IMMUN 441: Week 5 AC Quiz Section T cell signaling B cell signaling III. T cell development

I. T cell signaling

Signaling downstream of antigen receptors is mediated by a balance of kinases and phosphatases Protein ATP  ADP P T P Y

Other important players Signaling downstream of antigen receptors is mediated by a balance of kinases and phosphatases Kinase: Src Family: Src, Fyn, Lck, Lyn, etc. ZAP-70, Syk Csk Itk PKC-θ IκB kinase (IKK) MAP kinase cascade: Raf-Mek-Erk PI3 kinase Phosphatase: CD45 Calcineurin Other important players Scaffold proteins Adaptor proteins Grb2 Second messengers Ca2+ Small G proteins Ras family

Important signaling domains Protein Domain Found in Ligand Class Example of ligand SH2 Lck, ZAP70, Fyn, Src, Grb2, PLCɣ, Btk, Itk, PI3K phosphotyrosine pYXXZ SH3 Lck, Fyn, Src, Grb2, Btk, Itk proline PXXP PH PLCɣ, Akt, Btk, Itk phosphoinositides PIP3 PX P40phox , P47phox, PLD PIP2 PDZ CARMA1 C termini of proteins IESDV, VETDV C1 RasGRP, PKC-θ membrane lipid diacyl glycerol (DAG) phorbol ester NZF TAB2 polyubiquitin polyubiquitinated RIP, TRAF-6, NEMO

T cell signaling video

CD4 CD3 CD3 Lck Cd45, a phosphatase, dephosphorylates Lck, opening it up and allowing it to phorphorylate CD3 Balance of kinases and phosphatases that set the threshold for TCR signaling

CD4 CD3 CD3 Lck

CD4 CD3 CD3 ZAP70 Lck

CD4 LAT CD3 CD3 ZAP70 Lck SLP-76

CD4 LAT CD3 CD3 ZAP70 Lck SLP-76

CD4 LAT CD3 CD3 ZAP70 GADS Lck SLP-76

GADS scaffold bring them together, complex recruits PLC gamma CD4 LAT CD3 CD3 ZAP70 GADS SLP-76 Lck GADS scaffold bring them together, complex recruits PLC gamma

CD4 LAT CD3 CD3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck

B7 CD4 LAT CD28 CD3 CD3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck

B7 CD4 PIP2 LAT CD28 CD3 CD3 PI3K PLCγ PLCγ ZAP70 GADS SLP-76 Lck

PIP3 PLCγ B7 CD4 CD28 LAT PI3K ZAP70 Lck SLP-76 GADS SLP-76 Lck If you don’t have costimulation, what happens? Which TFs will you get? Which ones won’t you get?

CD4 PIP3 LAT CD3 CD3 Itk PLCγ PLCγ ZAP70 GADS SLP-76 Lck

PIP3 Itk PLCγ CD4 LAT ZAP70 Lck SLP-76 GADS SLP-76 Lck PIP3 recruits Itk PH domain, which can phosphorylate PLCgamma

CD4 PIP2 LAT CD3 CD3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck

DAG IP3 PLCγ CD4 LAT ZAP70 Lck SLP-76 GADS SLP-76 Lck Hydrolyzes pip2 into IP3 and DAG (which are second messengers) DAG diffuses into the membrane

IP3 PLCγ CD4 LAT ZAP70 Lck SLP-76 GADS SLP-76 Lck IP3 diffuses through cytosol to the ER Ca2+ Ca2+

IP3 PLCγ CD4 LAT ZAP70 Lck SLP-76 CD3 CD3 PLCγ Ca2+ Ca2+ Ca2+ Ca2+ GADS SLP-76 Lck Ca2+ Ca2+ Ca2+ Ca2+

IP3 PLCγ CD4 LAT ZAP70 Lck SLP-76 CRAC CRAC IP3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck Ca2+ Ca2+ Depletion of ER calcium leads to CRAC channel formation, Ca2+ Ca2+

IP3 PLCγ CD4 LAT ZAP70 Lck SLP-76 influx of extracellular calcium CD3 CRAC CRAC IP3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck Ca2+ Ca2+ influx of extracellular calcium Ca2+ Ca2+

IP3 PLCγ NFAT CD4 LAT ZAP70 Lck SLP-76 CD3 CD3 PLCγ Calmodulin Ca2+ CRAC CRAC IP3 PLCγ PLCγ ZAP70 GADS SLP-76 Ca2+ Lck Calmodulin Ca2+ Ca2+ NFAT Ca2+ Ca2+

IP3 PLCγ NFAT CD4 LAT ZAP70 Lck SLP-76 CRAC CRAC IP3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck Calcineurin Ca2+ Calmodulin Ca2+ Ca2+ NFAT Calcium bound calmodulin serves as a platform for the phosphatase calcineurin, which then dephorphorylates the TF NFAT Ca2+ Ca2+

IP3 PLCγ NFAT CD4 LAT ZAP70 Lck SLP-76 CD3 CD3 PLCγ Calcineurin CRAC CRAC IP3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck Calcineurin Ca2+ Ca2+ Ca2+ Calmodulin NFAT Ca2+ Ca2+

IP3 PLCγ IL-2 NFAT NFAT CD4 LAT ZAP70 Lck SLP-76 CRAC CRAC IP3 PLCγ PLCγ ZAP70 GADS SLP-76 Lck Calcineurin Ca2+ Ca2+ Ca2+ Calmodulin Translocates to the nucleates, where it can bind to its target loci, this TF alone not enough for IL-2 transcription Why is IL-2 important? Why do we not want only one TF initiating IL-2 transcription. Ca2+ Ca2+ IL-2 NFAT NFAT

DAG PLCγ IL-2 NFAT NFAT CD4 LAT ZAP70 Lck SLP-76 GADS SLP-76 Lck Now back to DAG, the secondary messenger important for NFkb and AP1 TF activation IL-2 NFAT NFAT

DAG PKCθ PLCγ IL-2 NFAT NFAT CD4 LAT ZAP70 Lck SLP-76 CD3 CD3 PLCγ GADS SLP-76 Lck IL-2 NFAT NFAT

DAG PKCθ PLCγ IL-2 NFAT NFAT CD4 LAT ZAP70 Lck SLP-76 CARMA1 BcL10 MALT-1 PKCθ PLCγ ZAP70 GADS SLP-76 Lck Recruits PKC theta, phosphorylates CARMA, recruits BCL10,MALt-1 IL-2 NFAT NFAT

PKCθ IKKα/β/γ IκB NFκB NFAT NFAT CD4 DAG LAT CD3 CD3 CARMA1 BcL10 MALT-1 PKCθ PLCγ PLCγ IKKα/β/γ ZAP70 GADS SLP-76 Lck IκB NFκB IL-2 NFAT NFAT

PKCθ IKKα/β/γ IκB NFκB NFAT NFAT CD4 DAG LAT CD3 CD3 CARMA1 BcL10 MALT-1 PKCθ PLCγ PLCγ IKKα/β/γ ZAP70 GADS SLP-76 Lck IκB NFκB Ikbeta is phorphorylated and degraded, allowing NFkb transocation to the nucleus IL-2 NFAT NFAT

PKCθ IKKα/β/γ NFκB NFAT NFAT CD4 DAG LAT CD3 CD3 CARMA1 BcL10 MALT-1 PKCθ PLCγ PLCγ IKKα/β/γ ZAP70 GADS SLP-76 Lck NFκB IL-2 NFAT NFAT

PKCθ IKKα/β/γ NFAT NFκB NFAT NFkB CD4 DAG LAT CD3 CD3 CARMA1 BcL10 MALT-1 PKCθ PLCγ IKKα/β/γ ZAP70 GADS SLP-76 Lck IL-2 NFAT NFκB NFAT NFkB

DAG PLCγ IL-2 NFAT NFκB NFAT NFkB CD4 LAT ZAP70 Lck SLP-76 CD3 CD3 GADS SLP-76 Lck IL-2 NFAT NFκB NFAT NFkB

DAG PLCγ IL-2 NFAT NFκB NFAT NFkB CD4 LAT ZAP70 Lck SLP-76 CD3 CD3 Ras-GRP PLCγ ZAP70 GADS SLP-76 Lck IL-2 NFAT NFκB NFAT NFkB

DAG PLCγ IL-2 NFAT NFκB NFAT NFkB CD4 Ras LAT ZAP70 Lck SLP-76 CD3 CD3 Ras-GRP PLCγ PLCγ ZAP70 GADS SLP-76 Lck IL-2 NFAT NFκB NFAT NFkB

DAG PLCγ IL-2 NFAT NFκB NFAT NFkB CD4 Ras LAT ZAP70 Lck SLP-76 CD3 CD3 Ras-GRP PLCγ PLCγ ZAP70 GADS SLP-76 Lck IL-2 NFAT NFκB NFAT NFkB

DAG PLCγ IL-2 NFAT NFκB NFAT NFkB CD4 Ras LAT Raf ZAP70 Lck SLP-76 CD3 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck IL-2 NFAT NFκB NFAT NFkB

DAG PLCγ MEK IL-2 NFAT NFκB NFAT NFkB CD4 Ras LAT Raf ZAP70 Lck SLP-76 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck MEK IL-2 NFAT NFκB NFAT NFkB

MEK ERK NFAT NFκB NFAT NFkB CD4 DAG Ras LAT CD3 CD3 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck MEK ERK Ras – Raf – Mek – Erk: MAP kinase cascade IL-2 NFAT NFκB NFAT NFkB

MEK ERK NFAT NFκB NFAT NFkB CD4 DAG Ras LAT CD3 CD3 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck MEK ERK c-Fos is induced by transcriptional activation (not present in unstimulated cells) IL-2 c-Fos NFAT NFκB NFAT NFkB

MEK JNK NFAT NFκB NFAT NFkB CD4 DAG Ras LAT CD3 CD3 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck MEK JNK IL-2 c-Fos NFAT NFκB NFAT NFkB

MEK JNK NFAT NFκB NFAT NFkB CD4 DAG Ras LAT CD3 CD3 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck MEK JNK c-Jun is activated directly by MAPK phosphorylation c-Jun IL-2 c-Fos NFAT NFκB NFAT NFkB

MEK JNK NFAT NFκB NFAT NFkB AP-1 CD4 DAG Ras LAT CD3 CD3 Ras-GRP Raf PLCγ PLCγ ZAP70 GADS SLP-76 Lck MEK JNK IL-2 c-Fos c-Jun NFAT NFκB NFAT NFkB AP-1

PIP2 IL-2 NFAT NFkB AP-1 IP3 DAG DAG DAG PLCγ IP3 PKCθ IKKα/β/γ Itk Ca2+ Ca2+ CD4 Ca2+ Ca2+ PIP2 Ca2+ Ca2+ Ca2+ LAT DAG DAG DAG CD3 CD3 CRAC CRAC PLCγ CARMA1 BcL10 MALT-1 IP3 ZAP70 GADS Lck (Raf) PKCθ IKKα/β/γ SLP-76 Itk (MEK) Ras-GRP Calmodulin IκB Calcineurin IP3 Ca2+ Calmodulin NFκB ERK JNK Ca2+ c-Jun NFAT Ca2+ Ca2+ Ca2+ IL-2 c-Fos NFAT NFkB AP-1

All factors converge at the IL-2 promoter Coincidence Detection: Need signal integration of all factors acting upstream of IL-2 in order for transcription to occur (Protects against aberrant activation of IL-2)

handout?

II. B cell signaling

BCR and TCR share conserved signaling elements

Differences in signal initiation

III. T cell development

MHC molecule expression by cell type Professional APCs Discussed MHCII expression in professional APCs previously in the context of initiating adaptive responses to pathogenic infections. Now, focus on MHCII expression and function in thymic epithelial cells – this enables the presentation of self antigen during T cell maturation in order to select against autoreactive TCR clones.

FACS plots on right: CD4+CD8+ → CD4

Organization of thymus facilitates T cell development

TCRβ rearrangement occurs in: DN thymocytes TCRα rearrangement occurs in: DP thymocytes

1. Beta selection RAG genes turned on

2. Positive selection/Lineage commitment RAG genes turned on Upregulate CD4 Strong Weak Signal Strength CD4 T cell CD8 T cell

3. Negative selection

TCRβ rearrangement occurs in: DN thymocytes TCRα rearrangement occurs in: DP thymocytes

Selection by thymic epithelium Positive Selection MHC TECs Selection by thymic epithelium Positive Selection TCRa/B Positively Selected Death by Neglect Negative Selection Origin of these cells is extremely important in understanding bone marrow chimeras Self Peptide Selection by BM-derived APCs Mφ / DC Survive Autoreactive T cells

Irradiation kills BM-derived cells but not thymic epithelial cells MHCa/b MHCa/b MHCa/b Positive Selection

Irradiation kills BM-derived cells but not thymic epithelial cells MHCa/b MHCa/b MHCa/b Positive Selection MHCa/b MHCa MHCb BM-derived cells: MHC a/b Thymic epithelium: MHCa/b BM-derived cells: MHC a/b Thymic epithelium: MHCa BM-derived cells: MHC a/b Thymic epithelium: MHCb Postiive selction is intrinsic to the thymic environment because only the T-cells coming out of the mouse are MHCa T-cells

Irradiation kills BM-derived cells but not thymic epithelial cells MHCa/b MHCa/b MHCa/b Positive Selection MHCa/b MHCa MHCb BM-derived cells: MHC a/b Thymic epithelium: MHCa/b BM-derived cells: MHC a/b Thymic epithelium: MHCa BM-derived cells: MHC a/b Thymic epithelium: MHCb Postiive selction is intrinsic to the thymic environment because only the T-cells coming out of the mouse are MHCa T-cells Predominantly MHCa Restricted T cells Predominantly MHCb Restricted T cells Mix of MHCa and MHCb Restricted T cells

Negative Selection Graft Rejected Graft Tolerated MHCa/b MHCa IR IR MHCb IR IR MHCa MHCa Skin Graft delete any T-cell that is strongly reactive during neg selection, thus eliminating any possible self reactive T-cells Graft Rejected Graft Tolerated

Positive Selection MHCa presenting TECs MHCa restricted T cells Positively Selected Death by Neglect

Positive Selection Negative Selection MHCa presenting TECs MHCa restricted T cells Positively Selected Death by Neglect Negative Selection MHCa restricted T cells MHC a/b presenting APCs That present self-peptide A and B Survive MHCa restricted MHCb tolerized T cells Autoreactive T cells

Questions?