We used six different experimental set-ups? ? 1) BIODEFENSE AND NEONATAL IMMUNITY Although there is a concerted national effort to find vaccines for potential bio-terrorism agents, little is being done to protect the nations infants. Most adult vaccines do not work for infants less than 6mo old, and many do not work for babies < 1yr. Because we cannot leave our children behind, we have been analyzing the immune responses of newborns.? A) The inhibitory effect of maternal antibodies: In 1996 we showed that newborn mice were immunologically competent (contrary to common opinion), as long as they were properly immunized (Science 271:1723-1726). We have now been looking at a second myth, namely that the presence of maternal antibody inhibits newborn responses. Last year we found that neonatal mice respond perfectly well to the model antigen Ovalbumin (OVA), even if they have received passive anti-OVA antibody from their mothers. The immunity in babies from immunized mothers lasts for at least 1yr, and does not fade more rapidly than that of babies from control mothers. Thus, in mice, maternal antibodies are not inhibitory. This year we have titered the OVA vaccine, used congenic strains to accurately follow the responses of the neonates, and shown that the antibody titers remain similar to those from unvaccinated mothers for more than 18 months..? B) anthrax: To see if our results with the model antigen, OVA, hold for real infections, we are studying the response to Anthrax. In preliminary studies to find the right vaccine dose, we found (astonishingly!) that the current test for adult immunity (neutralizing antibody) is misleading. Mice immunized with Anthrax protective antigen (PA) in adjuvant make high titers of antibody (as seen on ELISA tests) but no .neutralizing. antibody. Yet they are completely protected from challenge with live Anthrax. This result may radically change how to vaccinate individuals and will also change how we analyze the responses of newborn mice.? Further, we found that neonates immunized with Anthrax PA in adjuvant are protected from challenge, though some of them make no detectable antibody at all, suggesting that non-antibody protective mechanisms exist.? C) Neonatal responses to Measles: having found that neonatal mice respond perfectly well to both OVA and Anthrax, we turned to the measles vaccine to ask why neonatal humans do not respond well. We have been studying the vaccine variant of the Edmonston strain of Measles to see what properties make it a poor vaccine for children. ? ? 2) TRANSPLANTATION TOLERANCE When neonatal hearts are grafted into immunodeficient (RAG KO) mice and allowed to heal for 9mo, the recipients become populated with small numbers of T cells from the grafted hearts and become resistant to rejection. We are now analyzing the microchimeric T cell populations to see if they have a role in the induction/maintenance of tolerance. We have found (using small numbers of mice) that grafts from RAGKO mice, which can confer no chimerism, behave essentially like those from normal mice. We are in the process of confirming this result with larger numbers of mice to reach statistical significance.? ? 3) CD4 T CELLS CLEAR TUMORS In the process of studying how CD4 helper T cells collaborate with CD8 effectors to clear tumors, we found that the CD4 T cells are better at clearing tumors than CD8 cells. Against six out of six tumors, from five different tissues, CD4 effectors were more potent than CD8s. We have now found that this is true of another set of CD4 and CD8 T cells (OTI and OTII) Searching for the mechanism, we found that the CD4 T cells partner with NK cells. We are now studying this partnership to determine which cell does what. Thus far, it seems that IFN-gamma production by the CD4 T cells is involved, and macrophages are not. (Perez-Diez, et al (2007) CD4 Cells Can Be More Efficient at Tumor Rejection Than CD8 Cells Blood 109:5346? ? 4) DENDRITIC CELLS & IL-12p70 It is currently thought that dendritic cells become exhausted within 24 hours of stimulation by LPS. We have found that this is incorrect. The dendritic cells, while resistant to restimulation by LPS, can produce IL-12p70 when re-stimulated by activated T cells. We found that TH1 cells and TH0 cells can stimulate this production of IL-12p70 while TH2 cells cannot. Thus dendritic cells are not in control of immune effector class, but instead relay signals from the cells with which they are in contact. ? ? 5) IMMUNITY & HEALING: The Danger model led us to the view that tissues influence the effector class of immunity in order to prevent immune-mediated local damage. To determine whether wounded tissues are affected by the immune system, we punched small holes in the ears of mice and measured the healing rate. We found that young mice heal slowly while older mice, surprisingly, quickly regenerate both the epithelium and cartilage so well that there is eventually no visible scar. Histology suggested that the amount of inflammation is different between the two ages of mice. Thus we tested NSAIDs and found that they greatly inhibit the regeneration. Thus, surprisingly, inflammation seems to aid in the regeneration process. These results go against current thinking.? ? 6) TOLERANCE TO LATE-APPEARING ANTIGENS: One of the problems with the self-non-self model is that it does not account for tolerance to antigens that appear late in life, such as the milk proteins of lactating mothers (eg. alpha lactalbumin, beta lactoglobulin, casein etc.). To see if tolerance to these proteins is established in the thymus, or perhaps by fetal exposure during pregnancy or neonatal exposure during lactation, we have been using two different model systems in which we can obtain adult animals that carry a particular milk-protein gene but which have not previously encountered the protein, either across the placenta, or by drinking the milk as babies. ? A) SHEEP: Because fetal sheep have a 6 layer placenta, they receive no large maternal proteins. At birth, for example, they have no maternal antibody and need colostrum to obtain maternal protection. Lambs from sheep dairies (eg. Chattham Dairy, NY) are raised on cows milk replacer (as the sheep milk is used for cheese). We immunized such lambs after weaning and before puberty and found that, like the hALAC mice, they made good responses and did not manifest autoimmune disease when they were later bred and lactated. Biopsies from the udders showed no sign of infiltration or destruction. Thus the immune system has a mechanism to instigate tolerance to proteins that appear late in life, even after immunization.? B) HUMAN ALPHA LACTABLUMIN KNOCK-IN MICE: We obtained mice that had their own alpha lactalbumin (mALAC) genes replaced with the human allele (hALAC), and bred them for 22 generations to B10.Br. To make experimental animals, we bred normal B10.Br females to B10.Br hALAC males. The F1 progeny carry both human and mouse alleles of ALAC but have only been exposed to mALAC from their mothers. When immunized to hALAC after weaning but before puberty, these mice (both males and females) made good T cell and antibody responses. Thus they were not rendered tolerant by thymic expression. When bred, the immunized females showed no autoimmune reactions to their mammary tissue. Neither did the levels of antibodies to hALAC drop or the subclasses of antibodies change. Using pep-scan, we found that the carriers, which make good responses, are nveretheless somewhat tolerant, as they do not respond to a dominant peptide that stimulates non-carrier mice quite well.We are now in the process of making new T cell clones from immunized normal female mice, to make TCR Tg mice from these to follow the fate of immunized self-reactive T cells as the mice lactate through several pregnancies.
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