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T cell Activation in Vivo Carrie Miceli May 16, 2005 • Assigned reading: Drew M. Catron, Andrea A. Itano, Kathryn A. Pape, Daniel L. Mueller, and Marc K. Jenkins Visualizing the First 50 Hr of the Primary Immune Response to a Soluble Antigen Immunity 2004 21: 341-347. • Additional Reading: – Jenkins et al., In Vivo Activation of Antigen Specific CD4 T cells. Annual Review of Immunology 2001 Reinhardt and Jenkins Whole-body Analysis of T cell Responses Current Opinion in Immunology 2003 15:366-371 Germain and Jenkins. In vivo antigen presentation. Current Opinion in Immunology 2003 16: 120-125 Tracking antigen specific T cell dynamics in vivo • The problem: frequency of an ag specific T cell 1/105-1/106 • The dream solution: TCR transgenic mice with monoclonal specificity – Reality sets in, these mice are not normal – Antigen specific T cells are too abundant, no room for all cells to expand properly – Initial expansion followed by immediate crash – no productive immunity • The real solution (Marc Jenkins) – Adoptive transfer of TCR transgenic T cells into wt congenic – Seed TCR tg T cells at 0.3% (still not normal) – Availability of anti-TCR clonotypic antibody allows for identification of TCR tg T cells – Get expansion, homing, and differentiation of TCR tg T cells in response to specific antigen • MHC/peptide tetramers -an alternate solution (mark davis) OT-II B6.PL (Thy 1.1) OVA peptide-I-Ab SM1 B6.PL (Thy 1.1) Salmonella FliC peptide-I-Ab B6 (Thy 1.2) D3 T cell expansion 20-100X antigen w/ adj 10-20 antigen alone PM JK DL KM KH BH FC MO • Histology allows for in situ detection; flow allows for quantitation; activation and differentiation markers • Transferred T cells can be from knockout X TCR transgene • Recipient can be knockout to determine APC or tissue requirements – Can also adoptively transfer APC or specific B cells • Can take advantage of congenic strains of allelic CD45, CD8, or thy-1 mice • Can track number of cell divisions using CFSE • Can manipulate route of antigen delivery – Conditions of activation or inactivation (adjuvent or site and dose) • CFSE allows determination of number of cell divisions by flow cytometry. One can stimulate and monitor in vitro; adoptively transfer and monitor in vivo fate; or transfer and both stimulate and track fate in vivo Knock-out Wild-type Question answered using this approach. • Where do T cells first encounter antigen? • What are the molecular requirements for activation, homing to B cell zones, B cell help? • Do T cells die after several rounds of division? • Do they home to the tissues? • Do they become memory T cells? • Are those that home to tissues different from those that stay in lymphoid tissues, die or become memory Ts? KS CA TEa TCR recognizes Ea45-75 and can be detected with clonotypic ab Y-Ae ab recognizes surface pEa52-68/Iab. Soluble peptide labeled with RFP Ea 45-75 RFP TEa T cell pEa 52-68/ I-Ab Y-Ae AR Peptide-MHC II+ cells present at 4-14 hours are Langerhans cells that picked up soluble antigen from lymph. EaRFP Y-Ae B220 Peptide-MHC II+ cells present at >18 hours are dermal dendritic cells that picked up antigen at the injection site. AA SS In Situ Interactions Between Antigen-Specific CD4 T Cells and Peptide-MHC II+ APC T cells CD69 Y-Ae TEa B6 50 40 4 hours T cells Y-Ae 24 hours % T Cell/Y-Ae+ Cell Interactions 30 20 10 0 TEa 20 C57Bl/6 * 15 10 5 0 TEa JC C57Bl/6 DC Movie/subcuteneous injection • T cell in paracortex/T cell zone first encounters antigen (4 hrs post injection) on a LN resident Langerhans cell (migrated there earlier)…both antigen specific and non specific give it a try • T cell activated to express CD69 and lymphokines (and perhaps chemokine receptor responsible for interaction with interstitial dendritic cells) • Second wave of antigen presentation at 18 hrs by newly immigrated tissue/interstitial dendritic cells • T cells activated in the first wave specifically migrate to and interact with these immigrated interstitial dendritic cells and are stimulated to divide • Some leave to tissues, some migrate to B cell follicle to help In Situ Detection of IL-2 Production by Antigen-Specific CD4 T Cells No Antigen OVA/LPS, 12 h CJ In Vivo IL-2 Production by Naive CD4 T Cells Is Dependent on CD28 35 100 30 %CD86+ DC %IL-2+ OT-II Cells TLR4 Expression is Required for the Adjuvant Effects of LPS 25 20 15 10 5 80 60 40 20 0 0 0 2 4 6 8 0 Hours After Injection B10 OVAp i.v. B10 OVAp/LPS i.v. TLR4-/- OVAp i.v. TLR4-/- OVAp/LPS i.v. 2 4 6 8 In Vivo Clonal Expansion of Antigen-Specific CD4 T Cells Death or exodus? MMW MD RA EP HvB RZ OT-II Recipient, 5 Days After OVAp/IFA Injection in Tail LL OT-II DAPI Lymphokine Production Correlates with Cell Division History Starting CFSE Level Whole mouse movie • • • • • • • • • • • Spleen entry chemokine dependent, CD62L-independent Lymph node entry CCR7 dependent; CD62L dependent Free antigen flows through afferent lymph into T Cell area, taken up by resident Langerhans cells Dermal DC take up antigen at the injection site, migrate to T cell area in response to IL-1, TNF Naïve T cell activated by resident langerhans cell to express CD69, produce IL-2, more if B7 induced on APC Activated T cell seeks out migrating dermal dendritic cells, is reactivated T cells divide; some many times some few times T cells leave lymph node to efferent lymph Highly divided T cells lose CD62L, gain fPSGL-1 Highly dividing tissue homing (lungs and other tissues as well as specifically to site of injection). After APC death many T cells die there. Nonlymphoid seeking and effector lymphokine producing less dividing lymph node seeking; retain CD62L, produce IL-2 A productive primary CD4 T cell response to antigen in the presence of adjuvant-induced inflammation Jenkins Annual Rev of Immunol..2001 Response in the lymph nodes after subcutaneous injection of antigen plus adjuvant. This is the type of response that generates effector lymphokine-producing memory cells and is induced by microbes because they contain foreign proteins and molecules with adjuvant properties. Adjuvant molecules are recognized by pattern recognition receptors on cells of the innate immune system at the antigen injection site, causing the release of TNF- and IL-1. These cytokines signal the local tissue dendritic cells or monocytes to leave the tissue and migrate via an afferent lymphatic vessel to the draining lymph node after first ingesting antigen. During the migration process, these cells mature to produce peptide-MHC complexes from the ingested antigen and deliver these to the cell surface along with newly synthesized B7 molecules. After arriving at the lymph node, the dendritic cells crawl through the floor of the subcapsular sinus and present peptide-MHC complexes to naïve antigen-specific CD4 T cells in the T cell area. The T cells produce high levels of IL-2 and an unknown T cell growth factor, and they proliferate. This proliferation occurs in an IL-12-rich environment due to IL-12 produced by dendritic cells in response to the adjuvant. Those that divided the most and experienced the highest concentration of IL-12 lose CCR7, gain P-selectin ligand, and acquire the capacity for rapid IFN- production. Antigen-activated T cells that do not achieve this threshold number of cell divisions, or IL-12 concentration, remain CCR7+ and acquire rapid IL-2 production pot ential, but not the capacity for IFN- production. After leaving the lymphoid tissues during the primary response, the CCR7+ cells recirculate through lymphoid tissues like naïve T cells. In contrast, the CCR7- cells are excluded from lymph nodes and remain in the blood or enter nonlymphoid tissues that express P-selectin. As long as residual antigen is present to drive the survival of CCR7cells, then a second exposure to antigen will result in rapid production of effector lymphokines at the site of antigen entry either by CCR7- cells that happen to reside in that tissue or by CCR7- cells that are rapidly recruited from blood. As antigen is cleared from the body, CCR7- cells die or revert to the CCR7+ phenotype; in either case only CCR7+ memory cells remain. If antigen enters the body during this phase, then CCR7+ cells will be activated in the lymphoid organs and rapidly differentiate into CCR7-, effector lymphokine-producing cells capable of migrating to the site of antigen deposition. This process would be more efficient than the primary response because CCR7+ memory cells could achieve effector lymphokine production faster than naïve T cells, and extrinsic factors such as antibodies from the primary response would facilitate antigen presentation. Diagrammatic representation of an unproductive CD4 T cell response to antigen in the absence of inflammation. Presentation of an injected antigen is relatively inefficient because it depends on the low level of dendritic cell migration that occurs under noninflammatory conditions, or the small amount of antigen that leaks across the subcapsular barrier to be taken up by lymphoid–tissue resident dendritic cells. In either case, antigen presentation is carried out by dendritic cells that don’t express high levels of co-stimulatory ligands. Lack of the anti-apoptotic effects of inflammatory cytokines cause most of the T cells to die. The minimal signaling through CD28, IL-1 receptor, and receptors for differentiating cytokines such as IL12 and IL-4, experienced in the primary response would limit IL-2 and effector lymphokine production potential in any memory cells that survived. The combination of death of most of the expanded antigen-specific T cell Population and the functional defects in the survivors could explain the induction of peripheral tolerance by antigen administration in the absence of inflammation. Tracking antigen specific responses with MHC/tetramers Flow cytometric detection of antigen-specific T cells using fluorochrome labeled peptideMHC complexes. Refold soluble empty class I MHC antigens with a single antigenic peptide. Peptide/MHC complex is then biotinylated and mixed with streptavidin fluorochrome to produce a tetramer For class II MHC, need to covalently attach peptide Science 1996 Oct Phenotypic analysis of antigen-specific T lymphocytes Altman JD…... Davis MM. Figure 2. Correlation of antigen-specific staining in three of four patients with peptide-specific killing activity in CTL bulk cultures. Peripheral blood mononuclear cells from four healthy HIV-infected donors were separated as described (5). The CD4 counts for each patient at the time of analysis were as follows: patient 065, 410; patient 868, 330; patient 077, 270; and patient 606, 510. Two million peripheral blood cells from each patient were stained with anti-CD8a-CyChrome and phycoerythrin-labeled HLA-A2-Gag (solid line) or HLA-A2-Pol (dotted line) tetramers as indicated (A through D) (18). Software gates were set to display only CD8+ small lymphocytes. The percentages of CD8+ cells that were positive for either A2-Pol or A2-Gag within gates as displayed are included in each panel. The reproducibility of the A2-Pol+ and A2-Gag+ populations in patient 065 (A) was tested through analysis of five separate stains with each reagent; the standard deviations are reported. Bulk cultures were assayed for CTL activity (E through H) (19) on days 14 through 16 at an effector:target ratio of 50:1. Black bars, lysis of Gag-loaded targets; white bars, lysis of Pol-loaded targets.