PMC

Enhanced alloimmunization to HOD transfusion by PyV.OVA-II requires linkage of (OVA)_323-339 to HEL on the antigen. C57BL/6 mice were infected with PyV.WT or PyV.OVA-II and transfused with mHEL or HOD RBCs (uninfected and/or untransfused control groups were also included). One week after transfusion, sera were analyzed for anti-HEL IgG. Similar results were observed at 14 days (data not shown). A representative experiment with 5 mice/group is shown (mean ± SD depicted, with sera at 1:50 dilution); this experiment has been repeated twice (using C57BL/6 as well as C57BL/6 × B10.BR recipients) with similar results.

Prior infection with PyV.OVA-II significantly enhances alloimmunization to a subsequent HOD RBC transfusion. C57BL/6 mice were infected with wild-type polyoma virus (PyV.WT) or polyoma virus expressing (OVA)_323-339 (PyV.OVA-II). Additional control mice were uninfected. All groups received subsequent transfusion with HOD RBCs. Alloimmunization was assessed 2 weeks later by anti-HEL ELISA. (A) A representative experiment with 5 mice/group is shown, with mean ± SEM shown at sera dilutions of 1:50 to 1:50 000. (B) Enhancement was also evident by flow cytometric cross-matching with FVB or HOD RBC targets. These experiments have been reproduced 3 times (5 mice/group per experiment) with similar results.

Quantitative real-time PCR enumeration of viral genome copy number. DNA was extracted from splenic tissue taken 7 to 10 weeks after infection from recipient mice infected with wild-type polyoma virus (PyV.WT) or polyoma virus expressing (OVA)_323-339 (PyV.OVA-II) and then transfused with HOD RBCs. Quantitative RT-PCR was used to determine the polyoma virus genome copy number. No statistically significant difference in genome copy number between the 2 groups was seen. A compilation of data from 3 individual experiments (3-5 mice/group per experiment) is shown; uninfected mice had an undetectable viral genome copy number (data not shown).

Schematic of proposed enhanced alloimmunization. Response to infection. Peptides containing a polymorphism (designated by A) from a microbe will be processed and presented by host antigen-presenting cells; peptides presented in MHC II will be recognized by CD4⁺ T cells. However, in the absence of a B-cell epitope, the B cells will not be able to receive CD4⁺ T-cell help to generate an antibody response. Response to transfusion. Upon a second antigenic exposure (transfusion) that contains the same polymorphism A, the polymorphism in the blood group constitutes not only a CD4⁺ T-cell epitope but also a de novo B-cell epitope. Through receptor-mediated endocytosis, naive B cells phagocytose the polymorphism-containing blood group molecule. The polymorphism is then presented on the MHC II of B cells to the preformed helper or memory CD4⁺ T cells generated against the microbial infection. The B cells are then stimulated to differentiate into plasma cells that secrete antibodies against the polymorphism.

Enumeration of CD8⁺ T-cell antiviral responses to both PyV.WT and PyV.OVA-II. Intracellular cytokine staining was performed on splenocytes 7 to 10 weeks after infection with either wild-type PyV.WT or PyV.OVA-II; uninfected controls were also analyzed. Splenocytes were restimulated with and without a dominant MHC class I–restricted PyV peptide (LT_359-368) and stained with anti-IFNγ. Representative flow plots are shown (A), and compiled data are presented from a representative experiment (B). This experiment has been repeated 3 times (3-5 mice/group per experiment). In all cases, CD8⁺ T cells from infected mice expressed IFNγ upon peptide stimulation. In 2 of 3 experiments, a greater percentage of IFNγ-producing CD8⁺T cells was seen in PyV.OVA-II–infected mice compared with PyV.WT-infected mice; the data shown are from a representative experiment displaying this difference.

Experimental design and presentation of (OVA)_323-339 peptide after exposure to either PyV.OVA-II or HOD RBCs. Recipient mice were infected with wild-type polyoma virus (PyV.WT) or polyoma virus expressing a known T-cell epitope (ovalbumin (OVA)_323-339), PyV.OVA-II. Two weeks after infection, recipient mice were transfused with RBCs from control FVB donors (expressing no HEL epitope), mHEL donors (expressing HEL on RBCs), or HOD donors (expressing RBC-specific HEL fused to OVA). (A) Diagram of experimental design: Sera were collected prior to transfusion and at 7 and 14 days after transfusion. (B) Diagram of B- and T-cell epitopes expressed in PyV.WT and PyV.OVA-II, as well as in FVB RBCs, mHEL RBCs, and HOD RBCs. Presence of the (OVA)_323-339 epitope is indicated by the green line. (C) OT-II Thy1.1 splenocytes were adoptively transferred into C56BL/6 mice followed by infection with either PyV.WT (C left) or PyV.OVA-II (C right). At 8 days after infection, splenocytes were harvested and stained for CD4 and Thy1.1. (D) OT-II splenocytes were adoptively transferred into C56BL/6.PL-Thy1.1 mice followed 24 hours later by transfusion with either HOD RBCs or FVB RBCs. At 4 days after transfusion, splenocytes were harvested and stained for CD4 and Thy1.2. Groups included mice receiving OT-II cells alone (left), OT-II cells and HOD transfusion (right), or OT-II cells and FVB transfusion (bottom).