Augmented T-cell Proliferation Research Articles

Targeted inhibition of glycogen synthase kinase-3 using 9-ING-41 (elraglusib) enhances CD8 T-cell-reactivity against neuroblastoma cells

The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3β) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3β might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3β using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3β levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3β inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.

Pharmacologic Properties and Preclinical Activity of Sasanlimab, A High-affinity Engineered Anti-Human PD-1 Antibody.

Development of antagonistic mAbs that specifically target the immune checkpoint receptor, programmed cell death protein-1 (PD-1), is of great interest for cancer immunotherapy. Here, we report the biophysical characteristics and nonclinical antagonistic activities of sasanlimab (PF-06801591), a humanized anti-PD-1 antibody of IgG4 isotype. We show that sasanlimab binds selectively and with similar high potency to human and cynomolgus monkey PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, with no detectable Fc-dependent effector function. The binding of sasanlimab to human and cynomolgus PD-1 is associated with the formation of a stable complex, which is likely to be the main driver of this high-affinity interaction. In vitro, sasanlimab significantly augmented T-cell proliferation and cytokine production in mixed lymphocyte reaction and superantigen stimulation assays. In vivo, sasanlimab accelerated the incidence of GvHD by enhancing T-cell proliferation and cytokine secretion in a xenogeneic model of acute GvHD and halted the growth of MC-38 colon adenocarcinoma tumors in human PD-1 knock-in mice. Pharmacokinetic and toxicokinetic findings from cynomolgus monkey showed that sasanlimab was active and well-tolerated. Taken together, the data presented here support the clinical development of sasanlimab for the treatment of patients with advanced cancers as a single agent or in combination with other immunotherapies.

Ligand-inducible, prostate stem cell antigen (PSCA)-directed GoCAR-T cells in advanced solid tumors: Preliminary results with cyclophosphamide (Cy) ± fludarabine (Flu) lymphodepletion (LD).

2536 Background: Cell-surface protein PSCA is upregulated in many solid tumors and correlates with disease stage. BPX-601, an autologous T-cell product expressing a PSCA-CD3ζ CAR and a rimiducid (Rim)-inducible MyD88/CD40 co-activation switch to augment T-cell proliferation and persistence, is designed to have enhanced efficacy in solid tumors vs traditional CARs. This ongoing first-in-human study assesses safety, biologic, and clinical activity of BPX-601+Rim in PSCA+ cancers. Updated results, including those from patients (pts) who underwent LD with Flu/Cy, are presented. Methods: BP-012 is a 2-part, open-label trial. Part 1 is a 3+3 dose escalation of BPX-601 (1.25–5.0x106 cells/kg; Day [D] 0) given prior to a single, fixed Rim dose (0.4 mg/kg; D7) in pts with previously treated PSCA+ metastatic pancreatic, gastric, or prostate cancers with measurable disease. Results: As of Jan-22-2019, 15 pts have received BPX-601±Rim. Two pts at the highest cell dose received Flu/Cy for LD on D−5 to D−3 before BPX-601; LD after Flu/Cy was 96.6% and 84.3%. Thirteen pts received Cy alone on D−3; in these pts, LD ranged from 0–68.6%. Rapid cell expansion by D4 was observed in all pts with peak vector copy number 8.3-fold higher with Flu/Cy (n = 2) vs Cy LD (n = 13). Serum IP-10, IL-6 and TNFα increased > 2-fold from baseline in ≥1 pt in all Rim cohorts, with 3- to 20-fold Rim-dependent cell expansion in 6 pts. No CRS or DLTs were reported. After Rim, one Flu/Cy pt experienced a serious Grade 2 AE (encephalopathy) related to BPX-601+Rim that resolved with IV steroids; despite time-matched nonserious Grade 1 pyrexia, the pt had no other CRS symptoms. After BPX-601+Rim and ≥1 scan, best responses were 8 SD and 3 PD (1 non-evaluable). With a median follow-up of 9.8 wks, time to next treatment (tx) after BPX-601 ranged from 2.7–22.1 wks (n = 8) and ongoing tx-free intervals range from 9.1–30.1 wks (n = 4). Conclusions: BPX-601+Rim was well-tolerated with manageable safety and early evidence of enhanced CAR T-cell expansion and prolonged persistence after Flu/Cy vs Cy. Additional pts will undergo Flu/Cy LD prior to BPX-601 with single- and repeat-dose Rim. Clinical trial information: NCT02744287.

Novel Predictor Of Lenalidomide Response In Non-del5q MDS Reveals Linkage To Molecular Mechanism: First Characterization Of T-Cell Function In Cereblon Homozygous Deficient Mice

BackgroundDurable responses to lenalidomide (LEN, an immunomodulatory drug, IMiDTM) occur in both non-del5q and del5q Myelodysplastic Syndrome (MDS). In non-del5q MDS and multiple myeloma, modulation of the bone marrow microenvironment by an unknown mechanism has been theorized. IMiDs initiate numerous effects on the immune system, some of which may induce an anti-leukemic and anti-inflammatory response. The connection between the drug's immune modulating activity and its hematopoietic stimulatory activity in MDS is currently untested. We and others have shown that LEN expands T-cells and increases IL-2 (p<0.001) by substituting for CD28 co-stimulation. In support of this, we found that LEN activates a CD28-specfic transcription factor pCREB in the absence of extracellular ligation of the CD28 receptor suggesting that it is acting to suppress a pharmacologic target that is involved specifically in costimulatory signaling. Normally, several negative regulators provide immunotolerance to T-cells to limit self-reactivity and protect against autoimmune disease. Anti-cancer immunosurveillance is often circumvented through acquired resistance to costimulation. MethodsThe critical role of immune modulation in LEN-induced hematologic response was examined by testing the performance of a predictive biomarker in patients stratified by del5q status in a training cohort and in a blinded validation cohort. Molecular analyses were performed on sorted populations of cells and knock-down of genes in normal human T-cells. Finally, we demonstrate the relevance of cereblon, a molecular target of LEN, in the negative regulation of costimulatory CD28 signaling using a homozygous germline knockout mouse. ResultsComparison of sorted CD28+CD8+ and CD28-CD8+ human T-cells illustrates the importance of the expression of this receptor in LEN-induced T-cell activation. CD28- T-cells were found to be resistant to LEN. Accumulation of CD28- cells is a form of accelerated immunosenescence that is often associated with cancer and has been reported in MDS. To determine if CD28- T-cell accumulation is associated with LEN hematopoietic outcome in patients, we performed T-cell profiling before treatment in a training cohort (n=21) and a blinded validation cohort (n=35). Of the T-cell markers studied, a higher percentage of CD28- T-cells was associated with LEN hematopoietic failure in the training cohort (p=0.001). Using a R-Part-defined cutoff of CD28+ vs CD28- T-cells, the biomarker sensitivity was found to be 86%, specificity 82%, positive predictive value (PPV) 67%, and negative PV (NPV) 93% in non-del5q MDS patients, but did not improve response discrimination in del5q MDS. The association between CD28 and LEN response led us to hypothesize that the molecular target of LEN is involved in signaling by the CD28 receptor. A landmark study identified cereblon (CRBL), a RING-domain E3-Ubiquitin Ligase (UbL) to be the primary target of thalidomide and LEN. To examine the effect of CRBL on CD28 signaling, germ-line crbl knock-out mice (crbl-/-) were examined compared to wild-type (WT) C57BL6 mice. We found that crbl deficiency augments T-cell proliferation (Fig. 1A) and contextual activation of IL2 in the absence of CD28 co-stimulation similar to LEN. Examination of the bone marrow, thymus, and peripheral blood shows that crbl is a negative regulator of lymphopoiesis in the spleen and thymocyte with increased peripheral blood lymphocyte counts (Fig. 1B) and increased double negative (DN)-1 (P=0.01), DN3 (p=0.03), and reduced DN4 (p=0.005) thymocytes in crbl-/- compared to WT mice. [Display omitted] ConclusionsOur results indicate that immune reconstitution mediated by T-cell CD28 signaling is important for hematologic response in non-del5q MDS, but not del5q MDS, and is consistent with different molecular mechanisms of action in these two groups of patients. LEN may improve T-cell function by directly suppressing CRBL a novel E3-UbL regulator involved in lymphopoiesis, thymocyte development and CD28 co-stimulation. Our data suggests that CRBL acts similarly to the E3 UbL proteins Cbl-b, GRAIL, and ITCH that mechanistically induce immunotolerance. In response to LEN, T-cell functional activation may sustain anti-leukemic immunosurveillance or act to remove suppressive immature cell populations from the bone marrow microenvironment allowing hematologic improvement in non-del5q MDS. Disclosures:Epling-Burnette:Celgene: Research Funding. McDaniel:Celgene: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees.

Radiation Combined With Thermal Injury Induces Immature Myeloid Cells

The continued development of nuclear weapons and the potential for thermonuclear injury necessitates the further understanding of the immune consequences after radiation combined with injury (RCI). We hypothesized that sublethal ionization radiation exposure combined with a full-thickness thermal injury would result in the production of immature myeloid cells. Mice underwent either a full-thickness contact burn of 20% total body surface area or sham procedure followed by a single whole-body dose of 5-Gy radiation. Serum, spleen, and peripheral lymph nodes were harvested at 3 and 14 days after injury. Flow cytometry was performed to identify and characterize adaptive and innate cell compartments. Elevated proinflammatory and anti-inflammatory serum cytokines and profound leukopenia were observed after RCI. A population of cells with dual expression of the cell surface markers Gr-1 and CD11b were identified in all experimental groups, but were significantly elevated after burn alone and RCI at 14 days after injury. In contrast to the T-cell-suppressive nature of myeloid-derived suppressor cells found after trauma and sepsis, myeloid cells after RCI augmented T-cell proliferation and were associated with a weak but significant increase in interferon γ and a decrease in interleukin 10. This is consistent with previous work in burn injury indicating that a myeloid-derived suppressor cell-like population increases innate immunity. Radiation combined injury results in the increase in distinct populations of Gr-1CD11b cells within the secondary lymphoid organs, and we propose these immature inflammatory myeloid cells provide innate immunity to the severely injured and immunocompromised host.

Development of a Listeria monocytogenes-based vaccine against hepatocellular carcinoma

Live attenuated Listeria monocytogenes (LM) is a promising bacterial vector able to induce a T-cell response to tumor-associated antigens and demonstrates great potential for use in vaccine development. A novel recombinant LM-based vaccine (Lmdd (LM ΔdalΔdat)-MPFG (multiple peptide fusing genes)) was developed with the ability to express and secrete hepatocellular carcinoma (HCC)-related tumor-associated antigens fragments due to the insertion of hepatitis B virus (HBV)-X protein (HBx)-derived epitopes HBx(52-60) and HBx(140-148), the universal T-helper epitope, alpha-fetoprotein (AFP) epitope AFP(158-166), and melanoma antigen gene (MAGE)-3(271-279) into the HBV core protein. Following immunization with the Lmdd-MPFG vaccine, macrophages exhibited uptake of the bacteria; the vaccine was then nearly cleared 3 days after the first administration. It disappeared even more quickly following subsequent vaccinations. However, recombinant Lmdd-MPFG allowed for the full development of an antitumor response towards the human leukocyte antigen (HLA)-A0201 epitopes of MPFG. Each epitope stimulated an augmented T-cell proliferation and enhanced the supernatant level of interferon (IFN)-γ in vitro. In addition, IFN-γ-producing CD8(+) T cells as well as in vivo cytolytic activity were significantly increased in HLA-A2 transgenic mice. Additionally, the Lmdd-MPFG developed a strong antitumor response, as indicated by the significant resistance of immunized mice to MPFG-positive Hepa1-6 cell challenge in both a prophylactic and therapeutic setting. Tumor regression was accompanied by an enhanced cytotoxic T lymphocyte response and a decrease of regulatory T cells in the tumor. Collectively, these results suggest that utilizing attenuated LM as a vaccine vector, able to carry the MPFG gene, presents a potentially feasible strategy for prevention of HCC.

Specific microtubule-depolymerizing agents augment efficacy of dendritic cell-based cancer vaccines

BackgroundDamage-associated molecular patterns (DAMPs) are associated with immunogenic cell death and have the ability to enhance maturation and antigen presentation of dendritic cells (DCs). Specific microtubule-depolymerizing agents (MDAs) such as colchicine have been shown to confer anti-cancer activity and also trigger activation of DCs.MethodsIn this study, we evaluated the ability of three MDAs (colchicine and two 2-phenyl-4-quinolone analogues) to induce immunogenic cell death in test tumor cells, activate DCs, and augment T-cell proliferation activity. These MDAs were further evaluated for use as an adjuvant in a tumor cell lysate-pulsed DC vaccine.ResultsThe three test phytochemicals considerably increased the expression of DAMPs including HSP70, HSP90 and HMGB1, but had no effect on expression of calreticulin (CRT). DC vaccines pulsed with MDA-treated tumor cell lysates had a significant effect on tumor growth, showed cytotoxic T-lymphocyte activity against tumors, and increased the survival rate of test mice. In vivo antibody depletion experiments suggested that CD8+ and NK cells, but not CD4+ cells, were the main effector cells responsible for the observed anti-tumor activity. In addition, culture of DCs with GM-CSF and IL-4 during the pulsing and stimulation period significantly increased the production of IL-12 and decreased production of IL-10. MDAs also induced phenotypic maturation of DCs and augmented CD4+ and CD8+ T-cell proliferation when co-cultured with DCs.ConclusionsSpecific MDAs including the clinical drug, colchicine, can induce immunogenic cell death in tumor cells, and DCs pulsed with MDA-treated tumor cell lysates (TCLs) can generate potent anti-tumor immunity in mice. This approach may warrant future clinical evaluation as a cancer vaccine.

Phase II trial of high-dose interleukin-2 (IL-2) with priming and concomitant sargramostim (GM-CSF) in patients with advanced melanoma.

8560 Background: GM-CSF is a multifunctional cytokine that augments T-cell proliferation in the presence of antigen presenting cells (APC) and up-regulates the IL-2 receptor on T lymphocytes and mo...

Preclinical efficacy data of MGN1601, a tumor vaccine comprising 4-fold gene-modified and irradiated allogeneic tumor cells in combination with a DNA-based immunomodulator for the treatment of metastatic renal carcinoma.

e15067 Background: MGN1601 is a cell-based tumor vaccine for the treatment of mRCC. MGN1601 consists of two active pharmaceutical ingredients. One consists of allogeneic, human tumor cells, derived from RCC tissue, and transfected in-vitro with four different MIDGE vectors for transient expression of human IL-7, GM-CSF, CD80 and CD154. The other ingredient is the DNA-based immunomodulator dSLIM-30L1, mainly a TLR-9 agonist. Methods: Murine tumor models were used to evaluate the efficacy of a murine homologue of MGN1601 (mMGN1601). mMGN1601 was set up from Renca cells and transfected with 4 different MIDGE vectors for transient expression of mouse IL-7, GM-CSF, CD80 and CD154. For tumor inoculation in syngenic BALB/c mice Renca cells were used. For efficacy of the vaccine in a prophylactic setting, mice were vaccinated with mMGN1601 and thereafter challenged with Renca cells. In therapeutic settings Balb/c mice first received naïve Renca cells and were subsequently vaccinated four times with different test items, either the complete mMGN1601 or one out of three lacking different components. To address the mode of action, modulations of the immune system were monitored in NMRI mice (allogeneic setting). Results: Prophylactic setting: None of the vaccinated mice showed tumor growth, whereas all of the tumor-bearing mice of the control group died due to the tumor growth. Efficacy evaluation in the therapeutic setting revealed, that the complete tumor vaccine clearly achieved the most pronounced effects in tumor reduction, showing a survival of 92%, compared to 25% survival in the control group. The incomplete derivatives led to less pronounced therapeutic efficacy. The immunomodulatory effects of mMGN1601 in allogeneic NMRI mice generated a more than 3-fold increase of the cytotoxic activity of spleen cells, an augmented T-cell proliferation (> 6-fold) and an increase of CD4+, CD8+ and CD86+ cells at the site of vaccination (up to 20-fold). Conclusions: The murine homologue of MGN1601 showed excellent antitumor efficacy and significant pharmacological effects in murine models. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration MOLOGEN AG MOLOGEN AG MOLOGEN AG

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen-specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcgamma receptor (FcgammaR)-mediated antigen uptake and enhance antigen presentation resulting in augmented T-cell proliferation. We examined the impact of antigen presentation and T-cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene-deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcgammaR-deficient mice. Lung DC of WT, but not FcgammaR-deficient mice, induced increased antigen-specific CD4+ T-cell proliferation when pulsed with anti-OVA IgG immune complexes. Intranasal application of anti-OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen-specific T-cell proliferation in vivo. Finally, antigen-specific IgG in the serum of sensitized mice led to a significant increase of antigen-specific CD4+ T-cell proliferation induced by WT, but not FcgammaR-deficient, lung DC. We conclude that FcgammaR-mediated enhanced antigen presentation and T-cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.

Identification of Glucocorticoid-Induced TNF Receptor-Related Protein Ligand on Keratinocytes: Ligation by GITR Induces Keratinocyte Chemokine Production and Augments T-Cell Proliferation

Glucocorticoid-induced tumor necrosis factor (TNF) receptor-related protein ligand (GITRL) is a recently described co-stimulatory molecule expressed by antigen-presenting cells (APCs). Activated keratinocytes are known to engage intraepithelial T cells through co-stimulatory molecules. This study investigated the expression and function of GITRL in resting keratinocytes. We showed by immunofluorescence and flow cytometry that keratinocytes from Balb/C and C57Bl/6 mice, as well as PAM 212 murine cell line keratinocytes and human epidermal keratinocytes (HEK), express cell-surface GITRL. Stimulation of murine skin biopsies and HEK with GITR fusion protein (GITR: Fc FP) resulted in mRNA induction for chemoattractants: cutaneous T-cell-attracting chemokine (CTACK), thymus and activation-regulated chemokine (TARC), IL-8, monocyte chemoattractant protein-1 (MCP-1), and murine beta-defensin 3 (MBD3). Immunofluorescent studies on mouse biopsies treated with GITR: Fc FP confirmed corresponding TARC and MCP-1 protein production by keratinocytes. Chemokine induction was shown to be NF-kappaB-mediated. T-cell proliferation was enhanced by the addition of keratinocytes. This was reversed by pretreatment with an anti-GITRL antibody. We conclude that keratinocytes express GITRL, and that through this important co-stimulatory molecule, they have the potential to influence T-cell numbers in the skin through chemokine production and through a direct cell-cell effect on T-cell proliferation.

Mesenchymal Stem Cells Require a Sufficient, Ongoing Immune Response to Exert Their Immunosuppressive Function

Mesenchymal stem cells (MSC) have emerged to be one of the most promising candidates for cellular immunotherapy in solid organ transplantation because the reduction of conventional immunosuppression is highly desirable. However, little is known about the details of MSC-mediated immunomodulation and their clinical relevance. To address conflicting studies about the ability of MSC to suppress or augment T-cell proliferation, we introduce a transplantation-related rat model that allows studying the influence of MSC on alloproliferation. Hearts transplanted in a fully allogeneic transplantation model (LEW to ACI) were rejected earlier when recipients were pretreated with donor MSC, indicating activation of T cells in vivo. In additional co-culture experiments, T cells were differently affected by allogeneic MSC depending on the extent of previous activation: When conditions were rendered proinflammatory by adding high concanavalin A (ConA) concentrations or proinflammatory cytokines (interferon-gamma, interleukin-2, or tumor necrosis factor-alpha), MSC inhibited proliferation. Application of low doses of ConA or anti-inflammatory cytokines like IL-10 abrogated the suppressive effect of MSC. For application of MSC in solid organ transplantation, it will be important to further describe this switch effect of MSC function.

Targeting of antigens to B cells augments antigen-specific T-cell responses and breaks immune tolerance to tumor-associated antigen MUC1

AbstractB cells are antibody (Ab)–secreting cells as well as potent antigen (Ag)–presenting cells that prime T-cell activation, which evokes great interest in their use for vaccine development. Here, we targeted ovalbumin (OVA) to B cells via CD19 and found that a single low dose of anti–CD19-OVA conjugates, but not isotype mAb-OVA, stimulated augmented CD4 and CD8 T-cell proliferation and expansion. Administration of TLR9 agonist CpG could significantly enhance long-term T-cell survival. Similar results were obtained when the tumor-associated Ag MUC1 was delivered to B cells. MUC1 transgenic (Tg) mice were previously found to lack effective T-cell help and produce low-titer of anti-MUC1 Abs after vaccination. Targeting MUC1 to B cells elicited high titer of anti-MUC1 Abs with different isotypes, predominantly IgG2a and IgG2b, in MUC1 Tg mice. The isotype switching of anti-MUC1 Ab was CD4 dependent. In addition, IFN-γ–producing CD8 T cells and in vivo cytolytic activity were significantly increased in these mice. The mice also showed significant resistance to MUC1+ lymphoma cell challenge both in the prophylactic and therapeutic settings. We conclude that Ags targeting to B cells stimulate CD4 and CD8 T-cell responses as well as Th-dependent humoral immune responses.

DC-HIL is a negative regulator of T lymphocyte activation

AbstractT-cell activation is the net product of competing positive and negative signals transduced by regulatory molecules on antigen-presenting cells (APCs) binding to corresponding ligands on T cells. Having previously identified DC-HIL as a receptor expressed by APCs that contains an extracellular immunoglobulin (Ig)–like domain, we postulated that it plays a role in T-cell activation. To probe this function, we created soluble recombinant DC-HIL, which we observed to bind activated (but not resting) T cells, indicating that expression of the putative ligand on T cells is induced by activation. Binding of DC-HIL to naive T cells attenuated these cells' primary response to anti-CD3 antibody, curtailing IL-2 production, and preventing entry into the cell cycle. DC-HIL also inhibited reactivation of T cells previously activated by APCs (secondary response). By contrast, addition of soluble DC-HIL to either allogeneic or ovalbumin-specific lymphocyte reactions augmented T-cell proliferation, and its injection into mice during the elicitation (but not sensitization) phase of contact hypersensitivity exacerbated ear-swelling responses. Mutant analyses showed the inhibitory function of DC-HIL to reside in its extracellular Ig-like domain. We conclude that endogenous DC-HIL is a negative regulator of T lymphocyte activation, and that this native inhibitory function can be blocked by exogenous DC-HIL, leading to enhanced immune responses.

Shock Waves Increase T-cell Proliferation or IL-2 Expression by Activating p38 MAP Kinase

Shock waves were elicited by transient pressure disturbances, which could be used to treat musculoskeletal disorders. In present studies, we investigated whether the low-density shock waves (LDSWs), which are able to damage plasma membrane without impairing the vimentin or other organelles, might augment T-cell proliferation as well as IL-2 expression, and if mitogen activated protein kinase p38 (p38 MAPK) might be an underlying mechanism through which the LDSWs enhanced T-cell function. We found that the LDSWs increased activation of p38 MAPK in Jurkat T cells. The LDSWs alone didn't result in the T-cell proliferation and IL-2 expression. However, in combination with other stimuli, LDSWs could augment the T-cell proliferation and IL-2 expression. Inhibition of p38 MAPK using SB203580 reduced the stimulatory effects of the LDSWs, which indicated that the LDSWs enhanced IL-2 expression through a mechanism that involved p38 MAPK activation. We concluded that the p38 MAPK activation played a key role in the regulation of T cells function by the LDSWs.

IL‐7‐Dependent Homeostatic Proliferation in the Presence of a Large Number of T Cells in Gld Mice

In gld mice, CD4 and 8-double-negative (DN) T cells as well as naive and memory-phenotype T cells accumulate in the peripheral lymphoid organs. Although Fas ligand (L) defect accounts for the progressive accumulation of abnormal DN T cells, the existence of other mechanisms which may be involved in the defective homeostasis in gld mice has been unclear. In this study, we analyze T-cell homeostasis in gld mice using adoptive transfer systems. It was shown that a gld, but not C57BL/6 (B6), environment led to augmented proliferation of B6 T cells transferred without up-regulation of CD69. Thus, the augmented T-cell proliferation seemed to result from mal-homeostatic proliferation even in the presence of a large number of recipient T cells. T cells from lpr mice showed no significant proliferation in the B6 environment, suggesting that the absence of Fas-Fas L interaction was not responsible for the mal-homeostatic proliferation. Although similar levels of IL-7 mRNA were detected in gld and B6 spleens, the intensity of CD127 and the proportion of CD127+ cells in the T cells were significantly lower in gld mice than in B6 mice, suggesting that IL-7 excess in a gld environment is responsible for the abnormal proliferation of transferred T cells. The administration of anti-CD127 antibody inhibited the proliferation of transferred lymphocytes. Thus, IL-7-dependent proliferation seems to be involved in the abnormal proliferation of lymphocytes in gld recipients.

Pretreatment with glucocorticoids enhances T-cell effector function: possible implication for immune rebound accompanying glucocorticoid withdrawal.

Glucocorticoids (GCs) exert their immunosuppressive/antiproliferative effects largely through inhibition of cytokine expression, and paradoxically upregulate the expression of (proinflammatory) cytokine receptors on select nonlymphoid cells. Clinically, withdrawal of GCs was frequently associated with worsening of the outcome of heightened immunity disorders, thereby implicating enhanced cytokine and cytokine receptor expression as a possible consequence of acute/short-term GCs withdrawal. In view of the significance of this complication of GC therapy, we addressed the effect of GC withdrawal on cytokine receptor expression and subsequent T-cell effector function, using the proliferation of human T cells as biological readout. To mimic GC withdrawal, T cells were treated with GCs or controls, stimulated, and incubated for 16-20 h at 37 degrees C, washed, and reactivated for a further 4-48 h. Surface marker expression was assessed by FACS analysis, and proliferation was determined by measuring the cellular uptake of tritiated thymidine. Dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent manner, inhibited T-cell proliferation induced by anti-CD28 Ab + PMA. However, pretreatment of T cells activated with mitogens, cross-linking antibodies, or PMA + ionomycin ("CD3-bypass" stimulation regimen), but not resting T cells, with DEX or PRED resulted in a marked increase in IL-IR, IL-2R alpha, and IL-6R expression, which was accompanied by a significant enhancement in T-cell proliferation. This effect of GCs was neither stimulus specific nor did it result from alteration in cell viability, and was paralleled by augmentation in cytokine (rIL-2) effects on DEX-pretreated and preactivated T cells. Taken together, our results underline the dual effects of GCs in regulating T-cell activation and cytokine expression. In essence, GCs directly inhibited T-cell proliferation by suppressing cytokine production, and, by enhancing cytokine receptor expression, pretreatment with GCs augmented T-cell proliferation.

Human Follicular Dendritic Cells Inhibit Superantigen-Induced T-Cell Proliferation by Distinct Mechanisms

Follicular dendritic cells (FDCs) reside within germinal centers of secondary lymphoid tissue where they play a critical role in antigen-driven immune responses. FDCs express numerous adhesion molecules that facilitate cellular interactions with B and T cells within the germinal center microenvironment. Although human FDCs have been shown to influence B-cell development, very little is known about the ability of FDCs to regulate T-cell responses. To investigate this functional aspect of FDCs, highly enriched preparations were isolated by magnetic cell separation using the FDC-restricted monoclonal antibody HJ2. We found that isolated human FDCs inhibited proliferation of both autologous and allogeneic T cells, and were dependent on the number of FDCs present. Inhibition by FDCs was observed using two serologically distinct superantigens at multiple concentrations (Staphylococcus enterotoxin A and B). In contrast, B cells failed to inhibit, and often augmented superantigen-induced T-cell proliferation. Antibody-blocking studies showed that CD54 and CD106 were involved in the ability of FDC to inhibit T-cell proliferative responses. When FDCs and T cells were separated by a semipermeable membrane, the inhibitory effect was partially abrogated, demonstrating that in addition to cell-cell interactions, a soluble factor(s) was also involved in the process. The addition of indomethicin to cultures improved the proliferative response in the presence of FDCs, indicating that inhibition was mediated, in part, by prostaglandins. These results indicate that FDCs regulate T-cell proliferation by two molecular mechanisms and that FDC:T-cell interactions may play a pivotal role in germinal center development.

Hypertonic Saline Activates Protein Tyrosine Kinases and Mitogen-activated Protein Kinase p38 in T-cells

In previous in vitro studies, we have found that hypertonic saline (HTS) can augment T-cell proliferation and restore the function of suppressed T-cells. Our animal models have shown that HTS resuscitation reverses immunosuppression after hemorrhage and reduces mortality from sepsis. In the present study, we investigated if and how HTS may influence T-cell signaling and function on a subcellular level. Human peripheral blood mononuclear cells (PBMC) were used to determine the effect of HTS on T-cell interleukin 2 (IL-2) production and proliferation. Human Jurkat T-cells were used to study the effects of HTS on T-cell signal transduction, IL-2 mRNA transcription, and IL-2 expression. The effect of HTS on T-cell proliferation and IL-2 production was measured with PBMC and Jurkat T-cells. IL-2 mRNA transcription in HTS-treated Jurkat cells was measured by reverse transcriptase polymerase chain reaction. HTS-induced protein tyrosine phosphorylation in Jurkat T-cells was determined by immunoblotting with anti-phosphotyrosine antibodies. Expression in Jurkat cells of the mitogen-activated protein kinase p38 (MAPK p38), a signal transduction protein that is activated by osmotic stress, was determined by immunoblotting with anti-MAPK p38 antibodies. HTS-induced MAPK p38 activation in Jurkat cells was measured with an immune-complex kinase assay using ATF-2 as a substrate. Proliferation of activated human PBMC increased significantly upon addition of HTS to the culture medium. This effect of HTS was paralleled by enhanced IL-2 production of activated PBMC and Jurkat cells and IL-2 mRNA transcription of Jurkat cells. HTS exposure of Jurkat cells caused tyrosine phosphorylation of a number of cellular proteins. We found that Jurkat T-cells expressed MAPK p38 and that it was activated in the presence of HTS. All these effects of HTS on T-cell signaling and function were observed at NaCl concentrations that were within physiologically relevant levels (20-100 mmol/L hypertonicity). In T-cells, HTS triggers a signaling pathway that includes increased tyrosine phosphorylation of several cellular proteins and activation of MAPK p38. HTS alone does not result in IL-2 mRNA transcription, IL-2 expression, or T-cell proliferation. However, in combination with other stimuli, HTS augments T-cell IL-2 expression and proliferation. We speculate that HTS could "resuscitate" suppressed T-cells in trauma patients by circumvention of, or substituting for, blocked signaling pathways.

A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.