated by an anti-T cell receptor antibody-mediated killing assay. These patterns precisely correlate with those detected among developing T cells in vivo. Results may indicate that a programmed course of differentiation inherent to bone marrow cells may be triggered in the absence of a thymic environment.

Animals↗

Anti-Thy-1 plus complement-treated, cultured bone marrow cells resemble fetal thymocytes in killer cell function and marker expression.

Anti-Thy-1.2 plus complement treated bone marrow cells were tested after short-term culture for their ability to lyse allogeneic target cells. Significant lytic activity was generated after 9 days, and required both CAS and splenic or PEC feeders as culture supplements. Allogeneic as well as syngeneic-specific cytotoxic cells were generated polyclonally under such conditions, and could be separated by using limiting dilution protocols. When 65 clones were tested for lytic activity toward three targets bearing H-2k, H-2d, and H-2b haplotypes, respectively, only two clones lysed all three targets; 53 clones showed specificity toward one target only. Targets low in class I H-2 expression were lysed only minimally compared with high H-2 expressors. Allogeneic-kill by C57BL/6 bone marrow cells grown on AKR feeder cells was destroyed by treating effectors with anti-Thy-1.2, but not anti-Thy-1.1, antibody plus complement, suggesting 1) a de novo generation of surface Thy-1 during culture and 2) that effectors were derived from bone marrow, but not feeder, populations. Partial inhibition of kill occurred by treatment of effectors with anti-asialo-GM1 (approximately 80%), anti-Lyt-2 (approximately 60%), or anti-Ly-5.1 (approximately 30%) antibodies plus complement; treatment of effectors with anti-L3T4 or anti-NK-1.1 antibodies plus complement had no effect. When precursor populations were treated with either anti-Thy-1.2 alone or a combination of anti-Thy-1.2 and anti-Lyt-2 antibodies plus complement, killers were easily demonstrated. However, the addition of an anti-asialo-GM1 antibody plus complement treatment before culture abolished function. The characteristics of these effectors showed a resemblance to those described previously for day 14 to 17 fetal thymocytes, designated pCTL.

Animals↗

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus.

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.

Amino Acid Sequence↗

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins.

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.

Animals↗

Influenza-specific suppression: contribution of major viral proteins to the generation and function of T suppressor cells.

Suppressor cells were generated in BALB/c mice by two sequential injections of PR8 influenza virus (A/Puerto Rico/8/34[H1N1]) and were tested for their ability to inhibit proliferative cellular responses towards multiple viral and nonviral antigens. In this way, suppression specific to PR8 as compared with purified protein derivative (PPD) and keyhole limpet hemocyanin (KLH) antigenic responses was illustrated. Experiments involving adoptive transfer of suppression to naive hosts with subfractionated lymphocyte populations demonstrated that the suppressors were Lyt-2+ T cells. Two major questions were addressed with this system. First, a determination was made of which anti-viral protein proliferative responses were affected by the PR8-induced T suppressor (Ts) cells. Ts cells were found to inhibit proliferating cells with specificities for isolated hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix (M) antigens. Second, experiments were conducted to analyze the viral proteins contributing to the induction of PR8-specific Ts cells. Inoculations with either isolated HA or a combination of M + NP proteins induced T suppression specific to proliferative responses towards PR8. These experiments illustrate the contribution of external (HA and NA) as well as internal (M + NP) viral proteins to Ts cell generation and function.

Animals↗

Priming of virus-immune memory T cells in newborn mice.

Neonatal BALB/c mice can be primed at birth by intravenous inoculation of a small dose of A/Puerto Rico/8/34 (H1N1) (PR8) influenza virus. UV-inactivated PR8 virus, or PR8 virus complexed with monoclonal antibody to give a secondary cytotoxic T lymphocyte response when restimulated in vitro as adults. The frequency of responding T cells after secondary stimulation in vitro is approximately 40% of that found for adult mice primed intraperitoneally with a large dose of PR8 virus. The majority of the T cells generated from mice primed at birth or as adults are cross-reactive for H-2-compatible targets infected with the PR8 (H1N1) or A/Hong Kong/X31 (H3N2) viruses. Splenocytes from neonates receiving UV-inactivated vaccinia virus at birth give an augmented secondary cytotoxic T lymphocyte response when restimulated 8 days later in adoptive irradiated adult hosts. We found no indications of specific immunological unresponsiveness in mice exposed to either virus.

Animals↗

Characterization of the murine TH response to influenza virus hemagglutinin: evidence for three major specificities.

Several hemagglutinin (HA)-specific T cell hybridomas were generated from five PR8 influenza virus-primed BALB/c mice by fusion with the thymoma BW5147 and were tested for IL 2 (interleukin 2) production in response to a large panel of serologically distinct influenza viruses and HA-protein fragments. The hybridomas (provisionally categorized in the helper T cell lineage) could be placed into three major specificity groups based on their reactivity patterns toward 43 antibody-selected PR8 virus mutants that had been shown previously to differ from parental PR8 virus by defined (usually single) amino acid substitutions in the HA1 polypeptide. Three hybridomas with specificity 1 failed to respond to the PR8 mutant RV6 (Glu115----Lys), two with specificity 2 failed to respond to PR8 mutant DV4 (Ser136----Pro), and two with specificity 3 responded to all antibody-selected PR8 mutants without exception. The three reactivity patterns could be used along with further biochemical evidence to demonstrate the presence of three distinct T cell determinants on the HA1 polypeptide. Priming of mice with one of these T cell determinants in the form of a synthetic peptide induced a T cell subset able to proliferate in vitro in response to HA and intact PR8 virus. Further analysis of the T cell hybridomas for reaction with 17 natural influenza virus isolates of the H1 subtype revealed additional differences in fine nominal antigen specificity among these T cells. However, two pairs of T cells whose members were isolated from different mice exhibited indistinguishable fine nominal antigen specificities. The latter analysis showed that natural antigenic drift (i.e., accumulation of point mutations) in the HA occurring between the years 1934 and 1957 and again between 1977 and 1980 altered two T cell determinants but left one T cell determinant unchanged. The shifts from H1 to H2 or H3 subtypes resulted in the alteration of all three T cell determinants. The present findings suggest that 1) immunization with influenza virus induces HA-specific T cells that recognize determinants different from those seen by anti-HA antibodies, and 2) the HA-specific T cell repertoire of adult virus-primed BALB/c mice is more restricted than the B cell repertoire.

Animals↗

Photoaffinity reagents for use with pepsin and other carboxyl proteases.

Two compounds have been designed to serve as photoaffinity reagents for use with carboxyl proteases. 1,2-Epoxy-3-(4'-azido-2'-nitrophenoxy)propane has been synthesized and shown to react with porcine pepsin in the same fashion as the traditional inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane, while p-azidophenacyl bromide is similar to other phenacyl bromides in its reaction with pepsin. In combination with p-azido-alpha-diazoacetophenone, previously shown to resemble alpha-diazo carbonyl reagents in its reaction with pepsin, photoaffinity analogs are now available for all three of the widely-used carboxyl protease inhibitors.

Affinity Labels↗

Cross-reactivity patterns of vaccinia-specific cytotoxic T lymphocytes from H-2Kb mutants.

Limit-dilution cultures were used to select vaccinia-immune T-cell populations from bm1 and bm3 mutant mice that were not lytic for virus-infected targets expressing the Kb and Db MHC glycoprotein. Approximately 30% of virus-immune CTL were restricted in each case to Kbm1 and Kbm3, rather than to Db. Evidence of extensive cross-reactivity was found for these virus-immune CTL. Bm3 and bm11 mice sharing one amino acid mutation from wild-type but differing by a second mutation seen only in bm3 are the most cross-reactive pair in their presentation of vaccinia. The bm1 and bm10 pair with dissimilar mutations from wild-type affecting the same CNBr fragment are also largely cross-reactive. However, 30% cross-reactivity is also found for bm1 and bm3, which differ in separate CNBr fragments. That mutants expressing amino acid substitutions in the same region of the peptide tend to show more evidence of cross-reactivity does not necessarily mean the T cells see linear arrays of amino acids on the MHC glycoprotein. For instance, Kbm1 and Kbm10 differ for three amino acids, but bm1 T cells are highly lytic for bm10 virus-infected targets. However, there is no cross-reactivity for Kbm1 and Kb, which differ at only two amino acids. The key to further understanding may rest with defining the nature of the conformational differences among the Kbm1, Kbm10, and Kb glycoproteins.

Animals↗

Specific and nonspecific T-cell recruitment in viral meningitis: possible implications for autoimmunity.

Specific and nonspecific T-cell invasion into cerebrospinal fluid has been investigated in the nonfatal viral meningoencephalitis induced by intracerebral inoculation of mice with vaccinia virus. At the peak of the inflammatory process on Day 7 approximately 5 to 10% of the Lyt 2+ T cells present are apparently specific for vaccinia virus. Concurrently, in mice primed previously with influenza virus, 0.5 to 1.0% of the appropriate T-cell set located in cerebrospinal fluid is reactive to influenza-infected target cells. This vaccinia virus-induced inflammatory exudate may thus contain as many as 500 influenza-immune memory T cells. These findings are discussed from the aspect that such nonspecific T-cell invasion into the central nervous system during aseptic viral meningitis could result in exposure of potentially brain-reactive T cells to central nervous system components.

Animals↗

Cross-reactivity patterns of influenza-specific cytotoxic T lymphocytes from H-2Kb mutant mice.

Limit dilution cultures were used to test for influenza immune T cell populations from bm1 and bm3 mutant mice that were not lytic for virus-infected targets expressing the Kb and Db major histocompatibility complex glycoproteins. Both Kbm3- and Kbm1-restricted cytotoxic T cells were detected. Such effectors showed minimal cross-recognition of influenza on other mutant targets, except for the case of bm1 and bm10 targets. This is dissimilar to previous findings concerning vaccinia presentation in which bm3+bm11, bm1+bm9, and bm3+bm9 pairs each showed high cross-reactivity. These differences illustrate the role of the H-2K glycoprotein in immune responsiveness. Not only are multiple determinants on each H-2K glycoprotein involved in antigen presentation, they appear to play differential roles in the presentation of different viral antigens.

Animals↗

Patterns of isotype expression by B cell clones responding to thymus-dependent and thymus-independent antigens in vitro.

It was found that the Type 2 thymus-independent (TI-2) antigens bacterial levan, trinitrophenyl-Ficoll, and pneumococcal carbohydrate vaccine (PnC) stimulate clonal expansion and antibody secretion in splenic fragments from either hemocyaninprimed or unprimed irradiated recipients bearing B cells from unprimed donors. The in vitro stimulation with TI-2 antigens leads to the expression of isotype switching and provides a more balanced variety of isotypes than is usually observed in vivo. Still, some characteristic patterns of isotypes expressed in vivo to either TI-2 or thymus-dependent (TD) antigens are preserved in vitro. Frequencies of phosphocholine (PC)-reactive B cells responding to either PnC or to PC-hemocyanin (PC-Hy) suggest an appreciable overlap in populations responding to these TI and TD forms of antigen. The existence of a population responsive to either form of PC determinant is supported by the observation that many clones arising in the presence of both forms of antigen express patterns of isotypes that appear as summations of those distinct patterns shown by clones responding to only one form or the other. These data suggest that PC-Hy- and PnC-responding cells may derive from a linear rather than a branched pathway of B cell development and that expression of isotype switching over the lifetime of a developing B cell clone may be regulated in a manner dependent on the form of the stimulating antigen.

Animals↗

Capacity of influenza virus-monoclonal antibody mixtures to stimulate memory and cytotoxic T lymphocyte populations.

The magnitude of the cytotoxic T lymphocyte response in both immunologically naive mice and in mice primed with a serologically non-cross-reactive influenza A virus was greatly diminished if the virus used for challenge was first incubated with excess hemagglutinin-specific monoclonal antibody. However, the level of T cell stimulation was, when compared with virus stocks that are simply diluted, greater than would be expected from the infectivity titer remaining after exposure to the monoclonal antibody. Also, mice given mixtures of virus and monoclonal antibody were primed for a secondary cytotoxic T lymphocyte response, the frequency of memory T cells being approximately fourfold lower than that found for animals injected with virus alone. The possibility that such virus-antibody mixtures might be of value for immunization is worth further investigation.

Animals↗