CD44high CD62Llow Research Articles

Use of dirty mice to study skin resident memory T cells in cancer treatments and graft-versus-host-disease

Abstract Graft versus host disease (GVHD) is a significant cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). While donor T cells have been shown to be instrumental in disease pathogenesis, prior studies of host T cells are limited, as host T cells are assumed to be depleted by conditioning (chemotherapy ± irradiation) used in HSCT. Recent data suggest that host skin resident memory T cells survive HSCT conditioning in humans and contribute to GVHD, but deeper mechanistic interrogation is necessary. Herein, we developed a novel mouse model using dirty mice to study host skin resident memory T cells in chemotherapy and GVHD. Laboratory mice cohoused with pet-shop mice developed a robust memory T cell population including CD44high CD62Lhigh central memory T cells, CD44high CD62Llow effector memory T cells, and CD44high CD62Llow CD69+ CD103+ and CD103− skin resident memory T cells of both CD4+ and CD8+ T cell subsets. Similarly to human data, both CD4+ and CD8+ skin resident memory T cells in dirty mice survived induction chemotherapy and HSCT conditioning while other T cell populations in blood, lymph node and spleen were depleted. In a reduced intensity conditioning chemotherapy-based fully mismatched model of GVHD, dirty mice developed higher GVHD score than clean control mice, with some dirty mice developing early vitiliginous skin change that was not observed in clean controls. Taken together, dirty mice appear to be an ideal model to test skin resident memory T cells in cancer treatments and GVHD, and more broadly in inflammatory skin diseases. Supported by grants from Brigham Research Institute (126409)

Ovalbumin and Poly(i:c) Encapsulated Dendritic Cell-Targeted Nanoparticles for Immune Activation in the Small Intestinal Lymphatic System.

Here, antigen and adjuvant encapsulated dendritic cell-targeted nanoparticles for immune activation in the small intestinal lymphatic system to inhibit melanoma development are described. This strategy is demonstrated using chondroitin sulfate-coated nanoparticles (OPGMN) grafted with glycocholic acid and mannose for cationic liposomes encapsulated with ovalbumin as an antigen and polyinosine-polycytidylic acid as a cancer-specific adjuvant. OPGMN is absorbed in the gastrointestinal tract and delivered to the lymph nodes when orally administered. Oral delivery of OPGMN induces increased dendritic cell maturation compared to the intradermal route in the lymph node and induces T helper type 1 and type 2 responses, such as immunoglobulin G1 and G2c, interferon-gamma, and interleukin-2, in the blood. Repeated oral administration of OPGMN increases the population of CD3+ CD8+ T cells, CD44high CD62Llow memory T cells, and CD11b+ CD27+ natural killer cells in the blood. OPGMN completely prevents melanoma development in the B16F10-bearing C57BL/6 mouse model by reducing the population of CD4+ CD25+ Foxp3+ regulatory T cells in the blood. This strategy is expected to prevent the recurrence of tumors after various cancer treatments.

Encapsulation of Recombinant MOMP in Extended-Releasing PLGA 85:15 Nanoparticles Confer Protective Immunity Against a Chlamydia muridarum Genital Challenge and Re-Challenge.

Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.

CD153 VACCINE IS A NOVEL SENOTHERAPEUTIC OPTION FOR REMOVING SENESCENCE-ASSOCIATED T CELLS

Objective: Senotherapy targeting for senescent cells is designed to attenuate age-related dysfunction and promote healthy aging. Senescence-associated T cells (SA-T cells), defined as CD4+ CD44high CD62Llow PD-1+ CD153+ cells, accumulate in visceral adipose tissues (VAT) in obese individuals. Here, we show the long-lasting effect of using CD153 vaccination to remove SA-T cells from high-fat diet (HFD)-induced obese C57BL/6J mice. Design and method: The peptide vaccines for mouse CD153 consisted of a short peptide sequence conjugated to KLH (keyhole limpet hemocyanin) as a carrier protein to induce the specific CD153 antibody, and knowing the CD153 epitope information allowed five candidate peptide to be selected as antigens. The five peptide vaccines (30 microg of the CD153 peptide per mouse) were conjugated to KLH and coadministered with the Alum adjuvant (Alhydrogel) to seven-week-old male C57BL/6J mice twice at two-week intervals. CD153#D vaccine-induced antibodies effectively recognize the rmCD153 protein, and we selected the CD153#D vaccine as a candidate for further experimental studies. Results: The number of CD153+ SA-T cells increases gradually and systemically with age. Indeed, more CD153+ SA-T cells were observed in the splenic tissues of male C57BL/6J mice at 20 months of age than at 3 months of age (18.7 ± 0.5% vs. 3.8 ± 0.2%; p < 0.0001; n = 3 in each group). In VAT (viceral adipose tissue), CD153+ SA-T cells tended to increase more in elderly mice than in young mice (8.2 ± 1.0% vs. 4.3 ± 1.4%; p = 0.08; n = 3 in each group). We administered a CD153 peptide-KLH conjugate vaccine with Alhydrogel (CD153-Alum) or CpG oligodeoxynucleotide (ODN) 1585 (CD153-CpG) and confirmed an increase in anti-CD153 antibody levels that was sustained for several months. After being fed a HFD for 10–11 weeks, adipose SA-T cell accumulation was significantly reduced in the VAT of CD153-CpG-vaccinated mice, accompanied by glucose tolerance and insulin resistance. A complement-dependent cytotoxicity (CDC) assay indicated that the mouse IgG2 antibody produced in the CD153-CpG-vaccinated mice successfully reduced the number of SA-T cells. Conclusions: These results suggest that the CD153-CpG vaccine might be an optional tool for senolytic therapy.

PEGylated nano-Rehmannia glutinosa polysaccharide induces potent adaptive immunity against Bordetella bronchiseptica

Vaccines, in many cases, stimulate only too weak immunogenicity to prevent infection. Therefore, adjuvants are required during their preparation to boost the immune response. We herein developed a PEGylated nano-adjuvant based on Rehmannia glutinosa polysaccharide (RGP). The addition of PEG layer exhibits enhanced immune performance of the nano-RGP. Stimulation of dendritic cells (DCs) with PEGylated nano-RGP (pRL) led to increased proliferation and cytokine production (IL-6, IL-12, IL-1β and TNF-α). The pRL was internalized into DCs via a rapid and efficient method. The mice immunized with pRL exhibited enhanced antigen-specific serum IgG and Th1-(IFN-γ), Th2-(IL-4), and Th17-(IL-17, IL-6) cytokine production, contributing to a good anti-infection performance. Furthermore, the pRL could effectively deliver the antigen to the lymph nodes (LNs), activate DC in the LN and produce enhanced CD4+and CD8+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) as well as functional phenotypes. Our results revealed that pRL can act as a promising adjuvant with targeted delivery of antigen due to its effective activation and robust adaptive immunity induction of DCs.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

The CD153 vaccine is a senotherapeutic option for preventing the accumulation of senescent T cells in mice

Senotherapy targeting for senescent cells is designed to attenuate age-related dysfunction. Senescent T cells, defined as CD4+ CD44high CD62Llow PD-1+ CD153+ cells, accumulate in visceral adipose tissues (VAT) in obese individuals. Here, we show the long-lasting effect of using CD153 vaccination to remove senescent T cells from high-fat diet (HFD)-induced obese C57BL/6J mice. We administered a CD153 peptide-KLH (keyhole limpet hemocyanin) conjugate vaccine with Alhydrogel (CD153-Alum) or CpG oligodeoxynucleotide (ODN) 1585 (CD153-CpG) and confirmed an increase in anti-CD153 antibody levels that was sustained for several months. After being fed a HFD for 10–11 weeks, adipose senescent T cell accumulation was significantly reduced in the VAT of CD153-CpG-vaccinated mice, accompanied by glucose tolerance and insulin resistance. A complement-dependent cytotoxicity (CDC) assay indicated that the mouse IgG2 antibody produced in the CD153-CpG-vaccinated mice successfully reduced the number of senescent T cells. The CD153-CpG vaccine is an optional tool for senolytic therapy.

PD-1 inhibitors dependent CD8+ T cells inhibit mouse colon cancer cell metastasis.

BackgroundColon cancer is a common digestive tract malignancy which ranks as the third leading cause of cancer death worldwide. A current focus of anti-cancer research is harnessing the patient’s own immune system for therapy. Programmed cell death protein 1 (PD-1), an immune suppressor, is upregulated in various activated immune cells, such as T cells, and in viral infections and tumors.PurposeThe objective of this study was to investigate the function of PD-1 inhibitor on the metastasisi of mouse colon cancer cells.Patients and methodsIn the present study, we established an in situ colon cancer mouse model using the CT26 cell line. Hematoxylin-eosin (HE) staining was performed to detect colon cancer cell metastasis. The levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) in serum and mesenteric lymph nodes (MLNs) were detected by Enzyme-linked immunosorbent assay (ELISA). CD44high CD62Llow memory T cells, CD4+ FoxP3+ regulatory T cells, and IFN-γ and TNF-α levels in MLNs and spleen were detected by flow cytometry (FCM).ResultsWe found that anti-PD-1 therapy inhibited colon cancer cells metastasis to the small intestine, liver, and lung, and lengthened the survival time of mice. However, the depletion of CD8 suppressed the activity of anti-PD-1 antibodies. In response to anti-PD-1 immunotherapy, the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) in serum and mesenteric lymph nodes (MLNs) were significantly increased, while IL-6, IL-17, and transforming growth factor-β (TGF-β) were decreased. CD8 depletion had the opposite effect. In addition, anti-PD-1 treatment significantly increased CD44high CD62Llow memory T cells, decreased CD4+ FoxP3+ regulatory T cells, and increased IFN-γ and TNF-α levels in MLNs and spleen. Furthermore, anti-PD-1 treatment cannot exert these roles when CD8 is depleted.ConclusionThese results suggest that PD-1 inhibitors rely on CD8+ T cells to exert anti-tumor immunity in colon cancer.

The Chlamydia M278 Major Outer Membrane Peptide Encapsulated in the Poly(lactic acid)-Poly(ethylene glycol) Nanoparticulate Self-Adjuvanting Delivery System Protects Mice Against a Chlamydia muridarum Genital Tract Challenge by Stimulating Robust Systemic and Local Mucosal Immune Responses.

Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge.

SOCS3 treatment prevents the development of alopecia areata by inhibiting CD8+ T cell-mediated autoimmune destruction.

Alopecia areata is one of the most common autoimmune diseases resulting from T cell-mediated damage of hair follicles. CD8+ T cells infiltrate hair follicles and are responsible for destruction of hair follicles. However the underlying mechanisms for hair loss remain still obscure. In the present study, we identified that suppressor of cytokine signaling-3 (SOCS3), a classical inhibitor of cytokine signaling, significantly inhibits CD8+T cell maturation, interferon-γ (IFN-γ) production and alopecia areata. SOCS3 is downregulated in the skin of alopecia areata patients and murine autoimmune alopecia model. Furthermore, SOCS3 treatment prevents the development of alopecia areata in the graft model. SOCS3 decreases the CD44high CD62Llow effector memory CD8+ T cells, resulting in the decrease of IFN-γ production. The expression of Fas and major histocompatibility complex-1 (MHC I) is upregulated in skin from C3H/HeJ alopecia areata mice, and this increase is suppressed by SOCS3. The SOCS3 level is negative correlation with the Fas and MHC I level in patients with alopecia areata. These results suggest that SOCS3 treatment may be an effective strategy to treat autoimmune alopecia as well as to more generally prevent cytokine-dependent tissue destruction in inflammatory diseases.

Interferon-Gamma Signaling in the Pathogenesis of Idiopathic Pneumonia Syndrome Following Allogeneic Bone Marrow Transplantation

Idiopathic pneumonia syndrome (IPS) is a life-threatening acute noninfectious diffuse lung injury occurring after allogeneic bone marrow transplantation (BMT). We have recently shown that absence of STAT1 signaling in recipient APC leads to accelerated Graft Versus Host Disease (GVHD) and Transplant-associated mortality and morbidity after lethal irradiation in fully MHC-mismatched allogeneic or in non-irradiated P→F1 BMT models. We now addressed the impact of recipient Type II interferon signaling on the development of lung injury after allogeneic BMT using a non-irradiated P→F1 model. 1x107 BMCs plus 2.5x107 SPCs from C57BL/6 (B6) mice were i.v. injected into IFNγR-deficient CB6F1 (F1.GRKO) or CB6F1 wild type (WT) mice. F1.GRKO recipients had significantly reduced survival (MST 32 days post-BMT vs. not reached p<0.001, Log-rank test). Pulmonary function tests were performed 3 weeks post-BMT on anaesthetized mice that were tracheotomized and mechanically ventilated using a computer-controlled animal ventilator (Flexivent, SCIREQ). Static compliance was derived from plateau pressure measurements between total lung capacity and functional residual capacity, and broadband forced oscillations were used to determine airway resistance at baseline and following increasing doses of methacholine (3.1, 12.5 and 50 mg/ml). Static compliance and inspiratory capacity were reduced in F1.GRKO recipients when compared with WT mice (p<0.01). In addition, airway resistance, both at baseline and in response to methacholine, was increased in the KO recipient mice (p<0.05). Histopathology was evaluated on H&E, Trichrome and PAS stained lung sections to assess for inflammation, fibrosis and mucus hyperplasia, respectively. Lungs from the KO mice showed increased interstitial inflammation involving bronchioles, vasculature and parenchyma (pathology index 10 vs 1; p<0.001), fibrosis (fibrosis score 2.8 vs 0.15; p<0.05) and a trend for mucus hyperplasia/metaplasia. To further evaluate the cellular mediators of the observed lung injury, we assessed the cellular composition of the bronchioalveolar lavage fluid (BAL) at day 30 post-BMT. We observed a 10-fold increase in the total cell number of the BAL in F1.GRKO recipient mice compared to WT mice (4x105 vs.4x104, p<0.01); the majority of the cells in the BAL from wild type mice were macrophages/monocytes, while neutrophils predominated in the F1.GRKO mice. Importantly, more than 50% of the CD45+ BAL cells were donor derived in the F1.GRKO recipients, compared to only 10% in the wild type recipients. Furthermore, the absolute cell number of donor CD4+ and CD8+ cells in the knockout mice significantly outnumbered that in their wild type counterparts (p<0.01). Thus, our results suggest that absence of recipient IFN-γR signaling promotes accumulation and/or trafficking of donor neutrophils and T cells to the lungs. To better understand the underlying mechanisms of enhanced infiltration of donor cells in the lungs of IFNγR-/- mice, the activation, proliferation, and differentiation status of donor lymphocytes were assessed by Flowcytometry after BMT. In line with the lung infiltration, there was significantly increased donor cell engraftment in PBMC, spleen, lymph nodes, and bone marrow in F1.GRKO recipients (all with p<0.01); Moreover, there were also significantly increased activation (measured as CD44high CD62Llow cells), Th1 and Tc1 differentiation of donor CD4 and CD8 cells (p<0.01). Furthermore, enhanced Th17 differentiation, but reduced Foxp3+ Treg cell differentiation was noticed. Interestigly, we observed significantly lower PD-L1 expression on recipient APC in the F1.GRKO mice compared to wild type mice, suggesting that absence of PD-L1 expression may contribute to reduced contraction of activated donor T cells. Interestingly, in line with these findings we also observed significantly reduced PD-L1 expression when single cell suspension from lung tissues were analyzed by flow. Reduced PD-L1 expression was observed on both recipient CD45+, and CD45- cells in F1.GRKO when tested on day 22 post-BMT, meanwhile there was no difference in PD-L1 expression pattern in the cells isolated from small intestine. These results suggest that IFN-γ induced PD-L1 expression on hematopoietic and non-hematopoietic cells in the lung may be of significance in protecting recipients against acute donor-cell mediated lung injury and IPS. DisclosuresLentzsch:Celgene: Membership on an entity's Board of Directors or advisory committees; Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

PD-1 Signaling Has a Critical Role in Maintaining Regulatory T Cell Homeostasis; Implication for Treg Depletion Therapy By PD-1 Blockade

CD4+ Foxp3+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance. In the setting of allogeneic hematopoietic stem cell transplantation (HSCT), Treg recovery after transplant has been reported as a critical factor to suppress the onset of graft-versus-host disease (GVHD) and stabilize the immune condition. We previously demonstrated that low-dose IL-2 administration preferentially increased Treg in patients with active chronic GVHD and resulted in clinical improvement of the symptoms (NEJM 2011). In the clinical trial, IL-2 induced selective and rapid proliferation of Treg in the first week of therapy but proliferation subsequently returned to baseline levels despite continued daily administration of IL-2 (Sci Trans Med 2013). By detailed analysis of lymphocytes from patients receiving IL-2 therapy, we found that inhibitory molecule Programmed death-1 (PD-1) expression rapidly increased selectively on Treg 2 weeks after starting IL-2, suggesting PD-1 may work to suppress the excessive proliferation of Treg and stabilize homeostasis of this subset during IL-2 therapy (ASH 2014). However, the cellular mechanisms of inhibition of Treg proliferation to maintain Treg homeostasis has not been characterized. To tackle the issue, we here studied the impact and role of PD-1 expression on Treg by using murine IL-2 therapy model. B6 mice were administrated 5x103 IU IL-2 once a day for 14 days and CD4+CD25+Foxp3+ Treg were analyzed comparing with CD4+CD25-Foxp3- conventional CD4 T cells (Tcon) and CD8+ T cells. These subsets were further divided into subpopulations by the expression of CD44 and CD62L. The expressions of pSTAT5, Ki-67, Bcl-2, Fas, PD-1, CTLA-4, LAG3 and TIM-3 in each subset were also examined. As in human, low-dose IL-2 rapidly enhanced PD-1 on Treg, but not other inhibitory molecules including CTLA-4, LAG-3 and TIM-3. Low-dose IL-2 did not affect the expression level of any inhibitory molecules on Tcon and CD8T cells. To clarify the role of PD-1 expression on Treg, we compared PD-1-/- B6 with wild-type B6. PD-1-/- mice were administrated 5x103 IU IL-2 once a day for 28 days. In the first 7 days, IL-2 induced significantly higher levels of pSTAT5 expression in Treg in PD-1-/- cohort than WT cohort (MFI of pSTAT5; mean 4.8 vs 4.0, p<0.01). This resulted in the stronger proliferation and expansion of Treg in PD-1-/- cohort in the first week of IL-2. However, after 7 days, IL-2 expanded PD-1-/- Treg rapidly decreased and returned to baseline level, while IL-2 treated wild type Treg maintained to increase during the 4 weeks. We confirmed this phenomenon by another experimental system using the combination of low-dose IL-2 and anti-PD-1 antibody, and again found that the initial strong proliferation by IL-2 was just temporally and followed by the collapse of Treg homeostasis. To reveal the mechanism of Treg decrease in the absence of PD-1 signal, we assessed Annexin-V+ cells to detect apoptotic cells at day15. IL-2 treated PD-1-/- Treg showed significantly higher level of Annexin-V+ cells than WT Treg (mean 10.5% vs 4.6%, p<0.01). IL-2 treated WT Treg were predominantly CD44high CD62Lhigh central memory phenotype and this phenotype showed increase of PD-1 expression (%PD-1+ cells; mean 44.6% vs 31.7%, vs control, p<0.0001), in contrast, IL-2 treated PD-1-/- Treg showed the accumulation of CD44high CD62Llow effector memory phenotype, which showed high positivity of Annexin-V (mean 15.4% vs 6.4%, p<0.01). These data suggested that PD-1 modulates subset-balance of Treg and inhibits activation-induced-cell death (AICD). We also examined the expression of surface death receptor Fas and intracellular anti-apoptic protein Bcl-2 at day15 to investigate the pathway of apoptosis by lacking of PD-1 signal. IL-2 treated PD-1-/- Treg showed higher increase of Fas expression (mean MFI, 1.5 vs 1.1, p<0.05) and decrease of Bcl-2 expression (mean MFI, 2.6 vs 3.4, p<0.05) than IL-2 treated WT Treg after IL-2 therapy, suggesting that Fas and Bcl-2 might participate in the regulation of apoptosis. In conclusion, our findings indicate that the PD-1 pathway can limit over activation and proliferation of Treg and prevent apoptosis via Fas and Bcl-2 activity during IL-2 therapy. Our finding may implicate the development of Treg depletion therapy by blocking PD-1 signaling. DisclosuresNo relevant conflicts of interest to declare.

Study on the phenotype and the function of CD8αα＋TCRαβ＋regulatory T cells

Objective To investigate the phenotype and the immunoregulatory function of CD8αα＋TCRαβ＋regulatory T cells in peripheral blood samples from mice.Methods The distribution profile and the phenotype of CD8αα＋TCRαβ＋regulatory T cells in C57BL/6 mice were detected by flow cytometry.The cytokines released by CD8αα＋TCRαβ＋regulatory T cells upon the stimulation with anti-CD3 antibody were analyzed by cytometric bead array.The in vitro immunosuppressive activity of CD8αα＋TCRαβ＋regulatory T cells on activated CD4＋T cells was analyzed by using flow cytometry and carboxyfluorescein succinimidyl ester （ CFSE ） .An adoptive cell transfer assay was set up to evaluate the immunoprotective effects of CD8αα＋TCRαβ＋ regulatory T cells in a mouse model of experimental autoimmune encephalomyelitis （ EAE） .Results CD8αα＋TCRαβ＋regulatory T cells were detected in liver, spleen and peripheral blood samples collected from na？ve C57BL/6 mice.Compared with CD8αβ＋TCRαβ＋regulatory T cells, CD8αα＋TCRαβ＋regulatory T cells showed a memory-activated phenotype of CD25＋CD122high CD44high CD62Llow CD69high NK1.1＋DX5＋.CD8αα＋TCRαβ＋regulatory T cells could produce IL-2 after 24 hours stimulation with anti-CD3 antibody, followed by producing IFN-γ, TNF-α, IL-4, IL-17A and traces of IL-6 and IL-10. In vitro, CD8αα＋TCRαβ＋regulatory T cells specifically suppressed the proliferation of activated CD4＋T cells （ P〈0.01 ）.Moreover, they could delay the onset of EAE in mice and reduce clinical score （P〈0.01）.Conclusion CD8αα＋TCRαβ＋regulatory T cells were a unique population with immunoregula-tory function, which could be used as a potential therapeutic target in the treatment of autoimmune disease. Key words: CD8＋regulatory T cell Phenotype Immunosuppression EAE

Elimination of Progressive Mammary Cancer by Repeated Administrations of Chimeric Antigen Receptor-Modified T Cells

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

Vitamin C treatment of mouse bone marrow-derived dendritic cells enhanced CD8+ memory T cell production capacity of these cells in vivo

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4+ T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8+ T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ+ CD8+ T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44high CD62Llow CD8+ effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors.

Abstract 240: A Potential Role of Memory T cells in Hypertension

Immunological memory is an important component of the adaptive immune response to pathogenic stimuli. Effector memory T cells, which are CD44high CD62Llow, reside in various non-lymphoid tissues and serve as a first line defense against foreign antigens. We have previously shown that T cells are important in hypertension, but the role of memory T cells has not been defined. Thus we hypothesized that CD8+ memory T cells are component of this memory response. To test this hypothesis, mice initially received two weeks of ang II, were allowed to recover for 3 weeks and were then re-infused with low dose of ang II (140 ng/kg/min) that minimally raises blood pressure in naïve mice. This low-dose of ang II increased blood pressure to 137±6 mmHg in mice that had previously received a vehicle infusion, but to 172±12 mmHg in mice that had received ang II. In keeping with a memory T cell response, we found that angiotensin II causes a 5 to 10-fold increase in CD8+ CD62Lhigh/CD44high/CCR7+ central memory cells in the kidney while reducing these effector memory CD8+ T cells in the spleen. CD8+ memory T cells require co-stimulatory signaling between CD70 on macrophages and CD27 on T cells. We found that angiotensin II infusion increased CD70 mRNA in isolated kidney vessels by 5 to 10-fold. Similarly, flow cytometry revealed that angiotensin II increased macrophages expressing CD70 and CD8+ T cells expressing CD27 expression in the kidney. Thus, these studies indicate that repeated exposures to angiotensin II promote an immune memory response of CD8+ T cells and markedly enhance the hypertensive response to this octapeptide. The role of CD27 and CD70 signaling requires additional study but might serve as a therapeutic target.

Polyanhydride particle vaccine platform enhances early antigen-specific cytotoxic T cell responses (P4366)

Abstract Bioerodible nanoparticles have been shown to be useful as vaccine delivery vehicles. Previous work from this laboratory has shown that polyanhydride nanoparticles (NPs) can be used as an effective platform delivery system with the ability to modulate the release kinetics and to physicochemically stabilize protein antigens, activate dendritic cells, and elicit protective immunity to pathogens. The goal of this project was to evaluate the ability of the polyanhydride NPs to enhance cell-mediate immunity. Using a high frequency adoptive transfer model employing 3 x 106 OT I CD8+ T cells, we demonstrated significant expansion of ovalbumin (Ova)-specific CD8+ T cells that expressed an effector memory phenotype (CD44high CD62Llow) following immunization with Ova-loaded NPs. Induction of these effector CD8+ T cells resulted in the development of fewer and smaller tumors after implantation of EG7 tumor cells compared to mice immunized with Alum plus Ova or soluble Ova alone. To better model the naturally occurring low frequency of naïve CD8+ T cells, 3,000 OT I T cells were adoptively transferred prior to immunizing mice with one of three separate polyanhydride NP formulations (20:80 CPH:SA, 20:80 CPTEG:CPH, or 50:50 CPTEG:CPH). The results of these studies demonstrated that immunization with biocompatible and biodegradable 20:80 CPH:SA polyanhydride NPs effectively enhanced the induction of antigen-specific cytotoxic CD8+ T cells.

CD154 blockade alters innate immune cell recruitment and programs alloreactive CD8+ T cells into KLRG-1(high) short-lived effector T cells.

CD154/CD40 blockade combined with donor specific transfusion remains one of the most effective therapies in prolonging allograft survival. Despite this, the mechanisms by which these pathways synergize to prevent rejection are not completely understood. Utilizing a BALB/c (H2-Kd) to B6 (H2-Kb) fully allogeneic skin transplant model system, we performed a detailed longitudinal analysis of the kinetics and magnitude of CD8+ T cell expansion and differentiation in the presence of CD154/CD40 pathway blockade. Results demonstrated that treatment with anti-CD154 vs. DST had distinct and opposing effects on activated CD44high CD62Llow CD8+ T cells in skin graft recipients. Specifically, CD154 blockade delayed alloreactive CD8+ T cell responses, while DST accelerated them. DST inhibited the differentiation of alloreactive CD8+ T cells into multi-cytokine producing effectors, while CD40/CD154 blockade led to the diminution of the KLRG-1low long-lived memory precursor population compared with either untreated or DST treated animals. Moreover, only CD154 blockade effectively inhibited CXCL1 expression and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the absolute number of IFN-γ producing alloreactive CD8+ T cells, and intra-graft expression of inflammatory chemokines. These findings demonstrate that the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell expansion and differentiation and inhibited development of KLRG-1low memory precursor cells.

Most parasite-specific CD8+ cells in Trypanosoma cruzi-infected chronic mice are down-regulated for T-cell receptor-alphabeta and CD8 molecules.

The present study shows that CD8+ T lymphocytes expressing low levels of T-cell receptor (TCR)alphabeta, CD8 and CD3 accumulate in the spleen, blood, peritoneum and liver, but not in the lymph nodes of mice chronically infected with Trypanosoma cruzi. Analysis of spleen lymphocytes reveals that most CD8LOW TCRLOW T cells have an experienced phenotype (CD44HIGH CD62LLOW and CD45RA,B,CLOW). These cells have small size, lack activation markers such as CD69, CD25 and CD11b (Mac-1), and do not spontaneously secrete cytokines, suggesting they are at the resting state. When stimulated in vitro with T. cruzi-infected macrophages, TCRLOW CD8LOW T cells behave as parasite-specific memory cells, readily responding with interferon-gamma (IFN-gamma) production. Indeed, among parasite-activated CD8+ lymphocytes, IFN-gamma production was mostly due to TCRLOW CD8LOW cells. Upon in vitro stimulation with anti-CD3/CD28 monoclonal antibodies, down-regulated cells produce IFN-gamma and tumour necrosis factor-alpha, but not interleukin IL-10 or IL-4. Our results indicate that despite parasite persistence, most T. cruzi-specific experienced CD8+ cells are resting. Nevertheless, when encountering infected macrophages these cells differentiate to Tc1 effectors.