Maternal anti-HLA class I antibodies are associated with reduced birth weight in thrombocytopenic neonates.

In this comparative cross-sectional study, possible associations between maternal anti-HLA class I antibodies and birth weight in neonatal thrombocytopenia are explored. Although commonly detected in pregnancies and generally regarded as harmless, it has been suggested that such antibodies might be associated with fetal and neonatal alloimmune thrombocytopenia (FNAIT). As a link between FNAIT due to human platelet antigen 1a-specific antibodies and reduced birth weight in boys has previously been demonstrated, we wanted to explore whether maternal anti-HLA class I antibodies might also affect birth weight. To examine this, suspected cases of FNAIT referred to the Norwegian National Unit for Platelet Immunology during the period 1998-2009 were identified. Pregnancies where the only finding was maternal anti-HLA class I antibodies were included. An unselected group of pregnant women participating in a prospective study investigating maternal-fetal hemodynamics at the University Hospital North Norway during the years 2006-2010 served as controls. Twenty-nine percent of controls had anti-HLA class I antibodies. The thrombocytopenic neonates had a significantly lower adjusted birth weight (linear regression, P=0.036) and significantly higher odds of being small for gestational age (OR=6.72, P<0.001) compared with controls. Increasing anti-HLA class I antibody levels in the mother were significantly associated with lower birth weight and placental weight among thrombocytopenic neonates, but not among controls. These results indicate that maternal anti-HLA class I antibodies in thrombocytopenic neonates are associated with reduced fetal growth. Further studies are needed to test if placental function is affected.

Extraordinary variation in a diversified family of immune-type receptor genes.

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.

Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection.

Dendritic cells (DCs) elicit potent anti-tumoral T-cell responses in vitro and in vivo. However, different types of DC have yet to be compared for their capacity to induce anti-tumor responses in vivo at different developmental stages. Herein, we correlated the efficiencies of different types of monocyte-derived DC as vaccines on the resulting anti-tumor immune responses in vivo. Immature and mature DCs were separately pulsed with a peptide derived from tyrosinase, MelanA/MART-1 or MAGE-1 and a recall antigen. Both DC populations were injected every 2 weeks in different lymph nodes of the same patient. Immune responses were monitored before, during and after vaccination. Mature DCs induced increased recall antigen-specific CD4(+) T-cell responses in 7/8 patients, while immature DCs did so in only 3/8. Expansion of peptide-specific IFN-gamma-producing CD8(+) T cells was observed in 5/7 patients vaccinated with mature DCs but in only 1/7 using immature DCs. However, these functional data did not correlate with the tetramer staining. Herein, immature DCs also showed expansion of peptide-specific T cells. In 2/4 patients vaccinated with mature DCs, we observed induction of peptide-specific cytotoxic T cells, as monitored by chromium-release assays, whereas immature DCs failed to induce peptide-specific cytotoxic T cells in the same patients. Instead, FCS-cultured immature DCs induced FCS-specific IgE responses in 1 patient. Our data demonstrate that this novel vaccination protocol is an efficient approach to compare different immunization strategies within the same patient. Thus, our data define FCS-free cultured mature DCs as superior inducers of T-cell responses in melanoma patients.

Thioredoxin acts as a B cell growth factor in channel catfish.

To identify differentially expressed genes from channel catfish macrophages, a cDNA library from LPS-stimulated catfish macrophages was screened by subtractive hybridization. This screening yielded a 552-bp cDNA coding for catfish thioredoxin (CF-TRX). The deduced amino acid sequence revealed that CF-TRX contains 107 amino acids and is 59% homologous to human adult T cell leukemia-derived factor/TRX, originally described as an IL-2R alpha-inducing factor. Northern blot analyses showed that CF-TRX is expressed in catfish T and macrophage cell lines, but weakly in B cell lines. Similar results were also observed in Western blot analyses using a mAb specific for recombinant CF-TRX (rTRX). The use of rTRX in functional studies demonstrated that rTRX induces in vitro proliferative responses of catfish PBL that were synergistically enhanced by the addition of culture supernatants from catfish T cell lines. In addition, cell separation studies and flow cytometric analyses revealed that the cells proliferating in rTRX-stimulated cultures were mostly B cells. These results suggest that CF-TRX may have an important role in the activation and proliferation of channel catfish B cells.

Telomerase expression and telomere length in immortal leukocyte lines from channel catfish.

Normal channel catfish leukocytes readily undergo spontaneous in vitro immortalization yielding functionally active diploid cell lines. Since telomerase activation appears to be a critical step in the establishment of immortal mammalian cells, studies were undertaken to determine if and when telomerase expression occurs during the in vitro immortalization process of channel catfish leukocytes. To this end, freshly isolated peripheral blood leukocytes (PBL) from normal fish were shown to exhibit low to undetectable levels of telomerase activity and within four days after culture initiation showed dramatic increases in telomerase activity which typically remained high for at least four weeks. This activity then declined, concomitant with decreases in cellular proliferation and increases in cell death. Cells which escaped this culture "crisis" re-expressed high levels of telomerase activity indefinitely. Although telomerase activity was expressed early in the immortalization process, clonal cell lines derived from these cultures had relatively short telomeres. These results suggest that telomerase expression in catfish leukocytes is activation-induced, and its expression does not necessarily stabilize telomere length until a critically, albeit ill-defined, short length is reached.

Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish.

To determine the phenotypes of cytotoxic cells in channel catfish, clonal alloantigen-dependent leukocyte lines were established from mixed leukocyte cultures. Each clone was analyzed for expression of TCR alpha and beta genes by RT-PCR and for target cell specificity by 51Cr-release assay. Based on the above criteria, the following five different cell types were identified among the 19 clones analyzed: 1) TCR alphabeta+ allospecific cytotoxic cells, 2) TCR alphabeta+ nonspecific cytotoxic cells, 3) allospecific TCR alphabeta+ noncytotoxic cells, 4) TCR alphabeta- nonspecific cytotoxic cells, and 5) TCR alphabeta- allospecific cytotoxic cells. The demonstration of cloned, TCR alphabeta+, allospecific cytotoxic effectors provides the strongest evidence to date for the existence of cytotoxic T cells in fish.

T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes.

Herein are reported full length cDNA sequences for TCR alpha- and beta-chains of the channel catfish. Included are sequences belonging to four Valpha and six Vbeta families which share hallmarks in common with the Valpha and Vbeta genes of other species. Similar to the situation in other vertebrates, the catfish Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Calpha and Cbeta regions are shorter than those of mammals and the catfish Cbeta chain lacks a cysteine in its connecting peptide region. Two different catfish Cbeta cDNA sequences were identified, suggesting the existence of either two Cbeta loci or allotypes. Based on Southern blot analyses, each of the catfish TCR gene loci appear to be arranged in a translocon (as opposed to multicluster) organization with multiple V elements and a single or few copies of C region DNA. At the deduced amino acid level, the catfish Cbeta sequence exhibits 42% identity with the Cbeta of Atlantic salmon, 41% identity with the Cbeta of rainbow trout and 26% identity with Cbeta of the horned shark. The catfish Calpha amino acid sequence exhibits 44 and 29% identity with Calpha of the rainbow trout and southern pufferfish, respectively. TCRalpha and beta messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR alpha/beta expressing clonal lymphocyte line, designated 28S.1, has T-cell like function in that it constitutively produces a supernatant factor(s) with growth promoting activity. These findings should facilitate functional studies of fish TCRs and T cells in ways not previously possible with other 'lower' vertebrate models.

Functional and molecular characterization of teleost leukocytes.

The coupling of immunologically relevant in vitro assay systems, cell separation techniques, and the development of distinct clonal leukocyte lines has established the existence of T, B, natural killer, and accessory cell equivalents in teleosts. B cells are directly defined by monoclonal antibodies to teleost immunoglobulin (Ig) and identification of Ig H and L chain genes. As in mammals, fish B cells show Ig H-chain gene rearrangements, allelic exclusion, produce both membrane-bound and secreted forms of Ig, and transduce intracellular proliferative signals upon anti-Ig cross-linking. It has also been found that some fish B cells express a unique chimeric Ig chain with sequence homology to mammalian Ig delta. Teleost T cells are still indirectly defined as sIg- lymphocytes due to a lack of definitive surface markers. These mIg- lymphocytes are the responding cells in mixed leukocyte cultures, proliferate specifically to autologously processed and presented antigen, provide helper function for in vitro antibody responses, and produce interleukin-like factors upon activation. Recent identification of teleost T-cell receptor alpha and beta genes has now permitted the unequivocal genetic demonstration that some of these mIg- cells are bona fide T cells. It is anticipated that such long-term clonal cell lines will be indispensable tools for dissecting the physiology, biochemistry and molecular biology of teleost immune responses.

Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures.

In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.

Fish immunology: the utility of immortalized lymphoid cells--a mini review.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.

Anti-viral cytotoxic cells in the channel catfish (Ictalurus punctatus).

Cytotoxic cells isolated from the head kidney and peripheral blood of the channel catfish appear to represent distinct subpopulations of effector cells. Previous studies showed that the former lyse xenogeneic natural killer (NK) cell targets, whereas the latter preferentially lyse allogeneic cells. Here we extend these studies and present data suggesting a third class of cytotoxic effectors responsible for killing virus-infected allogeneic and autologous cells. Peripheral blood leukocytes (PBLs) freshly isolated from unimmunized catfish lyse uninfected allogeneic target cells as well as virus-infected allogeneic and autologous cells. Cell depletion and unlabeled ("cold") target inhibition studies discriminated between putative effector classes and supported the view that at least two populations of cytotoxic cells are present within peripheral blood leukocytes. One population lyses allogeneic targets, whereas a second population kills channel catfish virus (CCV)-infected cells. In addition, inhibitor studies demonstrated that early virus gene products are sufficient to render infected cells susceptible to lysis. These results suggest that channel catfish possess distinct populations of NK-like, PBL-derived cytotoxic cells capable of lysing allogeneic and virus-infected target cells.

Phylogeny of lymphocyte heterogeneity: cytotoxic activity of channel catfish peripheral blood leukocytes directed against allogeneic targets.

Channel catfish peripheral blood contains leukocytes that function as cytotoxic effectors directed against a variety of long-term cultured allogeneic, but not xenogeneic, targets. These effector cells are probably distinct from macrophages, B cells, and nonspecific cytotoxic cells. The cytotoxic activity of these effector cells was inhibited with monoclonal antibody 1H5. Although this reagent appears to react with a catfish cell surface molecule akin to the integrin LFA-1 present on the surface of nearly all leukocytes, it does not clarify the question as to whether or not these effectors are related to T cells.