Rachel S. Friedman

Deletional Tolerance Mediated by Extrathymic Aire-Expressing Cells

The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen–expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

A Synaptic Basis for Paracrine Interleukin-2 Signaling during Homotypic T Cell Interaction

T cells slow their motility, increase adherence, and arrest after encounters with antigen-presenting cells (APCs) bearing peptide-MHC complexes. Here, we analyzed the cell-cell communication among activating T cells. In vivo and in vitro, activating T cells associated in large clusters that collectively persisted for >30 min, but they also engaged in more transient interactions, apparently distal to APCs. Homotypic aggregation was driven by LFA-1 integrin interactions. Ultrastructural analysis revealed that cell-cell contacts between activating T cells were organized as multifocal synapses, and T cells oriented both the microtubule-organizing complex and interleukin-2 (IL-2) secretion toward this synapse. T cells engaged in homotypic interactions more effectively captured IL-2 relative to free cells. T cells receiving paracrine synaptic IL-2 polarized their IL-2 signaling subunits into the synaptic region and more efficiently phosphorylated the transcription factor STAT5, likely through a synapse-associated signaling complex. Thus, synapse-mediated cytokine delivery accelerates responses in activating T cells.

Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells.

Significance Alum has been used to improve the efficacy of vaccines since the 1930s. Here we show that alum acts in part via host DNA to increase the interaction time between T cells and APCs. Many vaccines include aluminum salts (alum) as adjuvants despite little knowledge of alum’s functions. Host DNA rapidly coats injected alum. Here, we further investigated the mechanism of alum and DNA’s adjuvant function. Our data show that DNase coinjection reduces CD4 T-cell priming by i.m. injected antigen + alum. This effect is partially replicated in mice lacking stimulator of IFN genes, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNase treatment impairs dendritic cell (DC) migration from the peritoneal cavity to the draining lymph node in mice immunized i.p. with alum. However, our data show that DNase does not affect accumulation of, or expression of costimulatory proteins on, antigen-loaded DCs in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNase does inhibit prolonged T-cell–DC conjugate formation and antigen presentation between antigen-positive DCs and antigen-specific CD4 T cells following i.m. injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DCs, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC class II presentation and better DC–T-cell interactions.

Induction of Human Immunodeficiency Virus (HIV)-Specific CD8 T-Cell Responses by Listeria monocytogenes and a Hyperattenuated Listeria Strain Engineered To Express HIV Antigens

ABSTRACT Induction of cell-mediated immunity may be essential for an effective AIDS vaccine. Listeria monocytogenes is an attractive bacterial vector to elicit T-cell immunity to human immunodeficiency virus (HIV) because it specifically infects monocytes, key antigen-presenting cells, and because natural infection originates at the mucosa. Immunization with recombinant L. monocytogenes has been shown to protect mice from lymphocytic choriomeningitis virus, influenza virus, and tumor inoculation.L. monocytogenes expressing HIV gag elicits sustained high levels of Gag-specific cytotoxic T lymphocytes (CTLs) in mice. We have examined the ability of Listeria to infect human monocytes and present HIV antigens to CD8 T lymphocytes of HIV-infected donors to induce a secondary T-cell immune response. Using this in vitro vaccination protocol, we show that L. monocytogenes expressing the HIV-1 gag gene efficiently provides a strong stimulus for Gag-specific CTLs in HIV-infected donor peripheral blood mononuclear cells.Listeria expressing Nef also elicits a secondary in vitro anti-Nef CTL response. Since L. monocytogenes is a pathogen, before it can be seriously considered as a human vaccine vector, safety concerns must be addressed. We therefore have produced a highly attenuated strain of L. monocytogenes that requiresd-alanine for viability. The recombinant bacteria are attenuated at least 105-fold. We show that when these hyperattenuated bacteria are engineered to express HIV-1 Gag, they are at least as efficient at stimulating Gag-specific human CTLs in vitro as wild-type recombinants. These results suggest that attenuatedListeria is an attractive candidate vaccine vector to induce T-cell immunity to HIV in humans.

Duration of antigen receptor signaling determines T-cell tolerance or activation

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4+ T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell–antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell–APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell–APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.

Amplification of Autoimmune Response through Induction of Dendritic Cell Maturation in Inflamed Tissues

Dendritic cells (DCs) are essential in T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans in type 1 diabetes. In this study, we investigated T cell induction of intra-islet DC maturation during the progression of the disease in both autoimmune-prone NOD and resistant C57BL/6 mice. We demonstrated steady-state capture and retention of unprocessed β cell-derived proteins by semimature intra-islet DCs in both mouse strains. T cell-mediated intra-islet inflammation induced an increase in CD40 and CD80 expression and processing of captured Ag by resident DCs without inducing the expression of the p40 subunit of IL-12/23. Some of the CD40high intra-islet DCs up-regulated CCR7, and a small number of CD40high DCs bearing unprocessed islet Ags were detected in the pancreatic lymph nodes in mice with acute intra-islet inflammation, demonstrating that T cell-mediated tissue inflammation augments migration of mature resident DCs to draining lymph nodes. Our results identify an amplification loop during the progression of autoimmune diabetes, in which initial T cell infiltration leads to rapid maturation of intra-islet DCs, their migration to lymph nodes, and expanded priming of more autoreactive T cells. Therapeutic interventions that intercept this process may be effective at halting the progression of type 1 diabetes.

Expansion of CD57 and CD62L-CD45RA+ CD8 T lymphocytes correlates with reduced viral plasma RNA after primary HIV infection.

OBJECTIVE CD8 T cells, expressing cell surface molecules distinct from those on resting and naive T cells, are increased in HIV infection. The association of increased CD38 and human leukocyte antigen DR (HLA-DR) CD8 T cells with poor prognosis has suggested that activated CD8 T cells may aggravate HIV infection. We examined whether other immunological parameters might influence the viral setpoint. DESIGN Peripheral T cells from nine untreated patients, obtained after primary HIV infection when plasma HIV had stabilized, were examined for proteins expressed in activated versus resting, memory versus naive, and cytolytic versus non-cytolytic T cells. METHODS The proportion of CD8 T cells that stain for CD38 and HLA-DR, CD28 and CD57 was compared with plasma viraemia and CD4 cell count. These parameters were also compared with the proportion of CD4 and CD8 T cells that express CD62L and CD45RA, present on naive cells and down-modulated in memory cells. Internal staining for the cytotoxic protein granzyme A was also examined. RESULTS An increase in CD38 and CD38 HLA-DR CD8 T cells correlated with increased plasma viral RNA (P < 0.00002, P < 0.03, respectively). An increase in CD8 T cells expressing granzyme A was associated with lower CD4 cell counts (P < 0.04). However, the expansion of CD57 and CD62L CD45RA+ CD8 T cells was associated with a lower viral setpoint (P < 0.01, P < 0.02, respectively). CONCLUSION Phenotypically defined activated CD8 T cells may have different functions in HIV infection. Activated CD8 T cells that are CD57 or CD62L(-)CD45RA+ may be beneficial, because their expansion in untreated patients correlates with a reduced viral setpoint after primary infection.

CD11c-Expressing B Cells Are Located at the T Cell/B Cell Border in Spleen and Are Potent APCs

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c+ B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c+ B cells form significantly more stable interactions with T cells than do FO cells. Together, these data identify a previously undescribed role for ABCs as potent APCs and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity.

Molecular and Immunological Evaluation of the Transcription Factor SOX-4 as a Lung Tumor Vaccine Antigen

The developmental transcription factor SOX-4 has been shown to be highly and differentially overexpressed in primary small cell lung carcinomas (SCLC). To examine the potential of SOX-4 for broad use as a lung cancer vaccine, we have evaluated the expression of SOX-4 in a panel of primary adenocarcinoma, squamous, and large cell tumor samples as well as in a panel of established small cell and non-small cell lung carcinoma tumor cell lines. SOX-4 mRNA is shown to be overexpressed in a substantial fraction of each of these lung tumor types. To examine the immunological potential of SOX-4, we have evaluated the presence of SOX-4-specific CD4 and CD8 T cells in PBMC of healthy donors and the presence of SOX4-specific Abs in sera from SCLC patients. We demonstrate the presence of both CD4 and CD8 T cells that recognize naturally processed epitopes derived from SOX-4 as well as the presence of SOX-4-specific Abs in sera from SCLC patients, but not in sera from healthy donors. The lung tumor-specific overexpression and demonstration of a comprehensive Ag-specific immune response specific for SOX-4 support the use of this molecule in the development of whole gene-, peptide-, or protein-based vaccination strategies against lung cancer. Furthermore, the identification of naturally processed T cell and Ab epitopes from SOX-4 provides valuable tools for the development of peptide-based vaccination strategies against lung cancer as well as to monitor SOX-4-specific responses in vaccinated patients.

Two-Photon Imaging of the Immune System: A Custom Technology Platform for High-Speed, Multicolor Tissue Imaging of Immune Responses

Modern imaging approaches are proving important for addressing contemporary issues in the immune system. These approaches are especially useful for characterizing the complex orchestration of immune responses in vivo. Multicolor, two-photon imaging has been proven to be especially enabling for such studies because of its superior tissue penetration, reduced image degradation by light scattering leading to better resolution, and its high image quality deep inside tissues. Here, we examine the functional requirements of two-photon imaging instruments necessary for such immune studies. These requirements include frame rate, spatial resolution and the number of emission channels. We use this discussion as a starting point to compare commercial systems and to introduce a custom technology platform that meets those requirements. This platform is noteworthy because it is very cost-effective, flexible and experimentally useful. Representative data collected with this instrument is used to demonstrate the utility of this platform. Finally, as the field is rapidly evolving, consideration is given to some of the cutting-edge developments in multiphoton microscopy that will likely improve signal strength, depth penetration and/or the experimental usefulness of this approach.