Immune response to MMTV infection

Hans Acha-Orbea¹ Alexander N. Shakhov¹ and Daniela Finke²

1Department of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland, ² Division of Developmental Immunology, Center for Biomedicine, Department of Clinical and Biological Sciences (DKBW), University of Basel, CH-4058 Basel, Switzerland

FIGURES

Figure 1. Comparison of endogenous mtv-loci from BALB/c, DBA/1 and outbred Swiss mice. This Swiss mouse colony was used to derive SPF mouse colonies by embryo transfer and forster nursing. It introduced MMTV(SW) into the SPF-derived BALB/c mice. A Southern blot after PvuII digestion of genomic DNA is shown. It cuts once inside the mtv-loci and generates 2 bands depending on the integration site per mtv-locus. The new mtv-53 locus is indicated to the left and the known mtv-loci to the right and the perfect correlation with deletion of T cells expressing TCR Vbeta 6, which is also deleted by the infectious MMTV(SW). Sequences of mtv-53 and MMTV(SW) were identical indicating a high likelihood that this new endogenous mtv locus can form sporadically infectious particles. .

Figure 2 A) Predicted superantigen structure. The transmembrane region is indicated in light blue. The nucleotide sequence shows 5 conserved initiation codons allowing the formation of superantigen proteins with shorter NH2 protein sequences (arrows left with amino acid position (aa)). Five potential N-linked glycosylation sites are indicated with black stars. The three furin cleavage sites (RARR, RGKR) are marked with arrows from the right with the indicated amino acid positions. At the COOH-terminal end the highly polymorphic is indicated (yellow). B) The MMTVs and mtv's, TCR Vbeta specificities and amino acid sequences of the polymorphic COOH terminal sequences of representative members of the 7 described families are indicated. Dashes represent sequences identical to the MMTV(SHN) sequence, dots stand for single amino acids lacking in comparison to this sequence introduced to achieve better alignment. Always the sequence of the upper member is indicated when two members are listed. The second member differs from the first in 6-9 amino acids in this COOH-terminal sequence whereas the rest of the molecule shows high sequence homology. Sequences were from (24-26, 32, 63, 64, 108, 109).

Figure 3. Stages of a normal T-cell-dependent B cell response. A) Antigen-presenting dendritic cells (DC) (yellow) enter the paracortex (P) of the draining lymph node from the periphery via lymphatic vessels and can transfer their antigen also to local DC. If they are issued from an infection they show an activated phenotype allowing priming of an effector T cell response. T and B cells are activated independently (1). T cells in the paracortex, B cells in the B cell follicles in the cortex (C). T cells make prolonged interaction with the dendritic cells to become effectors. This interaction has been termed synapse 1. B cells migrate to the cortical-paracortical junction after activation. B) Around day 3 after encountering DC, primed T cells migrate to the cortical-paracortical boundary to interact with B cells presenting the same peptide as the originally priming DC. This interaction is called synapse 2 and leads to two waves of B cell differentiation. They can differentiate directly into isotype switched plasmablasts migrating to the medulla (M) secreting IgM or switched isotypes of low affinity and being exported into the periphery or dying locally (2). They have a short lifespan. Alternatively, 1-3 B cells can initiate the formation of germinal centers (GC) in B cell follicles (3). There, they rapidly proliferate as centroblasts in the dark zone of the GC to reach 10.000 cells, switch their isotype and mutating their variable Ig regions. To survive they have to stop proliferating, take up their antigen from local stromal cells (follicular dendritic cells) in the light zone of the GC and receive a second time cognate help from T cells to rescue them from cell death and differentiate into long-lived memory or plasma cells. This interaction is called synapse 3. Long-lived plasma cells home to spleen or bone marrow (4) where they can survive life-long in niches. C) Germinal center formation. Lymph node sections were labeled with a follicular-dendritic cell-specific marker M2.

Figure 4. A) Natural transmission of MMTV from mother to babies. Infected mothers transmit MMTV to their babies during the first two weeks after birth. During lactation large amounts of viruses are produced through the action of steroid hormones. The mouse shown has a large mammary tumor but transmission occurs long before tumors appear. B) Histological sections of small intestine showing a Peyer's patches, the entry site for naturally transmitted MMTV. The follicle-associated epithelium (FAE) is indicated by arrows C) Immunohistological staining of dendritic cells in Peyer's patch and FAE (CD11c-positive dendritic cells are red). The dotted line indicates the FAE.

Figure 5. Activation and infection of dendritic cells and B cells. MMTV binds TLR 4 amongst other receptors. This interaction leads to activation of B cells and dendritic cells. A contribution of Ig crosslinking by MMTV has been observed (not shown). These interactions most likely allow entry into cell cycle and infection of the host cells.

Figure 6. Accelerated maturation of Peyer's patches after MMTV infection. Peyer's patches of BALB/c mice fed on wild type or MMTV(SW) infected mothers from birth were stained with IgD. Germinal center B cells in the germinal centers (GC) are IgD-, B cells outside GC IgD+. Note the better developed B cell follicles and early formation of GC in infected mice. In the second part of the Figure the superantigen-reactive Vbeta6+ T cells are labeled in blue. They accumulate after MMTV infection. In the lowest part the faster appearance of ICAM-1, a marker for mature B cell follicles, shows accelerated maturation of B cell follicles in infected mice.

Figure 7. Distribution of mtv-loci in commonly used mouse strains. In yellow the mtv-loci that express superantigens that can be presented by I-E and to a lesser extent by I-A, in light blue the strictly MHC class II I-E-dependent mtv-loci are shown. Black indicates mouse strains lacking MHC class II I-E. Expression of the indicated loci is indicated in dark blue with a + sign. Adapted from (9)

Figure 8. Classical summary of mls interaction (60). The four classical Mls-phenotypes mls a,b,c,d are shown. Arrows indicate in which direction strong mixed lymphocyte reactions are induced. The tip of the arrow points to the strain that vigorously responds to antigen presenting cells from the other strain.

Figure 9. T cell priming by MMTV superantigens. A) After infection of DC, superantigens and co-stimulation molecules are expressed by DC (CD80, CD86, CD40 as well as others). The superantigen associates with MHC class II and interacts with constitutively CD28 expressing CD4+ T cells expressing the corresponding TCR Vbeta. This interaction corresponds to synapse 1 in Figure 2A. (B). Activated T cells induce the expression of other co-stimulation molecules such as CD40L, which interact with CD40 expressed by DC.

Figure 10. Around day 3 T cells form the synapse 2 with activated B cells in the paracortical-cortical region of the lymph node. This again is induced by superantigen interactions. T cells secrete cytokines such as interferon g to induce isotype switching in B cells to IgG2a. Co-stimulation via CD40-CD40L is crucial at this step. As in classical response, direct differentiation into IgG2a secreting plasmablasts around day 5-7 after virus encounter is induced as well as formation of germinal centers. B) B cells from germinal centers as described in Figure 2 but again superantigen not antigen is driving rescue of plasma cells or memory cells independently on Ig specificity of the B cells. Germinal centers appear only a week after termination of the extrafollicular response. a NP-specific immune response. CD3+ T cells in blue, proliferating centroblasts in light red. IgD in brown) b) PNA staining of MMTV-induced germinal centers 22 days after virus injection. IgD (brown), proliferation (red), germinal center (PNA, blue) c) same as b) but blue indicates staining for CD3+ T cells. d) same as b) but blue indicates staining for superantigen-reactive TCR Vbeta 6+ T cells. Note that most T follicular T cells express the superantigen-reactive TCR. Reproduced with permission from (79))