I. I. T Cells T Cells II. Invariant TCR T Cells II. Invariant TCR T Cells

T Cells in Autoimmune and T Cells in Autoimmune and Infectious DiseaseInfectious Disease

T cells can account for 30% of the T cell T cells can account for 30% of the T cell infiltrate in MS lesions (Wucherpfennig, K. W., infiltrate in MS lesions (Wucherpfennig, K. W., et. al. PNAS 89:4588). et. al. PNAS 89:4588).

T cells expand from 5 to 17% of PBMCs in T cells expand from 5 to 17% of PBMCs in patients during acute stages of patients during acute stages of P. Falciparum P. Falciparum (Ho, M. et. al. Infect Immun. 62: 855–862). (Ho, M. et. al. Infect Immun. 62: 855–862).

Similar Similar T cell expansion has been observed T cell expansion has been observed in in M. TuberculosisM. Tuberculosis infection and Chron’s infection and Chron’s disease. disease.

KO Mice Reveal a Unique role KO Mice Reveal a Unique role for Gamma Delta T Cells for Gamma Delta T Cells

Infections agents can be lethal Infections agents can be lethal

Gram-positive Gram-positive Nocardia asteroidesNocardia asteroides Causes Causes Lethal Infection in Lethal Infection in T Cell KO Mice T Cell KO Mice

Tam et. al., Infection & Immunity, 2001 69:6165.

-/- KO

Lung Sections of Mice Infected with Lung Sections of Mice Infected with Aerosolized Gram-positive Aerosolized Gram-positive N. asteroidesN. asteroides

Neutrophils in -/- mice

B6

N. asteroides (dark blue stain)

Tam et. al., Infection & Immunity, 2001 69:6165.

KO mice reveal a unique role for KO mice reveal a unique role for Gamma Delta T cells Gamma Delta T cells

Certain infections can be lethal.Certain infections can be lethal.

Most infections reveal inflammatory defects in Most infections reveal inflammatory defects in KO mice.KO mice.

Pathological outcome is different than that of Pathological outcome is different than that of KO mice—which usually die upon infectious KO mice—which usually die upon infectious challenge. challenge.

Skin wound healing is impaired.Skin wound healing is impaired.

T Cells Distribute Differently Then T Cells Distribute Differently Then T Cells T Cells

Anatomically located lining the epithelial tissues Anatomically located lining the epithelial tissues

Small numbers (1-2% of cells) found in the lymphoid Small numbers (1-2% of cells) found in the lymphoid tissues, spleen and lymph nodestissues, spleen and lymph nodes

might play a role early in the immune responsemight play a role early in the immune response

Respiratory Tract

Small Intestine (IELs)

Skin (V5 V1)

Reproductive Tract

What do Gamma Delta TCRs What do Gamma Delta TCRs recognize? recognize?

CDR3 Length Distribution of Immune Receptor Chains

B cell receptor

T cell receptor

T cell receptor

Ag

MHC

Ag ?

Ed Rock, et. al.

What do Gamma Delta TCRs What do Gamma Delta TCRs recognize? recognize?

MHC class II, IEMHC class II, IEkk..

LBK5* T cell clone recognizes backbone residues of class II IEk and recognition is independent of the presented peptide.

*LBK5 was generated from immunization with non-MHC matched splenocytes.

Important residues for LBK5 stimulation

Significance of IESignificance of IEk k Recognition? Recognition?

Unlike MHC restricted Unlike MHC restricted T cell clones, peptide T cell clones, peptide does not confer reactivity of LBK5 to IEdoes not confer reactivity of LBK5 to IEkk..

Gamma delta T cells recognize antigens in a Gamma delta T cells recognize antigens in a fundamentally different way than fundamentally different way than T cells. T cells.

Gamma delta development seems to be normal Gamma delta development seems to be normal in b2m-/- and MHC II negative mice, which in b2m-/- and MHC II negative mice, which suggest that the many of the suggest that the many of the TCR ligands are TCR ligands are not conventional MHC molecules. not conventional MHC molecules.

What do Gamma Delta TCRs What do Gamma Delta TCRs Recognize? Recognize?

MHC class II, IEMHC class II, IEkk

HSV-gI envelope proteinHSV-gI envelope protein

HSV-1

gI envelope protein is left on the cell surface

Viral DNA

TgI4* T Cell Clone is Stimulated by Plate Bound HSV-gI Protein

Sciammas, R. et. al. J. Exp. Med 185:1969

*TgI4 was recovered from mice inoculated (I.V.) with HSV-1.

Cell lysis blocked by -TCR and -gI mAbs.

Significance of HSV-gI Interaction? Significance of HSV-gI Interaction?

Like B cell Ig receptors, Like B cell Ig receptors, TCRs can TCRs can recognize protein directly without need for recognize protein directly without need for processing and presentation upon MHC processing and presentation upon MHC molecules. molecules.

HSV-1

gI envelope protein is left on the cell surface

Viral cDNAV

C

V

C

T Cell

TCR

TgI4

Cytolitic activity

What do Gamma Delta TCRs What do Gamma Delta TCRs Recognize? Recognize?

MHC class II, IEMHC class II, IEkk

gI HSV envelope proteingI HSV envelope protein

Non-classical MHC T22/T10Non-classical MHC T22/T10

T22 Tetramer Binds High Frequencies of T Cells

T22

tet

ram

er

TCR

Crowley, M. et. al. Science 2000, 287:314

•T22 can’t present antigens because of its ‘truncated’ MHC barrel.

•T22 is upregulated on APCs upon CFA immunization or LPS stimulation.

•Affinity measurements can be made between G8 TCR and T22.

Non-immunized mice

Significance of T22 Recognition? Significance of T22 Recognition?

Self or ‘natural’ ligandSelf or ‘natural’ ligand

This ligand is not recognized by This ligand is not recognized by TCRs, TCRs, suggesting that the suggesting that the TCR recognize TCR recognize non-overlapping ‘cues’ through the non-overlapping ‘cues’ through the TCR.TCR.

T cell Status of immune system Frequency1

T10/T22- specific T cells ~ 1/250

MHC/peptide specific T cells(not primed) ~ 1/1,000,000

MHC/peptide specific T cells(Immunized; effector phase) ~1/2-1/100

Significance of T22 Recognition?

High frequencies = fast immune responses to ligand

What do Gamma Delta TCRs What do Gamma Delta TCRs recognize? recognize?

MHC class II, IEMHC class II, IEkk

gI HSV envelope proteingI HSV envelope protein

Non-classical MHC T22/T10Non-classical MHC T22/T10

Small phosphate antigens Small phosphate antigens

Small Alkyl-Phosphate Antigens are Secreted by Bacteria Small Alkyl-Phosphate Antigens are Secreted by Bacteria and Also Expressed in Mammalian Tissues. and Also Expressed in Mammalian Tissues.

Small phosphate antigens stimulate Small phosphate antigens stimulate human and monkey Vhuman and monkey V2 V2 V2 T cells 2 T cells (Shen, Science, 295:2255-8). (Shen, Science, 295:2255-8).

VV2 V2 V2 T cells expand (15-60%) in 2 T cells expand (15-60%) in response to a variety of infectious response to a variety of infectious agents. agents.

Reactivity is mediated through the Reactivity is mediated through the TCR and the CDR3 is important for TCR and the CDR3 is important for reactivity (Bukowski, J. I. 161:286)reactivity (Bukowski, J. I. 161:286)

Cell contact is required (Morita, Cell contact is required (Morita, Immunity 3:495). Immunity 3:495).

No processing is required (fixed cells + No processing is required (fixed cells + small phosphate antigen = stimulation) small phosphate antigen = stimulation) (Morita, Immunity 3:495). (Morita, Immunity 3:495).

Significance of Small PhosphateSignificance of Small Phosphate TCR Recognition? TCR Recognition?

Ligands can either be self or foreign. Ligands can either be self or foreign.

Small phosphates are produced by the Small phosphates are produced by the nucleotide salvage pathway, during nucleotide salvage pathway, during cellular stress (i.e. heat shock, starvation, cellular stress (i.e. heat shock, starvation, etc.) (Constant, P. Science 264:267).etc.) (Constant, P. Science 264:267).

Suggesting that Suggesting that T cells monitor cellular T cells monitor cellular stress. stress.

Other Reports of Other Reports of TCR Ligands TCR Ligands

MICA – a non classical MHC that is upregulated MICA – a non classical MHC that is upregulated on tumor cells and upon heat shock (Wu, J. et. on tumor cells and upon heat shock (Wu, J. et. al. J. I. 169:1236). al. J. I. 169:1236).

CD1c- No addition of antigen is required (Spada CD1c- No addition of antigen is required (Spada F et. al. JEM March 2000).F et. al. JEM March 2000).

Hsp60 has been reported to be stimulatory.Hsp60 has been reported to be stimulatory.

Qa-1b can stimulate Qa-1b can stimulate T cells. T cells.

T Cell Summary T Cell Summary

T cells can recognize self (MHC and MHC-T cells can recognize self (MHC and MHC-like) and foreign ligands (HSV-gI, small like) and foreign ligands (HSV-gI, small phosphate antigens).phosphate antigens).

Many of the ligands are up regulated on the Many of the ligands are up regulated on the surface of cells upon infection or stress. surface of cells upon infection or stress.

Taken together Taken together T cells might ‘see’ cell surface T cells might ‘see’ cell surface perturbations in homeostasis through their TCR perturbations in homeostasis through their TCR and regulate the immune response. and regulate the immune response.

Cell

LPS

VC

VC

T Cell

Small phosphate antigens

‘Non-homeostatic’ Cell

T22

Viral derived surface Antigens

II. Invariant TCR T cells. II. Invariant TCR T cells.

Invariant TCR T cells clonally/oligoclonally Invariant TCR T cells clonally/oligoclonally express TCRs with a limited express TCRs with a limited P/N/Junctional CDR3 diversity.P/N/Junctional CDR3 diversity.

High frequencies are found in specific High frequencies are found in specific tissues. tissues.

NK T cells, DETC NK T cells, DETC T cells, MAIT cells. T cells, MAIT cells.

Invariant NK T cells (iNK T Invariant NK T cells (iNK T cells) cells)

Defined as being reactive to CD1d.Defined as being reactive to CD1d.

Express the invariant VExpress the invariant V and limited V and limited V TCRs. TCRs.

iNK T cells represent the majority of T cells iNK T cells represent the majority of T cells found in the liver.found in the liver.

1-3% found in the spleen.1-3% found in the spleen.

Express markers found on NK cells.Express markers found on NK cells.

V TCR sequences of CD1d restricted iNK T cells

Mouse V14 P/N J281TGT GTG GTG GGC GCA C C TGT GTA GAT AGA GGT TCA GCC

1 TGT GTG GTG GGC GAT AGA GGT TCA GCC

CVVG DRGSA2 TGT GTG GTG GG G GAT AGA GGT TCA GCC

CVVG DRGSA3 TGT GTG GTG GTA GAT AGA GGT TCA GCC

CVV VDRGSA4 TGT GTG GTG GG T GAC AGA GGT TCA GCC

CVVG D RGSA

Human V24 P/N JQ

CVVS DRGST

Lantz, O. and Bendelac, A., J. Exp. Med. 180:1097, 1994.

What do the iNK T cells recognize? What do the iNK T cells recognize?

CD1d plus…. CD1d plus….

An unidentified endogenous self-glycolipid antigen. An unidentified endogenous self-glycolipid antigen.

-GlcCer synthase KO APCs fail to stimulate galcer/CD1d1 restricted NK T cells (Stanic et al. PNAS, 2003).

•Selected by CD1, CD8, CD4, positive cells in the thymus (Nat Immunol. 2:971).

-Galactosylceramide and iNK T cells

• CD1 family of B2m dependent class Ib proteins that can present a variety of lipid antigens to CD1 restricted T cells.

• 80% of NK T cells (NK1.1+) in both mouse and human express an invariant TCR (mV14-J281, hV24-JQ) that recognize CD1d loaded with -GalCer.

• Mammalians do not synthesize -GalCer which is derived from sea sponges.

-carbon linkage

-Galcer is likely a structural mimic of a ‘self’ or ‘natural’ ligand.

iNK T cells are specific for CD1d1 iNK T cells are specific for CD1d1 -Galcer-Galcer

0.1 1 10 100 1000Fluor

0.1

1

10

100

1000

PhyEry

13.8 84.8

0.029 1.35

0.1 1 10 100 1000Fluor

0.1

1

10

100

1000

PhyEry

12.6 87.4

0 2.09e-3

Loaded with a-Galcer

CD

1d t

etra

mer

TCR

Not preloaded

iNK T cell functioniNK T cell function

Rapidly (within 1-2 hours) secrete IFN-Rapidly (within 1-2 hours) secrete IFN- and/or IL-4 upon stimulation and/or IL-4 upon stimulation in vivoin vivo (Yoshimoto T (Yoshimoto T J. Exp. Med.J. Exp. Med. 179:1285). 179:1285).

JJ281-/- mice lack iNK T cells and have 281-/- mice lack iNK T cells and have delayed immune responses to a variety of delayed immune responses to a variety of infectious agents. infectious agents.

Dendritic Epidermal Dendritic Epidermal T cells T cells (DETCs) promote wound healing (DETCs) promote wound healing

-/-

B6

Jameson et. al. Science 296:747

What is the DETC TCR ligand?What is the DETC TCR ligand?

We don’t know….We don’t know….

Heat shocked keratinocytes stimulate Heat shocked keratinocytes stimulate DETC cells in a TCR dependent fashion DETC cells in a TCR dependent fashion (Haravan, W. Science 252:1430).(Haravan, W. Science 252:1430).

This ligand is not sensitive to trypsin This ligand is not sensitive to trypsin treatment (unpublished results), therefore treatment (unpublished results), therefore is most likely not a protein. is most likely not a protein.

Cell

Conserved in Mouse and Man?

iTCR (mouse)

Development is dependent upon: Ligand Location

Auto-reactive

Negatively selected?

NK T YesVa14-Ja281

CD4+,CD8+, CD1+ Thymocytes

? (similar to a-Galcer) Liver, Spleen Yes

?(No in most models)

MAIT YesVa19-Ja33

B cells, MR1+, Microbial Flora ?

Gut Lamina Propria Yes ?

DETC No Vg5 Vd1

Conformationally dependent fetal TCR expression

? (not a protein) Skin Yes

? (Yes, in some models)

Table summarizing invariant T cell phenotypes

First line of Defense First line of Defense

Invariant and gamma delta T cells respond Invariant and gamma delta T cells respond quickly to antigenic stimulus. quickly to antigenic stimulus.

Should these T cells be classified as being Should these T cells be classified as being apart of the innate immune system or not? apart of the innate immune system or not?