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Isolation and Characterization of Lymphocyte-Like Cells from a Lamprey

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Isolation and Characterization of Lymphocyte-Like Cells from a Lamprey Isolation and characterization of lymphocyte-like cells from a lamprey Werner E. Mayer*, Tatiana Uinuk-ool*, Herbert Tichy*, Lanier A. Gartland†, Jan Klein*, and Max D. Cooper†‡ *Max-Planck-Institut fu¨r Biologie, Abteilung Immungenetik, Corrensstrasse 42, D-72076 Tu¨bingen, Germany; and †Howard Hughes Medical Institute, University of Alabama, 378 Wallace Tumor Institute, Birmingham, AL 35294 Contributed by Max D. Cooper, August 30, 2002 Lymphocyte-like cells in the intestine of the sea lamprey, Petro- existence of the adaptive immune system in jawless fishes, myzon marinus, were isolated by flow cytometry under light- the identity of these cells has remained in doubt. The aim of the scatter conditions used for the purification of mouse intestinal present study was to exploit contemporary methods of cell lymphocytes. The purified lamprey cells were morphologically separation to isolate the agnathan lymphocyte-like cells to indistinguishable from mammalian lymphocytes. A cDNA library characterize them morphologically and by the genes they was prepared from the lamprey lymphocyte-like cells, and more express. than 8,000 randomly selected clones were sequenced. Homology searches comparing these ESTs with sequences deposited in the Materials and Methods databases led to the identification of numerous genes homologous Source and Preparation of Cell Suspension. Ammocoete larvae to those predominantly or characteristically expressed in mamma- (8–13 cm long) of the sea lamprey, Petromyzon marinus (from lian lymphocytes, which included genes controlling lymphopoiesis, Lake Huron, MI) were dissected along the ventral side to extract intracellular signaling, proliferation, migration, and involvement the intestine and the associated typhlosole (spiral valve). Cells of lymphocytes in innate immune responses. Genes closely related harvested by maceration of these tissues between two glass slides to those that in gnathostomes control antigen processing and were suspended in one part water and two parts PBS. Lamprey transport of antigenic peptides could be ascertained, although no intestinal cells with forward and sideward light-scattering char- sequences with significant similarity to MHC, T cell receptor, or Ig acteristics of a control population of intraepithelial lymphocytes genes were found. The data suggest that the evolution of lym- from the mouse intestine were identified and sorted by using phocytes in the lamprey has reached a stage poised for the the MoFlo cytometer (Cytomation, Fort Collins, CO). Sorted emergence of adaptive immunity. cells were prepared for electron microscopy by fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h before ertebrates with jaws (gnathostomes) possess an adaptive centrifugation and resuspension in the same buffer. For light- V(anticipatory) immune system that is absent in nonverte- microscopic evaluation, sorted cells were coated onto glass slides brates (1). The hallmark of the system is its capacity to anticipate by using a cytocentrifuge, stained with Wright stain, washed, and an encounter with any protein or carbohydrate foreign to the photographed. body and to retain a memory of this encounter. This memory Construction of cDNA Library. Total RNA was extracted from 1 ϫ enables the organism to respond in an accelerated manner after 6 reexposure to the same antigenic stimulus. The adaptive immune 10 sorted cells with the help of the RNeasy mini kit (Qiagen, system is comprised of a complex array of different cell types and Hilden, Germany). To construct a directional cDNA library with high representation of 5Ј ends, the SMART cDNA library- molecules intricately interacting with one another. In this array, ͞ three cell types and three types of molecules are central to the construction kit (BD Biosciences CLONTECH, Heidelberg, immune-system function. These include T and B lymphocytes as Germany) was used according to manufacturer protocol. Briefly, Ϸ200–300 ng of total RNA were reverse-transcribed by the well as the dendritic cells, all derived from a common hemo- ͞ poietic stem cell (2), and the T cell receptor (TCR), B cell PowerScript reverse transcriptase (BD Biosciences CLON- TECH), and the transcript was subjected to cDNA amplification receptor (BCR, Ig), and MHC molecules. Lymphocytes of jawed in 25 cycles of long-distance PCR. The cDNA was size- vertebrates are small cells in which the nucleus, characterized by fractionated by chromatography on CHROMA SPIN-400 col- a densely compacted, coarse chromatin, occupies Ϸ90% of the umns, and fractions containing cDNA larger than 500 bp were cell volume, and the cytoplasm is limited to a thin rim (3). They pooled and directionally inserted into the ␭TriplEx2 vector. possess the ability to respond to stimuli by enlarging their volume Ligated DNA was packaged into phage particles by using the (lymphoblast transformation) and dividing repeatedly. Although Gigapack Gold in vitro packaging kit (Stratagene, Amsterdam) some of the activated lymphocytes terminally differentiate into and transfected into the Escherichia coli strain XL1-Blue (BD effector cells, others return to the state of small, resting cells (1). Biosciences͞CLONTECH), which then were plated. A total of The proliferative phase multiplies those lymphocytes that ex- 2 ϫ 106 independent plaque-forming units was obtained. Not all press TCR or Ig receptors specific for the stimulating antigen and inserts represented full-length cDNA sequences, presumably thus expands them into clones of cells that express the same because of incomplete DNA synthesis and preferential amplifi- receptor. The return to the morphology of small lymphocytes cation of small molecules in the PCR step used to increase the marks the generation of memory cells. The differentiated lym- yield of cDNA. Insert sizes ranged from 500 bp to 4 kb, the phocytes can kill infected target cells (cytotoxic T lymphocytes), majority being between 500 bp and 1.5 kb. assist other cells in their development (helper T lymphocytes), or become Ig (antibody)-secreting plasma cells (B lymphocytes). Analysis of Individual Clones. The unamplified library was plated at TCR, Ig, and MHC receptors have been found thus far only a dilution yielding single, well isolated plaques. A total of 9,312 in jawed vertebrates. Attempts to demonstrate their presence in clones was picked manually and resuspended in 500 ␮l of lambda living jawless vertebrates, represented by lampreys and hagfishes, dilution buffer (100 mM NaCl͞10 mM MgCl ͞35 mM Tris⅐HCl, have failed (4). On the other hand, small cells with darkly 2 staining nuclei and scanty cytoplasm have been observed in histological sections prepared from either lamprey or hagfish Abbreviations: TCR, T cell receptor; BCR, B cell receptor. tissues (5). Because of the failure to obtain clear evidence for the ‡To whom correspondence should be addressed. E-mail: [email protected]. 14350–14355 ͉ PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.212527499 Downloaded by guest on September 28, 2021 Fig. 3. Transmission electron-microscopic views of lamprey lymphocyte-like cells (A) and mouse intestinal epithelial lymphocytes (B). The cells contain large nuclei (N) with heterochromatin forming a peripheral rim adjacent to the nuclear envelope. The nuclei are surrounded by a thin layer of cytoplasm Fig. 1. Fractionation of lamprey intestine cells by light-scattering character- (Cy) containing several small spherical mitochondria (M). istics using flow cytometry: forward (180°) and sideward (90°) light-scatter diagrams of lamprey typhlosole cells (A) and mouse intestinal intraepithelial lymphocytes (IEL, B). The populations containing small lymphocyte-like cells Sequence Analysis. are encircled. Insert sequences were subjected to BLASTX searches (6) against the nonredundant protein database at the National Center for Biotechnology Information (NCBI). Immu- nologically relevant sequence matches to database entries were pH 7.5͞0.01% gelatin). Insert DNA from 1 ␮l of the phage ␮ examined in more detail. By using the NCBI BLASTCLUST suspension was PCR-amplified in a 25- l reaction volume with program, the sequences were grouped into clusters of identical the primer pair TriplEx2LD5 (5Ј-CTCGGGAAGCGCGCCAT- Ј Ј or overlapping sequences. In this step, 1,735 sequences were TGTGTTGGT-3 ) and TriplEx2LD3 (5 -ATACGACTCAC- combined into clusters containing between 2 and 210 sequences. TATAGGGCGAATTGGCC-3Ј) by either HotStar Taq poly- merase (Qiagen) or the Expand long-template PCR system Results and Discussion (Roche Diagnostics) using the manufacturer’s buffer systems. Physical Characteristics of Lamprey Lymphocyte-Like Cells. The Cycling conditions in a PTC-200 Peltier thermal cycler (MJRe- typhlosole, an invaginated spiral valve spanning the length of the search and Biozym, Hessisch Oldendorf, Germany) were: initial lamprey intestine, was the primary tissue source for the cells in denaturation at 94°C for 2 min (Expand long-template PCR this analysis. Cells derived from this tissue were analyzed in system) or 15 min (HotStar Taq polymerase), followed by 10 parallel with mouse intestinal epithelial cells by automated flow cycles of denaturation at 94°C for 10 sec, primer annealing at cytometry. The lamprey typhlosole contains a discrete subpopu- 65°C for 30 sec, and elongation at 68°C (Expand long-template lation of cells with light-scatter characteristics that closely re- PCR system) or 72°C (HotStar Taq polymerase) for 6 min. semble those of the lymphocytes in the mouse intestinal
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