Elizabeth Ingulli

Distinct Dendritic Cell Populations Sequentially Present Antigen to CD4 T Cells and Stimulate Different Aspects of Cell-Mediated Immunity

Peptide:MHC II complexes derived from a fluorescent antigen were detected in vivo to identify the cells that present subcutaneously injected antigen to CD4 T cells. Skin-derived dendritic cells (DCs) that acquired the antigen while in the draining lymph nodes were the first cells to display peptide:MHC II complexes. Presentation by these cells induced CD69, IL-2 production, and maximal proliferation by the T cells. Later, DCs displaying peptide:MHC II complexes migrated from the injection site via a G protein-dependent mechanism. Presentation by these migrants sustained expression of the IL-2 receptor and promoted delayed type hypersensitivity. Therefore, presentation of peptide:MHC II complexes derived from a subcutaneous antigen occurs in two temporally distinct waves with different functional consequences.

Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cells for the study of T-cell activation in vivo

Summary: Adoptive transfer of TCR‐transgenic T cells uniformly expressing an identifiable TCR of known peptide/MHC specificity can be used to monitor the in vivo behavior of antigen‐specific T cells. We have used this system to show that naive T cells are initially activated within the T‐cell zones of secondary lymphoid tissue to prohferate in a B7‐dependent manner. If adjuvants or inflammatory cytokines are present during this period, enhanced numbers of T cells accumulate, migrate into B‐cell‐rich follicles, and acquire the capacity to produce IFN‐7 and help B cells produce IgG2a. If inflammation is absent, most of the initially activated antigen‐specific T cells disappear without entering the follicles and the survivors are poor producers of IL‐2 and IFN‐γ Our results indicate that inflammatory mediators play a key role in regulating the anatomic location, clonal expansion, survival and lymphokine production potential of antigen‐stimulated T cells in vivo.

Development of a Novel Transgenic Mouse for the Study of Interactions Between CD4 and CD8 T Cells During Graft Rejection

The goal of this study was the development of a system in which the cooperative interactions between CD4 and CD8 T cells specific for defined peptides from a single minor histocompatibility antigen could be studied. A transgenic mouse strain that expresses chicken ovalbumin (Act‐mOVA) on the surface of all cells in the body was produced as a source of tissues containing such an antigen. Skin grafts from Act‐mOVA donors were rapidly and completely rejected by wild‐type recipients, but only when both CD4 and CD8 T cells were present. CD4 T cells by themselves caused an incomplete form of rejection characterized by rapid but partial contraction of Act‐mOVA grafts. CD8 T cells alone caused complete rejection of Act‐mOVA skin grafts but only after a long delay. Adoptively transferred ovalbumin‐specific TCR‐transgenic CD4 and CD8 T cells were stimulated by Act‐mOVA graft antigens and CD8 T‐cell accumulation in the grafts was enhanced by specific CD4 T cells. These findings, together with the fact that the ligand for ovalbumin peptide‐specific CD8 T cells can be detected in Act‐mOVA tissues with an MHC‐restricted antibody, make this an ideal system for the study of cooperation between CD4 and CD8 T cells.

Mechanism of cellular rejection in transplantation.

The explosion of new discoveries in the field of immunology has provided new insights into mechanisms that promote an immune response directed against a transplanted organ. Central to the allograft response are T lymphocytes. This review summarizes the current literature on allorecognition, costimulation, memory T cells, T cell migration, and their role in both acute and chronic graft destruction. An in depth understanding of the cellular mechanisms that result in both acute and chronic allograft rejection will provide new strategies and targeted therapeutics capable of inducing long-lasting, allograft-specific tolerance.

In Situ Analysis Reveals Physical Interactions Between CD11b+ Dendritic Cells and Antigen-Specific CD4 T Cells After Subcutaneous Injection of Antigen

In situ staining techniques were used to visualize physical interactions between dendritic cell subsets and naive Ag-specific CD4 T cells in the lymph node. Before injection of Ag, CD8+ dendritic cells and naive OVA-specific CD4 T cells were uniformly distributed throughout the T cell-rich paracortex, whereas CD11b+ dendritic cells were located mainly in the outer edges of the paracortex near the B cell-rich follicles. Many OVA-specific CD4 T cells were in contact with CD8+ dendritic cells in the absence of OVA. Within 24 h after s.c. injection of soluble OVA, the OVA-specific CD4 T cells redistributed to the outer paracortex and interacted with CD11b+, but not CD8+ dendritic cells. This behavior correlated with the uptake of OVA and the presence of peptide-MHC complexes on the surface of CD11b+ dendritic cells, and subsequent IL-2 production by the Ag-specific CD4 T cells. These results are consistent with the possibility that CD11b+ dendritic cells play a central role in the activation of CD4 T cells in response to s.c. Ag.

Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo

Flow cytometry was used to study signaling events in individual CD4 T cells after antigen recognition in the body. Phosphorylation of c-jun and p38 mitogen-activated protein kinase was detected within minutes in all antigen-specific CD4 T cells in secondary lymphoid tissues after injection of peptide antigen into the bloodstream. The remarkable rapidity of this response correlated with the finding that most naive T cells are in constant contact with dendritic antigen-presenting cells. Contrary to predictions from in vitro experiments, antigen-induced c-jun and p38 mitogen-activated protein kinase phosphorylation did not depend on CD28 signals and was insensitive to inhibition by cyclosporin A. Our results highlight the efficiency of the in vivo immune response and underscore the need to verify which signaling pathways identified in vitro actually operate under physiological conditions.

Cutting Edge: In Vivo Identification of TCR Redistribution and Polarized IL-2 Production by Naive CD4 T Cells

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.

CD4 T cell-dependent conditioning of dendritic cells to produce IL-12 results in CD8-mediated graft rejection and avoidance of tolerance.

Rejection of ectopic heart transplants expressing OVA requires OVA-specific CD4 and CD8 T cells. In the absence of CD4 T cells, OVA-specific CD8 T cells proliferate and migrate to the graft, but fail to develop cytolytic functions. With CD4 T cells present, clonal expansion of the CD8 T cells is only marginally increased but the cells now develop effector functions and mediate rapid graft rejection. In the presence of CD4 T cells, Ag and B7 levels do not increase on dendritic cells but IL-12 production is up-regulated, and this requires CD154 expression on the CD4 T cells. OVA-specific CD8 T cells lacking the IL-12 receptor fail to differentiate or mediate graft rejection even when CD4 T cells are present. Thus, CD4 T cells condition dendritic cells by inducing the production of IL-12, which is needed as the “third signal” for CD8 T cell differentiation and avoidance of tolerance.

MHC class II deprivation impairs CD4 T cell motility and responsiveness to antigen-bearing dendritic cells in vivo

The role continuous contact with self-peptide/MHC molecules (self ligands) in the periphery plays in the function of mature T cells remains unclear. Here, we elucidate a role for MHC class II molecules in T cell trafficking and antigen responsiveness in vivo. We find that naïve CD4 T cells deprived of MHC class II molecules demonstrate a progressive and profound defect in motility (measured by real-time two-photon imaging) and that these cells have a decreased ability to interact with limiting numbers of cognate antigen-bearing dendritic cells, but they do not demonstrate a defect in their responsiveness to direct stimulation with anti-CD3 monoclonal antibody. Using GST fusion proteins, we show that MHC class II availability promotes basal activation of Rap1 and Rac1 but does not alter the basal activity of Ras. We propose that tonic T cell receptor signaling from self-ligand stimulation is required to maintain a basal state of activation of small guanosine triphosphatases critical for normal T cell motility and that T cell motility is critical for the antigen receptivity of naïve CD4 T cells. These studies suggest a role for continuous self-ligand stimulation in the periphery for the maintenance and function of mature naïve CD4 T cells.

The Adjuvant Effects of Mycobacterium tuberculosis Heat Shock Protein 70 Result from the Rapid and Prolonged Activation of Antigen-Specific CD8+ T Cells In Vivo

Heat shock protein 70 (hsp70) is a potent adjuvant that links innate and adaptive immune responses. To study how hsp70 activates naive CD8+ T cells in vivo, we tracked Ag-specific CD8+ T cells in mice immunized with a fusion protein containing chicken OVA linked to hsp70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). On a molar basis, OVA.TBhsp70 was several hundred times more effective than OVA peptide plus CFA in eliciting specific CD8+ T cell responses. Immunization with OVA.TBhsp70 activated >90% of detectable OVA-specific CD8+ T cells within 3 days and led to the persistence of cytotoxic effectors for at least 17 days. These studies demonstrate that the potent adjuvant effect of M. tuberculosis hsp70 results from the relatively complete, rapid, and durable activation of Ag-specific CD8+ T cells.