Joseph Schwager

A SECOND IMMUNOGLOBULIN LIGHT CHAIN ISOTYPE IN THE RAINBOW TROUT

Abstract A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription – polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Cκ and Cλ isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.

Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus.

Sequences of immunoglobulin (Ig) cDNA clones of Xenopus laevis show that at least three different VH families are expressed in association with different JH elements and different isotypes of Ig constant regions. In genomic Southern blot analysis, the VH probes for each family hybridize to a distinct set of multiple DNA fragments. In contrast, the genomic JH elements and the IgM constant region gene are localized in a single DNA fragment of approximately 15 kb. Genomic VH elements contain regulatory sequences similar to those in VH genes of shark, fish and mammals and have a leader peptide sequence that contains an intron; they encode the VH region until residue 95 and have heptamer–23‐bp–nonamer motifs similar to the rearrangement signal sequences (RSS) in all other vertebrate VH elements. The six genomic JH elements so far sequenced have a nonamer–23‐bp–heptamer motif at their 5′ end. These RSS motifs imply the existence of DH elements. The comparison of cDNA clones that contain similar constant regions but different VH regions or JH elements suggest rearrangement events. This is shown by Southern blot analysis of erythrocyte and B cell DNA with a JH probe. Thus, the overall organization of the Xenopus Ig gene locus is similar to that of mammals but strikingly different from shark.

Modulation of interleukin production by ascorbic acid

We studied the influence of ascorbate (vitamin C) on peripheral blood mononuclear cells (PBMC) of pigs with hereditary deficiency in ascorbate synthesis. Groups of animals were depleted of, or supplemented with dietary ascorbate for up to 5 weeks. B lymphocytes and T lymphocyte subsets differed in the two experimental groups only marginally and transiently as determined by analysis of cell surface markers. The proliferative response of PBMC to B and T lymphocyte mitogens was lower in depleted as compared to supplemented animals. Interleukin (IL)-2 and IL-6 were determined by bioassays and were secreted within few hours after mitogenic activation of PBMC which contained normal physiological concentrations of ascorbate. IL-2 production peaked at about 24 h of in vitro culture after Con A activation, but it lasted for 2-3 days after PWM activation. The production of IL-2 and IL-6 were compared during systemic depletion and supplementation with ascorbate. Depleted PBMC produced IL-2 which accumulated in cultures instead of being rapidly consumed by IL-2 dependent cell growth. This suggests that cellular ascorbate influences the production of IL-2. Secretion of IL-6 by mitogen activated PBMC was also affected by prolonged dietary ascorbate depletion. The results suggest that ascorbate levels exert an early effect on immune homeostasis via reactive oxygen intermediates (ROI)-dependent expression of interleukin genes, since the transcription factor NF-kappa B is sensitive to ROI and regulates the expression of interleukin genes.

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.

Maturation of the mitogen responsiveness, and IL2 and IL6 production by neonatal swine leukocytes.

The maturation of the immune system of neonatal piglets was studied by following changes in the phenotypic composition and function of blood-borne leukocytes. The proportion of mature T and B lymphocytes decreased in the first week of birth and the circulating cells had poorly developed capacities to respond to mitogens and to secrete interleukins. From the end of the first week, however, there was a steady increase in the proportion of mature T cells (CD4+ and CD8+) and B cells in blood until 6-7 weeks after birth, when the study was ended. By 3-4 weeks, the relative proportions of different lymphocyte subsets resembled an adult-type composition. As they increased in prevalence, lymphocytes also developed capacities to proliferate and secrete interleukins. Proliferative responses to T-cell and B-cell mitogens reached adult levels within 2 weeks and 4-5 weeks, respectively. Blood leukocytes produced large quantities of IL6 by 1-2 weeks after birth and IL2 by 2-3 weeks. In contrast to lymphocyte patterns, the myeloid and granulocyte lineages were dominant at birth but then declined steadily. Unlike lymphocytes, the monocytes, macrophages and granulocytes appeared to be fully functional from the time of birth and exhibited a strong oxidative burst after appropriate stimulations. The magnitude of this response remained constant over the first 6-7 weeks. These results indicate that the first 3-4 weeks of post-natal life are a particularly susceptible interval for newborn piglets because constitutive and functional components necessary for specific cellular immune responses remain immature. This deficit may be offset by non-specific cellular mechanisms and maternally derived antibodies.

T cell migration during development: homing is not related to TCR V beta 1 repertoire selection.

T cell precursors enter the chick thymus in three waves during embryonic life. Each wave of thymocyte precursors colonizing the thymus gave rise to a similar TCR V beta repertoire in thymus, spleen and intestine both in terms of V beta 1 and J beta usage as well as in the length of V beta‐D beta‐J beta junctions. Seventeen V beta 1s were utilized, and a new J beta segment was found. In the progeny of the third wave, more nucleotides were deleted at the 5′ end of the J beta segment, but the overall size of the CDR3 was conserved by a concomitant increase of N nucleotide addition at the V beta‐D beta‐J beta junctions during rearrangement. This CDR3 modification was observed in the spleen but not in the intestine, implying that progeny of the third wave migrate preferentially to the spleen, a possibility that was confirmed by adoptive cell transfers into congenic chickens. Very low frequencies of non‐productive rearrangements in the intestine suggested that negative selection may occur in this organ. The present analysis indicates that V beta 1+ T cells in spleen and intestine are primarily of thymic origin, this colonization of both organs occurs in waves and is not characterized by preselection of the TCR V beta 1 repertoire.

Evolutions of the Immune System

Determining the relative importance of the elements of the immune sytem is a major task to which comparative studies may help first in pointing to key elements and second in suggesting their phylogenetic origin. These comparative analyses are complicated by many difficulties due to the following facts. 1) The immune system coevolved with the other physiological systems of the organism and its evolution should not be considered as the evolution of a single independent entity. 2) With respect to the history of the system one has a very incomplete and perhaps misleading view of the phylogeny of the Animal Kingdom. 3) The immune system with what we know of its genetic multiplicity is going to be a complex evolving unit with probably an enormous amount of flexibility enabling some of its elements to evolve in a few generations perhaps as fast as in the history of the species, not to mention its somatic evolution during ontogeny.

Relation Between the Heavy Chain Complementarity Region 3 Characteristics and Rheumatoid Factor Binding Properties

Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecificity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3s (15.3+/-2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3s of smaller length (11.3+/-2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3s and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.

Thymocyte emigration during chicken ontogeny: A key step for the development of the lymphoid system?

T cell precursors enter the chicken thymus in three waves during embryonic life. Adoptive cell transfers showed that peripheral organs such as intestine and soleen are also colonized bv waves of vS and al3 thymocytes. Since embryonic hemopoietic tissues, bone m&row and spleen, as well as TCR negative thymocytes are ineffective sources for intestinal T cells, these latter are primarily thymic migrants during chicken development. Tissue section analysis indicates that ~6 T cells enter the intestinal epithelium at all villus levels and suggests thereafter a comigration of y6 intraepithelial lymphocytes toward the villus tip. Ontogeny of TCR p gene recombination and expression showed that transcription of unrearranged genes precedes the first recombination event which can be either the VD or the DJ step. Each wave of thymocyte precursors gives rise to a similar TCR 9~1 repertoire in thymus, spleen and intestine both in terms of Vpl and Jp usage as well as in the length of Vpl-Dp-J junctions. In the progeny of the third wave, more nucleotides are de eted at the 5’ end of the Jp segment, but P the overall size of the CDR3 is conserved by a concomitant increase of N nucleotide addition at the Vpl-Dp-Jo junctions during rearrangements. This CDR3 modification is observed in the spleen but not in the intestine, implying that progeny of the third wave migrate preferentially to the spleen. Finally, differentiation of each wave of thymocyte precursors is similar, particularly emergence of the

Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals

T cell population occurs 2 to 3 days earlier than Vpl T cell population. Thus, the colonization of the thymus in discrete waves and the difference of kinetics between y6 and cc!3 thymocyte differentiation have for consequence an interspersed emigration of ~6 and o.p T cells, whereas overall thymocyte emigration appears continuous. REGULATION OF TCR-CD3 CELL SURFACE EXPRESSION