Novel Targeting to XCR1+ Dendritic Cells Using Allogeneic T Cells for Polytopical Antibody Responses in the Lymph Nodes

Vaccination strategy that induce efficient antibody responses polytopically in most lymph nodes (LNs) against infections has not been established yet. Because donor-specific blood transfusion induces anti-donor class I MHC antibody production in splenectomized rats, we examined the mechanism and significance of this response. Among the donor blood components, T cells were the most efficient immunogens, inducing recipient T cell and B cell proliferative responses not only in the spleen, but also in the peripheral and gut LNs. Donor T cells soon migrated to the splenic T cell area and the LNs, with a temporary significant increase in recipient NK cells. XCR1+ resident dendritic cells (DCs), but not XCR1− DCs, selectively phagocytosed donor class I MHC+ fragments after 1 day. After 1.5 days, both DC subsets formed clusters with recipient CD4+ T cells, which proliferated within these clusters. Inhibition of donor T cell migration or depletion of NK cells by pretreatment with pertussis toxin or anti-asialoGM1 antibody, respectively, significantly suppressed DC phagocytosis and subsequent immune responses. Three allogeneic strains with different NK activities had the same response but with different intensity. Donor T cell proliferation was not required, indicating that the graft vs. host reaction is dispensable. Intravenous transfer of antigen-labeled and mitotic inhibitor-treated allogeneic, but not syngeneic, T cells induced a polytopical antibody response to labeled antigens in the LNs of splenectomized rats. These results demonstrate a novel mechanism of alloresponses polytopically in the secondary lymphoid organs (SLOs) induced by allogeneic T cells. Donor T cells behave as self-migratory antigen ferries to be delivered to resident XCR1+ DCs with negligible commitment of migratory DCs. Allogeneic T cells may be clinically applicable as vaccine vectors for polytopical prophylactic antibody production even in asplenic or hyposplenic individuals

Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies

<div><p>Aim</p><p>Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells.</p><p>Results</p><p>Rat TECs were subdivided on the basis of phenotype into three subsets; ED18⁺ED19^+/−keratin 5 (K5)⁺K8⁺CD205⁺ class II MHC (MHCII)⁺ cortical TECs (cTECs), ED18⁺ED21⁻K5⁻K8⁺<i>Ulex europaeus</i> lectin 1 (UEA-1)⁺CD205⁻ medullary TECs (mTEC1s), and ED18⁺ED21⁺K5⁺K8^dullUEA-1⁻CD205⁻ medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18⁺ED19^+/−K5⁺K8⁺ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII⁺CD103⁺ but negative for TEC markers, including CD205. Those in the medulla were MHCII⁺CD103⁺ and CD205⁺ cells were found only in the TEC-free area.</p><p>Conclusion</p><p>Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.</p></div

Expression of TEC-related markers in thymi of rats and mice.

<p>Expression of TEC-related markers in thymi of rats and mice.</p

Phenotype of TEC subsets in rats and mice.

#<p>: not tested.</p><p>Phenotype of TEC subsets in rats and mice.</p

Characterization of cortical thymic epithelial cells.

<p>Frozen sections of rat (A-C), and mouse (E) thymus, and a cytosmear from isolated rat thymic nurse cells (D) were subjected to multicolor immunofluorescence staining. The following antibodies were used: Alexa488-conjugated ED18, Alexa488-conjugated ED19, anti-rat CD45 antibody followed by Alexa594-conjugated anti-mouse IgG antibody, anti-K5 antibody followed by AMCA-conjugated anti-rabbit IgG antibody, anti-K8 antibody followed by Alexa-594 conjugated anti-chicken IgY antibody, Alexa647-conjugated anti-rat MHCII antibody and Alexa647-conjugated anti-mouse MHCII antibody.</p

Primary antibodies used in this research.

#<p>: conjugated with either biotin conjugation kit (Dojindo) or Alexa conjugation kit (Invitrogen) in house, ^a produced at VU University Medical Center (Amsterdam, Netherland), ^b Assay Biotechnology (Sunnyvale, CA, USA), ^c Abcam (Cambridge, United Kingdom), ^d BD Pharmingen (Franklin Lakes, NJ, USA), ^e Biolegend (San Diego, CA, USA), ^f donated by Dr. M. Brenan, ^g produced at Rockefeller University (NY, USA), ^h ECACC, ⁱ donated by Dr. G. Kraal, ^j eBioscience (San Diego, CA, USA), ^k Sigma-Aldrich (Saint Louis, MO, USA), ^l Invitrogen (Camarillo, CA, USA), ^m Vector Labs (Burlingame, California, CA, USA), ⁿ Jackson Immunoresearch (West Grove, Pennsylvania, USA)</p><p>Primary antibodies used in this research.</p

Immunohistological analysis of thymic epithelial cell-associated molecules in the thymi of rats and mice.

<p>Double immunoenzyme staining for molecules of interests (blue) and tissue frameworks (type IV collagen, brown) Cortical (subcapsular) and medullary epithelium-free areas unique to rats are indicated by “cEFA” and “mEFA”, respectively. Epithelium-containing areas in the medulla are indicated by “mECA”. The antibodies used were anti-K5 followed by anti-rabbit IgG, anti-K8 followed by anti-chicken IgY, and anti-mouse CD205 followed by anti-rat IgG. Anti-rat MHCII and anti-rat CD205 followed by biotin-conjugated anti-mouse IgG, and biotin-conjugated anti-mouse MHCII, and biotin-conjugated ED18/ED19/ED21 were further reacted with anti-biotin antibody. Secondary antibodies and anti-biotin antibody were all conjugated with alkaline phosphatase and developed with the Vector Blue substrate kit. Tissue frameworks were stained with anti-type IV collagen antibody followed by peroxidase-conjugated anti-rabbit IgG and developed with 3, 3′-diaminobenzidine substrate.</p