THREE DIFFERENT IgD CELL POPULATIONS IN CHANNEL CATFISH, ICTALURUS PUNCTATUS Eva-Stina Edholm*, James L. Stafford, Manoranjan Sahoo, Eva Bengtén, Norman W. Miller and   Melanie Wilson.

Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS, USA 39216-4505

IgM and IgD are currently the only immunoglobulin (Ig) molecules identified in catfish. While catfish IgM resembles mammalian IgM, catfish IgD is quite different. It is a chimeric molecule consisting of a rearranged VDJ region spliced to the Cµ1 domain, followed by seven Cδ domains and either a transmembrane or secreted tail. To study catfish IgD function, monoclonal anti-IgD antibodies were generated. Flow cytometry and cell separation techniques demonstrate the presence of three IgD+ populations in peripheral blood leukocytes (PBL). Two are B lymphocyte-like populations with one expressing messages for the membrane forms of IgM and IgD (IgM+/IgD), and the other only expressing the membrane form of IgD (IgM-/IgD+). Moreover, PCR protocols amplify appropriate rearranged Ig messages from both populations indicating that they contain functional B cells. In contrast to the situation in mammals where IgM-/IgD+ B cells are rare, IgM-/IgD+ cells can represent as much as 60% of the total PBL in some catfish. The third population consists of IgM-/IgD+

granular-like cells of unknown origin that do not express Igd message, but appear to be armed with exogenous IgD via an IgD-binding receptor. At the present, the function of the granulocytes is unknown, however they degranulate in response to cross-linking of the surface bound IgD. Supported by NIH grant R01 AI19530 United States Department of Agriculture (2006-35204-16880) "US Veterinary Immune Reagent Network".

Four representative staining profiles were observed in PBL from different catfish. Panels show scatter profiles for each type, followed by histograms and scatter plots of designated regions after anti-IgM or anti-IgD staining. The FACScan was calibrated between each run using chicken red blood cells to eliminate instrumental variation.Type 1 consists almost exclusively of small, non-complex cells, with some cells staining at a high intensity for IgM and some cells staining at a lower intensity for IgD.Type 2 contain cells similar to those observed in Type I, plus a population of slightly larger and more internally complex cells (R2) that stain at a medium intensity for IgD.Type 3, while similar to Type II, contains an additional population of cells, highly granular in appearance that stain with a high intensity for IgD.Type 4 resembles Type 2 as far as scatter profiles and anti-IgM staining. However no IgD staining was observed.

  

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Fig 1A. Schematic representation of IgD Catfish IgD is a chimeric molecule consisting of a rearranged VH region spliced to a Cµ1 domain exon, seven Cδ domains and a TM.

1B. IgD in different speciesRed blocks indicate a Cµ1 or Cµ1-like domain

IgD+ cell populations Different IgD+ cell populations were detected using anti-IgM and anti-IgD mAbs in magnetically activated cell sorting (MACS). Cells from a Type 2 fish were (A) first sorted using positive selection with anti-IgM and (B) the IgM- fraction was then positivelyselected with anti-IgD; Cells from a Type 3 fish (C) were negatively sorted with anti-IgM. Fractions from each selected population were stained with Giemsa/Dip stain and analyzed by FACS and RT-PCR.IgM+/IgD+ cells express message for secreted (sec) and membrane (mem) IgM and IgD. Some TCR message is also detected and may be due to T cells that express IgM via an FcR.IgM-/IgD+ lymphocyte-like cells express message for both IgDsec and IgDmem. Type 3 negatively selected IgM-/IgD+ did not express message for IgD, although they are >80% surface positive for IgD (data not shown). Phenotypically they have a more granulocyte appearance.

Fish type 2

Anti-IgM

Ant

i-IgD

Representative Type 2 PBL double stained with anti-IgM and anti-IgD show the different IgD expressing populations IgM-/IgD+ (75%), IgM+/IgD+ (13%), IgM+/IgD- (1%), and IgM-/IgD- (11%).

Anti-IgM and Anti-IgD double staining

Isolation and characterizations of IgD+ cells

Is IgD expressed with IgL chains?

IgD may be expressed on the surface without associated IgL chains:Fish Type 2 PBL and MAC-Sorted IgM-/IgD+ Type 2 cells were analyzed by FACS for IgL staining (IgL-G and IgL-F). Total PBL contained both IgM, IgD and IgL chain positive cells; no staining above background was observed for anti-IgM, anti-IgL-F and anti-IgL-G in IgM-/IgD+ cells. In addition, IgL staining was not observed on negatively selected IgM-/IgD+ granulocytes from fish Type 3 (data not shown).

Induction of IgD staining in Type 4 PBL

IgD staining is inducible PBL isolated from Type 4 fish were stimulated with AL-5 media alone (control) or supplemented with Con A (25µg/ml) or LPS (100 µg/ml). Cells were examined on day 2 for surface IgD staining, and scatter profiles and histograms are shown. No induction was observed in control or LPS stimulated cultures, while the ConA stimulated cultures were positive for IgD staining.

Induction of IgD staining in response to culture supernatantsBy day 3, IgD- PBL from fish Type 4 cultured in AL-5 media supplemented with 10% (v/v) supernatants collected from 3B11 B-cells and 42TA macrophages exhibit anti-IgD staining. In contrast, supernatants from 28S.3 T cells or Mixed leukocyte cultures (MLC) did not induce surface IgD staining.

Possible function of IgD on granulocytes

A. Cross-linking of surface bound IgD on the negatively sorted IgM-/IgD+ granulocyte population from fish Type 3 resulted in cell degranulation. PBL were negatively selected with anti-IgM using MACS. Fractions were Giemsa stained before and after treatment with anti-IgD and goat-anti-mouse Ig. B. Proposed mechanism for IgD expression on catfish granylocytes. It is hypothesized that IgD protein is acquired from serum and expressed on the surface of these granular cells via a FcR/IgD-R type of receptor. Upon cross-linking these cells release granules. It may be that these granulocytes are functionally analogous to mammalian mast cells or basophils.

• Three populations of IgD+ cells have been identified, two lymphocyte populations that differentially express IgM (IgD+/IgM+) and (IgD+/IgM-), and a granular cell population.

• IgD may be expressed on the cell surface without IgL chains.

• IGHD1 encodes for the IgDmem form and IGHD3 encodes for IgDsec

• IgD on the B cell surface is associated with CD79 molecules.

• No IgD message is found in the granular cell population.

• Cross-linking surface bound IgD results in granulocyte degranulation.

• Anti-IgD staining on granulocytes is inducable.

• Whether IgD on granulocytes is the IgDsec encoded by IGHD3 needs to be verified, it may be that a shorter form of IgD lacking the TM and CYT is produced from IGHD1.

• Based upon these unique and novel findings our hypothesis is that mIgD, like mIgM, functions as a B cell antigen receptor and that sIgD pre-arms granular effector cells via an FcδR and serves as a functional bridge between adaptive and innate immunity.

Conclusions

Since there are no existing anti-IgL-σ or IgL-λ mAbs, cDNA from Type 2 PBL and MACSorted IgM+/IgD+ and IgM-/IgD+ cells were analyzed in RT-PCR using primers specific to V and C domains of IgL-λ and IgL-σ. No IgL chain message was detected in the IgM-/IgD+ cell population. Whether other Vλ or Vσ familes exist in catfish is currently unknown.

IgD is associated with CD79 molecules on the B cell surfaceA. Clonal catfish 3B11 B cells were nucleofected with combinations of epitope tagged constructs; IgD (Flag) and CD79a and CD79b (HA). 36 hours post nucleofection, cells were analyzed by FACS for surface expression of tagged protein(s). Cells nucleofected with surface IgD alone did not express the construct on the surface while triple transfected cells successfully expressed surface IgD. B. IgD and CD79 association was demonstrated by co-immunoprecipitations. Cells were lysed in mild detergent and selected using anti-HA tag or anti-FLAG tag mAbs. Resultant proteins were visualized using anti-FLAG-HRP in Western blot analysis. Anti-trout Igµ (1.14) is isotype matched control.

IgD association with CD79 on B cells

5’-end analysis of IgD Potential antigen-binding variability of IgD expressed in isolated IgM-/IgD+ B cells was investigated using 5’-RACE. All IgDmem (IGHD1, see below) transcripts contained the Cµ1 domain and a functionally rearranged VDJ region. No preferential V, D or J segment usage was observed. IgDsec (IGHD3, see below) transcripts were found expressed either spliced to ϕCµ2/ϕCµ3 or with a leader (L) immediately upstream of the Cδ1.

IgD 5’- end variability

Development of anti-IgD mAb (7D11)• Anti-Delta mAb was made against recombinant (r) IgD domain 2 generated

in either procaryotic (E.coli) or eukaryotic (Sf9; insect) systems

• Hybridomas were screened by Flow cytometry (FACS) using freshly isolated PBL

• rIgD protein was purified on Magne-His nickel columns and refolded by stepwise dialysis in the presence of a redox pair

Four IgL isotypes exist in catfish; IgL-F, IgL-G, IgL-σ, IgL-λ.They are 21-32% identical to each other at the amino acid level.

Program # 863Abstract # D382 863.4

The IGH locus contains several IGHD genes. Three IgD genes have been identified (IGHD1, IGHD2 and IGHD3). IGHD1 is located at the 3’ end of the locus immediately downstream of IGHM1 and encodes for the membrane form of IgD (δTM). IGHD3 and IGHD2are located downstream of IgM pseudogenes; IGHD3 encodes for a functional secreted form of IgD (δs) found in catfish sera. To date it is unclear if IGHD2 is functionally expressed. The leader (L) exon used by IGHD3 is shown in green.

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Four types of IgD expression profiles