OP0042 BLOCKING OF CD103+ TISSUE RESIDENT MEMORY T CELLS (TRM) AS A THERAPEUTIC STRATEGY IN SJOGREN’S SYNDROME

Background:Tissue-resident memory T cells (TRM), are a recently identified T cells population featuring tissue localization and expression of markers of tissue homing, CD69 and CD103. Recently, the expansion of CD8+ TRMs and their involvement in the sialadenitis was described in a murine model of SS. However, CD4+ and CD8+ TRM’s functional relevance in pSS is still not fully understood, and the TRM therapeutic targeting unexplored.Objectives:The study aimed to address the role of CD4+ and CD8+ TRMs in the pathogenesis of pSS and to explore the therapeutic targeting of the tissue residency marker of TRM CD103.Methods:An animal model of experimental (ESS) obtained by immunization of female C57BL/6 mice (n=10) with salivary glands (SG) protein extract and Freund’s complete adjuvant used to investigate the dynamic of infiltration of SG by CD4+ and CD8+ TRMs, their frequency, and the impact of CD103 blockade. For the therapeutic intervention, at 10-weeks post-immunization, the salivary gland was cannulated via Wharton’s duct, and an anti-CD103 neutralizing antibody or vehicle-injected. The mice’s saliva flow rate was assessed, and SGs were analyzed by Flow-cytometry and immunohistochemistry (IHC).The frequency and localization of TRMs was analyzed in minor SG of sicca syndrome (nSS) and pSS patients (n=39) by flow cytometry and IHC. The expression of genes involved in the tissue retention of TRMs was assessed in SG by RT-PCR.Results:Upon the ESS progression, a significant progressive increase in CD45+CD103+ cells frequency was observed from 5wk to 20wk post-immunization (p<0.001), where the CD8+ were the most abundant, followed by CD4+. Consistently, CD103+CD8+ T cells were detected within the lymphocytic infiltration of SG from ESS mice. Sorted purified SG CD10+CD3+CD8+ T cells showed higher Granzyme B, TNF-alpha expression compared to CD103-CD3+CD8+ at both mRNA and protein levels. Notably, ESS mice treated with anti-CD103 showed improvement in salivary function (p<0.05) and reduced lymphocytic infiltrations measured as focus score (FS) (p<0.01) and area-fraction (p<0.01). Consistently, anti-CD103 treatment consistently reduced CD103+ cells and IFN-gamma+, Granzyme B+, and TNFa+ CD8+ cells. We next performed phenotypic analysis of CD45+CD103+ immune cells in the SG of pSS patients observing an increase in both with CD8+CD103+CD69+ and CD4+CD103+CD69+ (p<0.05). Finally, IHC showed that the expansion of TRMs in pSS salivary glands was accompanied by a down-regulation of E-cadherin glandular expression and their migration outside the epithelium in the context of inflammatory infiltrates. SG of patients with pSS showed a significant up-regulation of BLIMP1, KFL-2, and S1PR1 and down-regulation of ITGB2. CXCL9 and CXCL10, and IL-15 involved in the tissue recruitment and long-term survival of TRMs were significantly modulated in pSS salivary glands.Conclusion:TRM are expanded and activated in the SG of pSS and ESS, participating in the organization of tissue inflammation. Although the mechanisms behind this expansion are still not fully understood, CD103 could be a valuable novel therapeutic target to prevent lymphocytic infiltrations and glandular destruction in Sjogren syndrome.Disclosure of Interests:None declared

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Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽

10.1182/blood-2018-99-115638 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2952-2952

Author(s):

Takahiro Miyazaki ◽

Peiwen Kuo ◽

Mekhala Maiti ◽

Palakshi Obalapur ◽

Murali Addepalli ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cynomolgus Monkey ◽

Memory T Cells ◽

Granzyme B ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

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Retrograde migration supplies resident memory T cells to lung-draining LN after influenza infection

Journal of Experimental Medicine ◽

10.1084/jem.20192197 ◽

2020 ◽

Vol 217 (8) ◽

Cited By ~ 4

Author(s):

J. Michael Stolley ◽

Timothy S. Johnston ◽

Andrew G. Soerens ◽

Lalit K. Beura ◽

Pamela C. Rosato ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Memory T Cells ◽

Influenza Infection ◽

Granzyme B ◽

Lymphatic Vessels ◽

Cd8 T Cell ◽

Lung Infection ◽

Cell Responses ◽

Resident Memory T Cells

Numerous observations indicate that resident memory T cells (TRM) undergo unusually rapid attrition within the lung. Here we demonstrate that contraction of lung CD8+ T cell responses after influenza infection is contemporized with egress of CD69+/CD103+ CD8+ T cells to the draining mediastinal LN via the lymphatic vessels, which we term retrograde migration. Cells within the draining LN retained canonical markers of lung TRM, including CD103 and CD69, lacked Ly6C expression (also a feature of lung TRM), maintained granzyme B expression, and did not equilibrate among immunized parabiotic mice. Investigations of bystander infection or removal of the TCR from established memory cells revealed that the induction of the TRM phenotype was dependent on antigen recognition; however, maintenance was independent. Thus, local lung infection induces CD8+ T cells with a TRM phenotype that nevertheless undergo retrograde migration, yet remain durably committed to the residency program within the draining LN, where they provide longer-lived regional memory while chronicling previous upstream antigen experiences.

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