Patricia Taylor

CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28.

A monoclonal antibody (mAb) specific for the 93-104 segment of pigeon cytochrome c (cyt) was shown to block interleukin 2 production and proliferation by pigeon cyt-specific T cells in response to the pigeon cyt 81-104 peptide using either the LK35.2 B cell hybridoma or normal splenocytes as antigen-presenting cells (APC). The mAb inhibited the response to soluble peptide antigen presented by metabolically inactive paraformaldehyde-fixed APC but not the response to APC that were pre-pulsed with Ag. These results suggest that the mAb blocked the formation of peptide-major histocompatibility complex (MHC) class II molecule complexes at the cell surface but did not displace the peptide once bound to the MHC class II molecule. As determined by direct binding experiments using labeled peptide, the major means of free peptide association with live APC was fluid-phase endocytosis. No free peptide associated directly with the MHC class II molecule at the cell surface near 0 degrees C since APC pulsed with peptide on ice did not activate cyt-specific T cells. The mAb enhanced the association of the radiolabeled peptide with APC at 4 degrees C apparently by binding of the peptide-mAb complex to Fc receptors. By stripping molecules from the LK35.2 cell surface using a nonspecific protease it was shown that the peptide-mAb complexes were not internalized either at 4 degrees C or 37 degrees C. Since the mAb was found to stably bind the peptide at pH levels below that of endosomes (pH 5.5-6.2) even if the peptide-mAb complexes were taken up by fluid-phase endocytosis, it is likely that the peptide would not be able to associate with MHC class II molecules inside the APC. This mAb appears to inhibit T cell activation by blocking the formation of peptide-MHC class II molecule complexes at the cell surface and by interfering with uptake of the peptide into endosomes. Therefore, it is different from other antibodies that have been reported to block T cell receptor recognition of preformed peptide/MHC class II molecule complexes.

Abstract The process by which bone marrow is rejected by host T cells has not been able to be directly visualized to date. To study the process of allogeneic bone marrow rejection and the effects of therapeutic interventions, we created 2 models that allow us to 1) quantify the specific expansion of host-type alloreactive T cells early post-transplant in response to allogeneic BM infusion and to 2) image host T cells in vivo during BM rejection. For the first model, 2C and TEa lymph node (LN) cells were adoptively transferred into syngeneic C57BL/6 (B6) Rag deficient mice on d-2. 2C CD8+ and TEa CD4+ T cell receptor transgenic T cells are reactive against BALB/c alloantigen. Mice were irradiated with 200 cGy on d-1 and BALB/c BM was infused on d0. Controls included mice that received 2C/TEa LN cells but no BM. Ten days later, spleen analysis revealed that 2C CD8+ and TEa CD4+ T cells had expanded 322-fold and 33-fold (ave of 6 exp.), respectively, in mice receiving BALB/c BM compared to controls that did not receive BM. Expanded T cells were activated as determined by flow cytometric parameters and cell surface antigens. Data indicate that host alloreactive T cell expansion was inhibited by >95% by combined, but not single, costimulatory pathway blockade. Studies are in progress to analyze in vitro host anti-donor responses of adoptively transferred T cells. To visualize the response of host T cells to donor BM in vivo, we developed a rejection model for imaging involving the adoptive transfer of green fluorescent protein (GFP) T cells (obtained from GFP transgenic mice) into syngeneic non-GFP B6 recipients immediately following sublethal irradiation (500 cGy). Allogeneic BALB/c or syngeneic B6 BM was infused the following day. The syngeneic BMT controls allowed for the distinction of homeostatic vs alloreactive expansion of GFP+ T cells. Transplanted mice not receiving GFP T cells served as negative controls to verify lack of autofluorescence. Cohorts were imaged d4 to d18 post BMT. By d4, low numbers of GFP+ cells were evident in femoral BM cavity, peripheral and mesenteric LNs, spleen, Peyer’s patches (PP) and to a lesser extent, lung. By d7, massive expansion of GFP+ cells could be visualized throughout the body of recipients of allogeneic BM. LNs, spleen, PP (peri-follicular area) and BM cavity increased dramatically in GFP intensity from d4 to d7. On d7 to d14, there were large foci of GFP+ cells in the lung, liver, skin, gingiva, kidney, uterus, and colon in allogeneic BMT recipients. Compared with allogeneic BMT recipients, syngeneic BMT recipients had greatly reduced numbers of GFP+ T cells in lymphoid organs and only rare cells were noted in liver, kidney, skin, BM and gingiva. In both allogeneic and syngeneic BMT recipients, lengths of ileum were diffusely infiltrated while other sections contained discrete foci of GFP+ cells. These imaging data provide a vivid illustration of the massive expansion and multi-organ distribution of host anti-donor T cells in vivo. Recent generation of GFP+ 2C and GFP+ TEa mice will permit the imaging of alloantigen-specific T cells during a rejection response. Additional imaging experiments are planned to study the fate of GFP+ BM transferred to allogeneic recipients under conditions of engraftment vs rejection. These models provide a unique platform for the testing of therapeutic interventions.

Abstract ICOS, a CD28/CTLA-4 family member, is expressed on activated T cells. ICOS Ligand, a B7 family member, is constitutively expressed on B cells, monocytes and some T cells. Through the use of blocking anti-ICOS mAb and ICOS deficient (−/−) mice, we found that ICOS:ICOSL interactions play an important role in GVHD and BM graft rejection. Anti-ICOS mAb (given d-1 to d28 post BMT) significantly delayed or reduced mortality at 2 different T cell doses in a full MHC-disparate GVHD model. ICOS−/− T cells led to delayed or reduced mortality at 3 different cell doses compared to wild-type T cells. ICOS−/− CD4+ or CD8+ T cells infused into class II- or class I-disparate recipients, respectively, revealed that ICOS:ICOSL interactions regulate both CD4+ and CD8+ T cell alloresponses. Anti-ICOS inhibited GVHD in a CD28-independent fashion. Anti-ICOS inhibited GVHD mediated by either stat 4−/− or stat 6−/− T cells indicating that the ICOS pathway regulates both Th2 and Th1-mediated GVHD. In contrast to blockade of the B7:CD28/CTLA-4, CD40L:CD40 or the OX40:OX40L pathway, anti-ICOS mAb inhibited GVHD even when delayed until d5 post BMT, a time when substantial T cell expansion has occurred. A TCR transgenic model of GVHD was used to further study effects of ICOS:ICOSL blockade. All CB6 F1 recipients of anti-host alloreactive 2C CD8+ and TEa CD4+ T cells succumbed to GVHD mortality by d18 after transfer of cells. In contrast, 88% of anti-ICOS-treated mice survived long-term. Evaluation of spleens early after transplant revealed that anti-ICOS mAb reduced the number of TEa CD4+ cells by 44% and 2C CD8+ cells by 83%. Green fluorescent protein (GFP) 2C CD8+ and GFP TEa CD4+ T cells were infused into irradiated CB6 F1 mice and irrelevant or anti-ICOS mAb was administered. Mice were imaged on d4, 7 and 12 after T cell transfer. By d7, pronounced infiltration of GFP+ cells was noted in the peripheral and mesenteric LN, spleen, Peyer’s patches (PP), skin, gingiva, liver, kidney, lung, ileum, and colon of GVHD control mice. In contrast, there were fewer GFP+ cells in the spleen, ileum, colon, kidney, lung, skin and gingiva of anti-ICOS-treated mice, although there was no decrease in GFP+ cells in LNs or PP. To study the role of host ICOS expression in BM graft rejection, wild-type or ICOS−/− mice were sublethally irradiated and given allogeneic BM and evaluated for donor chimerism at 6 weeks post BMT. Five of 10 wild type mice engrafted (ave − 26% donor) in contrast to all 10 of ICOS−/− mice (ave − 71% donor). Collectively, these data indicate that ICOS:ICOSL interactions play an important role in GVHD, whether mediated by CD4+ Th1 or Th2 T cells or CD8+ T cells. Importantly, blockade of ICOS:ICOSL after initiation of alloresponses inhibited GVHD, in contrast to blockade of other costimulatory pathways, suggesting that the ICOS pathway may be a novel therapeutic target in primed transplantation situations. Anti-ICOS interfered with expansion of donor T cells in the spleen early after transplant and reduced the number of effector cells in several GVHD target tissues. These data suggest this pathway may be indicated for therapeutic targeting for the inhibition of GVHD and BM graft rejection.