360 Delivery Of Augmented Il-2 Receptor Signals To Cd81 T CellsReplaces The Requirement For Helper Function In TheGeneration Of Primary But Not Secondary Cd81 T CellResponses

L. E. Cheng1, P. D. Greenberg2; 1University of California, San Francisco,

San Francisco, CA, 2University of Washington, Seattle, WA.

RATIONALE: The induction of CD81 T cell responses to many antigens,

including the gag epitope of the FBL-3 erytholeukemia cell line (gagFBL),

requires CD41 T cell help (Th). One CD41 Th function thought to play a

major role in this process is the activation of antigen presenting cells (APC)

via CD40 ligation which leads to the upregulation of the costimulatory lig-

ands CD80/86.

METHODS: Since costimulation of CD81 T cells enhances production of

IL-2, we hypothesized that the augmentation of autocrine IL2R signalling

via a chimeric GM-CSF/IL2R in CD81 T cells might be sufficient to re-

place the requirement for CD41 Th in the generation of primary and sec-

ondary anti-gagFBL responses.

RESULTS: Immunization of mice possessing GMIL2R1CD81 T cells led

to the generation of gagFBL-specific CD81 T cells. This response was ab-

sent in wild-type controls. The GMIL2R increased the proliferation and

survivability of CD81 T cells in vivo. Despite generation of a primary re-

sponse, expression of the GMIL2R did not lead to memory responses upon

antigen rechallenge.

CONCLUSIONS: These data suggest that the provision of adequate IL2R

signals in the context of T cell activation may be all that is required to in-

duce a primary CD81 T cell response to CD41 Th-dependent antigens.

While provision of IL2R signals was sufficient to overcome the require-

ment for Th function during the primary response, additional Th functions

may be necessary for the generation of memory CD81 T cells.

Funding: NIH

361 Up-regulation of Granzyme B Gene Expression by ChemicalAllergens

R. J. Dearman, H. T. Caddick, C. J. Betts, I. Kimber; Syngenta CTL,

Macclesfield, UNITED KINGDOM.

RATIONALE: Selective T helper (Th) 1 and Th2 type responses are in-

duced following repeated topical exposure of BALB/c strain mice to chem-

ical contact and respiratory allergens, respectively. Gene expression

profiles following exposure of mice to the chemical respiratory allergen tri-

mellitic anhydride (TMA) have been analyzed using oligonucleotide mi-

croarrays. The most strongly up-regulated gene was the T cell cytolytic

molecule granzyme B. Changes in granzyme B expression in tissue iso-

lated from mice treated with TMA, or with the contact allergen dinitro-

chlorobenzene (DNCB), have been measured using real time PCR.

METHODS: Mice were exposed to 10% TMA, to 1% DNCB or to vehicle

alone on the dorsum of both ears. Draining lymph nodes were excised 24 to

120h later, total RNA prepared and changes in gene expression quantified

by real time PCR. Levels of granzyme B were normalized against the

housekeeping gene HRPT and expressed as fold changes relative to

naı̈ve (untreated) controls.

RESULTS: Treatment with TMA resulted in a marked increase in gran-

zyme B expression (fold changes of 5.71/-2.4, 20.71/-1.9 and 9.01/-

0.9 induced 48, 72 and 96h after exposure, respectively, n53). Less marked

changes were observed for DNCB-activated cells (fold changes of 3.511/-

0.1, 5.61/-0.7 and 3.111/-1.0 induced 48, 72 and 96h after exposure, re-

spectively). Less than 2fold changes were observed throughout the time

course for tissue derived from vehicle-treated animals.

CONCLUSIONS: Selective up-regulation of granzyme B gene expression

has been observed following exposure to the respiratory chemical allergen

TMA, consistent with recent observations that granzyme B may be the

preferential mechanism for Th2 cell death.

362 The MAb CHO-131 Identifies a Subset of CutaneousLymphocyte-Associated Antigen T cells Enriched in P-Selectin Binding Cells

Z. Ni; University of Minnesota, St. Paul, MN.

RATIONALE: T cells utilize the vascular adhesion molecules E-selectin

and P-selectin to enter inflamed skin. A phenotypic marker that directly

distinguishes human T cells enriched in P-selectin binding activity has

not been described. The mAb CHO-131 and P-selectin bind an identical

glycan moiety consisting of a sialylated and fucosylated oligosaccharide

properly positioned on a core-2 O-glycan. We examined whether CHO-

1311 T cells are enriched in P-selectin binding T cells and their effector

activities.

METHODS: A direct comparison of CHO-1311 and CHO-131- CLA1T

cells binding to E- and P-selectin, effector and central memory cell com-

position, and distribution in inflamed skin was performed.

RESULTS: CHO-131 stains a population of effector and memory of T

cells in the peripheral blood that represent a subset of cutaneous lympho-

cyte antigen (CLA1) T cells (; 46%), detected by the mAb HECA-452.

CHO-1311 CLA1 T cells revealed a significantly greater P-selectin, but

not E-selectin, binding activity, and they were considerably enriched in

psoriatic lesions. Unlike the peripheral blood of healthy individuals,

HECA-452 and CHO-131 stained a similar proportion of T cells in psori-

atic lesions, indicating a trafficking advantage by CHO-1311 T cells.

CONCLUSIONS: We conclude that the CHO-1311 T cell population is

enriched in P-selectin binding cells and at particular skin diseases. These

findings should provide new insights into the regulation and function of

skin homing T cells.

Funding: National Institutes of Health

363 IL-12/TGF-b1 Regulation of TCR Signal Transduction in HumanMemory T Cells Associated with Malignant and Non-malignant Chronic Inflammatory Microenvironments

L. Broderick, R. B. Bankert; University at Buffalo, Buffalo, NY.

RATIONALE: T cells persisting in the microenvironments of primary hu-

man non-small cell lung tumors, synovial fluid of rheumatic joints and na-

sal polyps demonstrate similar phenotypes identifying effector memory T

cells yet these cells remain unresponsive to the increasing tissue pathology.

METHODS: Effector memory T cells were isolated from the primary, hu-

man, non-small cell lung tumors, nasal polyps and synovial fluid of rheu-

matic joints and evaluated for their response to TCR stimulation via

CD31CD28 cross-linking by nuclear translocation of NF-kB and NFAT

under confocal microscopy. Response to pulsation with cytokines includ-

ing TNF-a and IL-12, and low pH elution of surface proteins was examined

to delineate the nature of the TCR anergy.

RESULTS: Effector memory T cells from these chronic inflammatory mi-

croenvironments were found to persist in a TGF-b1 -mediated anergic

state. Using IL-12, effector memory T cell anergy can be reversed both

in vitro and in vivo resulting in the activation, proliferation and T cell me-

diated eradication of tumor cells from the xenografts.

CONCLUSIONS: Effector memory T cells persist in chronic inflamma-

tory microenvironments of malignant and non-malignant tissues by a re-

versible, regulatory mechanism mediated by TGF-b1.

Funding: National Research Service Award Training Grant AIO7614

J ALLERGY CLIN IMMUNOL

JANUARY 2007

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