Cells In Mixed Lymphocyte Culture Research Articles

Abstract 5316: SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response

Abstract Background: Monoclonal antibodies targeting the co-stimulatory receptors CD137 (4-1BB) and CD40 have displayed encouraging activity against a wide range of solid tumors in preclinical animal models. However, their clinical development as monotherapy has been hindered by dose-related liver toxicity, leading to limited efficacy at safe doses. To address this challenge and bolster tumor-specific immunity by orchestrating the interaction between dendritic cells (DCs) and cytotoxic T cells, we introduce SM2256 - a bispecific conditional CD40 and 4-1BB dual agonist. SM2256 physically bridges DCs and T cells, enhancing T cell priming and reactivation. In preclinical animal studies, SM2256 exhibited both excellent tolerability and potent anti-tumor activity. Methods: SM2256 was engineered by fusing high-affinity, non-ligand-blocking VHH domains targeting 4-1BB and CD40 to a silenced IgG1 Fc backbone. This fusion resulted in a tetravalent bispecific antibody capable of conditionally activating CD40 and 4-1BB upon mutual receptor binding. We assessed the binding specificity and affinity of SM2256 to human CD40 and 4-1BB using ELISA and flow cytometry. The target-dependent activation of CD40 and 4-1BB was confirmed via reporter assays and co-culture systems using primary DCs and T cells. In vivo safety and anti-tumor efficacy were evaluated in a syngeneic transplantation model using the MC38 cancer cell line in B-h41BB/B-hCD40 double humanized mice. Results: SM2256 exhibited sub-nanomolar binding affinity to recombinant human and cynomolgus monkey CD40 and 4-1BB, activating 4-1BB-NFκB-luc and CD40-NFκB-luc reporter cell lines at picomolar concentrations in a strictly target-dependent manner. Furthermore, SM2256 physically facilitated the interaction between DCs and T cells, leading to increased expression of DC co-stimulatory ligands and elevated secretion of cytokines by T cells in mixed lymphocyte cultures. In a syngeneic tumor transplantation model, SM2256 significantly restrained the growth of MC38 tumors in B-h41BB/B-hCD40 double humanized mice. Conclusions: SM2256 is a novel conditional CD40 and 4-1BB dual agonist that demonstrates promising preclinical efficacy against solid tumors while boasting an improved safety profile when compared to existing monoclonal antibodies. Citation Format: Wenjing Song, Simin Yang, Yi Wei, Shihao Lu, Huiqin Geng, Xiaodan Liu, Hong Zhou, Siyao Xie, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5316.

Volatile Phases Derived from Serum, DC, or MLC Culture Supernatants to Deduce a VOC-Based Diagnostic Profiling Strategy for Leukemic Diseases.

Volatile organic compounds (VOCs) reflect the metabolism in healthy and pathological conditions, and can be collected easily in a noninvasive manner. They are directly measured using electronical nose (eNose), and may qualify as a systemic tool to monitor biomarkers related to disease. Myeloid leukemic blasts can be transformed into leukemia-derived dendritic cells (DCleu) able to improve (anti-leukemic) immune responses. To profile immunological changes in healthy and acute myeloid leukemic (AML) patients' ex vivo cell cultures, we correlated the cell biological data with the profiles of cell culture supernatant-derived VOCs. DC/DCleu from leukemic or healthy whole blood (WB) were generated without (Control) or with immunomodulatory Kit M (Granulocyte macrophage-colony-stimulating-factor (GM-CSF) + prostaglandin E1 (PGE1)) in dendritic cell cultures (DC culture). Kit-pretreated/not pretreated WB was used to stimulate T cell-enriched immunoreactive cells in mixed lymphocyte cultures (MLC culture). Leukemia-specific adaptive and innate immune cells were detected with a degranulation assay (Deg) and an intracellular cytokine assay (InCyt). Anti-leukemic cytotoxicity was explored with a cytotoxicity fluorolysis assay (CTX). VOCs collected from serum or DC- and MLC culture supernatants (with vs. without Kit M pretreatment and before vs. after culture) were measured using eNose. Compared to the Control (without treatment), Kit M-pretreated leukemic and healthy WB gave rise to higher frequencies of mature (leukemia-derived) DC subtypes of activated and (memory) T cells after MLC. Moreover, antigen (leukemia)-specific cells of several lines (innate and adaptive immunity cells) were induced, giving rise to blast-lysing cells. The eNose could significantly distinguish between healthy and leukemic patients' serum, DC and MLC culture supernatant-derived volatile phases and could significantly separate several supernatant (with vs. without Kit M treatment, cultured vs. uncultured)-derived VOCs within subgroups (healthy DC or leukemic DC, or healthy MLC or leukemic MLC supernatants). Interestingly, the eNose could indicate a Kit M- and culture-associated effect. The eNose may be a prospective option for the deduction of a VOC-based profiling strategy using serum or cell culture supernatants and could be a useful diagnostic tool to recognize or qualify AML disease.

Vitamin D3 and zinc synergistically induce regulatory T cells and suppress interferon-γ production in mixed lymphocyte culture

Although vitamin D3 (cholecalciferol) and zinc are known to individually shift the immune response towards tolerance, little is known about the effect of their combined administration. This work contributes to understanding the combined action of zinc and vitamin D3 in different in vitro models for immune reactions. Zinc and vitamin D3 in combination boosted the differentiation into Foxp3+CD4+ T cells (Treg). Vitamin D3 alone reduced the percentage of CD4+T-bet+ T cells (TH1). In mixed lymphocyte culture (MLC), therapeutic concentrations of vitamin D3 and zinc in combination suppressed interferon-γ (IFN-γ) secretion more strongly than the single agent treatment. This effect was also detected for a combination of subtherapeutic concentrations of both vitamin D3 and zinc. Vitamin D3, even at nanomolar concentrations, increased intracellular zinc levels. PBMC (peripheral blood mononuclear cells) of individuals at risk of zinc deficiency responded to vitamin D3 treatment with a higher mRNA expression of Zip13. In PBMC, both agents reduced activation-induced IL-17 secretion. In summary, this study shows, for the first time, a vitamin D3-induced upregulation of CD4+Foxp3+ T cells in MLC. The data propose a model where zinc augments the effect of vitamin D3 in certain therapeutic and subtherapeutic concentrations. Lower concentrations of both vitamin D3 and zinc could be used for effective treatment, thus reducing possible side effects from vitamin D3 and zinc. Vitamin D3 and zinc in combination may be a promising and cheap option to treat dysregulated immune response in various conditions.

Mechanisms of CD8+ T-Cell Resistance to Post-Transplantation Cyclophosphamide (PTCy)

Despite the lymphocyte reducing effects of PTCy after hematopoietic cell transplantation (HCT), CD8+ T-cell numerical reconstitution is rapid and diverges from CD4+ T-cell reconstitution. To understand this rapid reconstitution and explore possible mechanisms of CD8+ T-cell resistance to PTCy, we used mixed lymphocyte culture (MLC) of healthy donor CD3+ T cells with irradiated HLA-mismatched CD3− peripheral blood mononuclear cells as previously published (Kanakry CG et al., STM, 2013). Accordingly, cells in MLCs were treated on day 3 with the active Cy analog mafosfamide (Maf) 7.5 ug/ml or from days 0-7 with cyclosporine (CsA) 600 ng/ml or rapamycin (Rap) 15 ng/ml. We found that all drugs reduced CD8+ T-cell numbers at day 7. However, the relative impact on CD8+ subsets differed by treatment. Maf preserved a similar distribution of memory and naive subsets compared with untreated MLCs, unlike CsA and Rap which preferentially spared naives. Percentages of mucosal associated invariant T (MAIT) and phenotypically stem cell memory CD8+ T cells were increased after Maf. Proliferation was reduced after Maf but persisted at lower levels, distinct from CsA or Rap, which abolished proliferation. Mechanisms of resistance to Cy include expression of aldehyde dehydrogenase (ALDH), the major in vivo detoxifying enzyme for Cy. Multidrug transporter (MDR) effluxing also may play a role. At day 3 of MLC, all assessed CD8+ T-cell subsets increased expression of ALDH1A1 from undetectable expression in donor T cells. In a Rhodamine-123 efflux assay, all CD8+ T-cell subsets also increased MDR activity at day 3 compared with donor cells. Both ALDH1A1 expression and Rhodamine-123 effluxing declined at day 7. Increased effluxing by CD8+ T cells also was seen in clinical HCT from donor and recipient day 0 to recipient day +3 blood samples. In MLC, pharmacologic inhibition of ALDH with diethylaminobenzaldehyde or MDR with PK11195 sensitized CD8+ T cells to Maf, with combined inhibition leading to death of nearly all cells, suggesting both mechanisms may contribute to CD8+ T-cell resistance. To dissect the underlying molecular mechanisms, we explored the relative impact of proliferative state and cytokines on MDR activity. Effluxing was largely restricted to those cells that either had not proliferated or had proliferated in MLC only a single generation; most T cells that had proliferated ≥2 generations were not effluxing. Exogenous IL-4, IL-7, or IL-15 reduced effluxing, but IL-2, IL-6, IL-9, IL-17, IL-21, or IL-23 did not affect it. Conversely, exogenous IL-2 modulated ALDH1A1 expression in a dose-dependent manner: low doses (0.1-1 IU/ml) increased expression, while high doses (≥100 IU/ml) reduced it. Our work provides insight into the impact of PTCy on CD8+ T-cell subsets and the biology of chemotherapy resistance pathways relevant for understanding the dynamics of CD8+ T-cell immune reconstitution after PTCy.

Interleukin-12 (IL-12p70) Promotes Induction of Highly Potent Th1-Like CD4(+)CD25(+) T Regulatory Cells That Inhibit Allograft Rejection in Unmodified Recipients.

In rat models, CD4+CD25+ T regulatory cells (Treg) play a key role in the induction and maintenance of antigen-specific transplant tolerance, especially in DA rats with PVG cardiac allografts (1, 2). We have previously described generation of alloantigen-specific Treg (Ts1), by culture of naïve natural CD4+CD25+ Treg (nTreg) with specific alloantigen and IL-2 for 4 days. These cells express mRNA for IFN-γ receptor (ifngr) and suppress donor but not third party cardiac allograft rejection mediated by alloreactive CD4+ T cells at ratios of <1:10. Here, we show that Ts1 also expressed the IL-12p70 specific receptor (il-12rβ2) and that rIL-12p70 can induce their proliferation. Ts1 cells re-cultured with rIL-12p70 alone or rIL-12p70 and recombinant interleukin-2 (rIL-2), suppressed proliferation of CD4+ T cells in mixed lymphocyte culture at <1:1024, whereas Ts1 cells re-cultured with rIL-2 and alloantigen only suppressed at 1:32–64. The rIL-12p70 alloactivated Ts1 cells markedly delayed PVG, but not third party Lewis, cardiac allograft rejection in normal DA recipients. Ts1 cells re-cultured for 4 days with rIL-12p70 alone, but not those re-cultured with rIL-12p70 and rIL-2, expressed more il-12rβ2, t-bet, and ifn-γ, and continued to express the markers of Ts1 cells, foxp3, ifngr, and il-5 indicating Th1-like Treg were induced. Ts1 cells re-cultured with rIL-2 and alloantigen remained of the Ts1 phenotype and did not suppress cardiac graft rejection in normal DA rats. We induced highly suppressive Th1-like Treg from naïve nTreg in 7 days by culture with alloantigen, first with rIL-2 then with rIL-12p70. These Th1-like Treg delayed specific donor allograft rejection demonstrating therapeutic potential.

Interleukin-2 (hrIL2) Primed T cells display Reduced Alloproliferative Capacity In Vitro Due To Induction Of FOXP3+ Regulatory Cells

IntroductionProphylactic donor lymphocyte infusions (pDLI) after allogeneic transplantation contribute to immune restoration and reduce viral infections. Furthermore, we have recently shown that pDLI in patients with high risk leukemia significantly reduces the relapse rate, however, they were associated with a relatively high incidence of Graft versus Host Disease (GvHD)(BBMT 2013;19:75-81). Strategies to minimize GvHD without compromising the effect of pDLI against leukemia are needed. IL2 plays dual role in immune responses, contributing to both the generation of effector T cells and the maintenance of regulatory T cells (Tregs). Recently, low dose interleukin-2 (IL-2) therapy has been advanced as a potential immune modulator able to modulate the immune response to aid transplant tolerance and to suppress GvHD through expansion of Tregs (N Engl J Med. 2011; 2055-66). We investigated the impact of priming DLI with low dose IL2 on the proliferative responses to allo-stimulation in vitro. MethodsCD3+ T cells purified from healthy individuals by MACS negative selection were primed (p-T cells) or unprimed (np-T cells), with or without (control) 100 U/ml hrIL2 (Proleukin, Novartis) for 7 days. Composition of T-cell cultures was analyzed by flow cytometry for: a) the percentage of T regulatory cells (CD4+/CD25high/Foxp3+/Helios+, b) their differentiation (CD28/CD27), c) their immune exhaustion (Programmed cell death 1, PD1). In vitro alloproliferative capacity of the p-T cells was analyzed with CFSE cell proliferation assay by using them as responder cells in mixed lymphocyte cultures (MLC), with irradiated allo-PB mononuclear cells as stimulators. ResultsIn vitro priming of T-cells with IL-2 (p-T cells) in contrast to np-T or control cells: 1) increase the numbers of CD4+CD25highFoxp3+/Helios+ cells (n=8, 3.3%±0.7 mean±SEM vs 1.01%±0.22, p=0.004 και 1.4%±0.42, p=0.006). Increased levels of Foxp3 expression was also confirmed by Real Time PCR (n=2,1.25AU±0.15 vs 0.29AU±0.04, p=0.028 και 0.26AU±0.07, p=0.024). 2) did not affect the proportion of CD28+/CD27+ non late-differentiated cells (n=3, 60%±0.15 vs61%±0.04, p=0.91 και 59%±0.08, p=0.024). 3) did not cause immune exhaustion through PD1 expression (n=6, 13.3%±1.9 vs 8.1%±2.1, p=0.76, και 14%±2.2, p=0.68). 4) significantly decreases their response rate to allo-stimulus in MLC (n=8, 45%±0.5 vs65%±0.2, p=0.006 και 64%±0.2, p=0.008). The p-T cells regained their alloproliferative capacity after FACS-sorting removal of CD4+/CD25high Tregs. ConclusionsOur results show that ex vivo priming of T cells with low dose of IL-2 reduces their in vitro alloproliferative capacity. This reduction is not due to late differentiation or immune – exhaustion of T cells but to selective induction of Foxp3+ cells with immunomodulatory properties in the culture. It remains to be seen whether IL2-primed DLI is safe and effective in transplant patients. Disclosures:No relevant conflicts of interest to declare.

Low Antigen-specific T-cell Response in Pulmonary Tuberculosis is Associated with Impaired Phenotype and Functions of Interferon-α Induced Dendritic Cells

The phenotype and functions of monocyte-derived dendritic cells (DCs), generated upon stimulation with GMCSF and IFN-α, were investigated in pulmonary tuberculosis (TB). Patient INF-DC cultures were characterized by increased number of CD14+ cells, lowered level of CD25+ cells and increased expression of B7-H1. Besides, DCs produced enhanced levels of IL-6 and IL-10 and differed by reduced secretion of INF-γ and INF-α. Patient IFN-DCs had lower capacity to stimulate allogeneic T-cell proliferation and induce CD3+IFN-γ+ T cells but increased ability to activate CD3+IL-4+ T cells in mixed lymphocyte culture. The most pronounced increase of IL-10 production, as well as the decrease of allostimulatory activity and the alteration of T1/T2 stimulatory activity of DCs were registered in patients with low PPD-induced proliferative response. The data obtained evidence the tolerogenic phenotype of monocyte-derived IFN-DCs and the association of DC impairment with decreased antigen-specific T cell response in TB patients.

Contribution of Dehydroepiandrosterone Sulfate and Progesterone to In Vitro Regulation of Tolerogenic Activity of IFN-α-Induced Dendritic Cells

Dehydroepiandrosterone sulfate and progesterone exhibited an immunomodulatory effect on the tolerogenic characteristics of IFN-α-induced dendritic cells. The hormone effects depended on the initial level of allostimulatory activity of dendritic cells in mixed lymphocyte culture. However, dehydroepiandrosterone sulfate significantly more often stimulated allostimulatory activity by attenuating the tolerogenic properties of dendritic cells, while progesterone potentiated their tolerogenic potential. The capacity of the hormones (dehydroepiandrosterone sulfate and progesterone) to attenuate tolerogenic activity of dendritic cells was associated with reduction of FasL expression on these cells, while the increase in tolerogenic activity was associated with the increase in the percentage of CD123(+) dendritic cells, and under conditions of modification with dehydroepiandrosterone sulfate it was associated with increased B7-H1 expression. Possible contribution of indolamine-2,3-dioxygenase and prostaglandin E2 to stimulation of tolerogenic characteristics of dendritic cells modified with dehydroepiandrosterone sulfate and progesterone, respectively, was demonstrated.

Interleukin-32, α1 Anti-Trypsin (AAT-1) and Graft-Versus-Host-Disease

AbstractAbstract 522Graft-versus-host disease (GVHD) is an important complication of allogeneic hematopoietic cell transplantation (HCT). The role of various cell populations, cytokines and chemokines, present pre and post-transplantation, in the development of GVHD has been studied extensively.We investigated the potential role of Interleukin (IL)-32 in alloreactivity and GVHD. IL-32 is the protein product of the NK4 transcript first reported in IL-2 activated T lymphocytes and natural killer cells. IL-32 has pro-inflammatory and pro-apoptotic properties and induces expression of TNF-α in several cell targets. We used one-way mixed lymphocyte cultures (MLC) as a simple in vitro model of GVHD to determine IL-32 expression upon alloactivation. The α and γ isforms of (IL)-32 protein were 2-fold upregulated in allogeneic MLC compared to autologous controls (n=4, p=0.037). Concurrently, the concentrations of TNF-α, IL-6 and IL-8 in the allogeneic MLC supernatants were, as expected, upregulated significantly. This finding led us to evaluate IL-32 expression as a potential marker for GVHD after allogeneic HCT in 45 patients with either active acute GVHD or chronic GVHD, and 16 patients who did not show clinical evidence of GVHD. IL-32 mRNA levels in peripheral blood unsorted white blood cells, correlated with acute GVHD (RT-PCR values expressed as mean +/− SEM of the ratio of expression of IL-32/GUS-B = 1.01, p=0.011, vs control values IL-32/GUS-B = 0.23) but not with chronic GVHD, (IL-32/GUS-B = 0.26, p=0.16) (Figure 1). As the serine protease neutrophil proteinase 3 (PR3) has been shown to serve as activator of IL-32, and to process several inflammatory cytokines, including IL-32, TNF-α and IL-8, we postulated that the addition of the serine protease inhibitor α-1 anti-trypsin (AAT), would interfere with the processing of IL-32 by PR3, and as a result would lead to decreased proliferation of cells in MLC, and reduced cytokine production. To test this hypothesis, MLCs were treated with AAT at concentrations of 1–20 ug/ml and expression of IL-32 and PR3 were determined. In MLCs treated with AAT at the optimal concentration of 5 ug/ml, added to cultures on alternate days for a period of 7 days T lymphocyte proliferation was suppressed when compared to vehicle-treated MLC, (mean CPM=33.000 versus CPM=67.000; p=0.012). Concurrently there was a 2.5 fold decrease in IL-32 and PR3 protein levels (n=4, p=0.023). CONCLUSION: IL-32 is upregulated in patients with acute GVHD. Determination of IL-32 in patients with suspected GVHD may support the diagnosis and the decision to treat.AAT interferred with T cell activation and the release of cytokines, including IL-32, suggesting that administration of AAT may have therapeutic potential in patients with acute GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High dose cyclophosphamide therapy in solid tumors: Therapeutic, toxic, and immunosuppressive effects

Cyclophosphamide (CY) in a total dose of 120 mg/kg was given over 2 days on one to three occasions to 12 patients with a variety of nonlymphoid solid tumors. Two of 10 patients with measurable disease had a partial response. One had embryonal rhabdomyosarcoma and the other had ovarian carcinoma. In the 20 treatment courses, the mean leukocyte count was less than 1,00O/mm3 for 7 days. Thrombocytopenia was variable with a mean nadir of 87,90O/mm3 on day 12. There were four instances of local infection, one of bacteremia, and 11 of fever of undetermined origin. Weight gain of 2 or more kg occurred after 12, and EKG changes compatible with cardiotoxicity after six courses of CY. Serial skin testing with recall antigens revealed a frequent transient loss, and in three patients a later overall gain, in responsiveness. All patients, were immunized with Vi antigen before CY, and with Vi and sheep red blood cells (SRBC) after CY. Specific immune tolerance to Vi, with a satisfactory response to SRBC, was found in six of 12 patients. Patients' responses to the lymphocytes of 2 ABO compatible normal subjects were measured in mixed lymphocyte culture (MLC) before and after CY. Patients were given 500 ml of whole blood from one of the subjects prior to CY. Following CY, the patients' responses to the blood donors' cells were not significantly lower than their responses to nondonor cells. Thus, tolerance to HL-A antigens was not demonstrable in RILC. Three patients had an increase from subnormal to normal reactivity to nondonor cells in MLC.

CD4+CD25+TREGS ACTIVATED IN VITRO WITH IL-2 AND IL-12P70 SUPPRESS REJECTION OF FULLY ALLOGENEIC GRAFTS IN ANIMALS WITH NO OTHER IMMUNOSUPPRESSION.

Hall, B. M.1; Verma, N. D.1; Boyd, R.1; Robinson, C. M.1; Tran, G. T.1; Wang, C.2; Bishop, G. A.3; Hodgkinson, S. J.1 Author Information

Effect of Dehydroepiandrosterone Sulfate on Maturation and Functional Properties of Interferon-α-Induced Dendritic Cells

We studied the effect of adrenal cortex hormone dehydroepiandrosterone sulfate on maturation and functional activity of interferon-alpha-induced dendritic cells. Dehydroepiandrosterone sulfate stimulated differentiation and maturation of interferon-alpha-induced dendritic cell, which manifested in a decrease in the number of CD14(+)cells and increase in the ratio of mature CD83(+)dendritic cells expressing costimulatory molecules (CD80 and CD86). The induction of dendritic cell differentiation after treatment with dehydroepiandrosterone sulfate was accompanied by an increase in the production of interferon-gamma. At the stage of dendritic cell maturation, the effect of dehydroepiandrosterone sulfate manifested in a 4-fold increase in tumor necrosis factor-alpha production. Dehydroepiandrosterone sulfate had little effect on the production of Th2/antiinflammatory cytokines at the stages of differentiation and maturation of interferon-alpha-induced dendritic cells. Dehydroepiandrosterone sulfate increased the ability of dendritic cells to stimulate Th1 cytokine production by T cells (interferon-gamma). This hormone had no effect on the ability of interferon-alpha-induced dendritic cells to activate CD3(+)IL-4(+)T cells in mixed lymphocyte culture.

ICOS/B7h Bidirectional Signal to Induce Secretion of IL-1β and IL-17 (46.3)

Abstract B7h is constitutively expressed by antigen presenting cells (APC) and binds ICOS expressed by activated T cells. ICOS triggering costimulates T helper (TH) cell activation and modulates their cytokine secretion, whereas the effect of B7h triggering in APC is unknown. This work investigated B7h reverse signalling in dendritic cells (DC). We induced DC maturation with LPS in the presence or absence of a soluble ICOS-Ig fusion protein; then, both cytokine secretion and allostimolatory activity were evaluated. B7h stimulation substantially modulated LPS-induced DC maturation by influencing cytokine secretion. The most striking effect was 80-fold increase of IL-1b secretion, but other changes were 3-fold increase of IL-10 and 2-fold decrease of TNF-a. In IL-1b secretion, B7h triggering acted as a "second signal" by activating caspase-1, required to cleave pro-IL-1b whose synthesis was induced by LPS. B7h triggering on LPS-activated DC also modulated their capacity to activate allogeneic T cells in mixed lymphocyte cultures (MLC) by decreasing the proliferative response and changing cytokines secretion, including 20-fold decrease of IFN-g and 5-fold increase of IL-17. This effect was partly ascribable to the cytokine milieu secreted by DC and particularly to the high IL-1b levels, known to be involved in differetiation of human Th17 cells. However, ICOS triggering too played a role in IL-17 secretion since blocking of the B7h/ICOS interaction inhibited IL-17 secretion in MLC with DC. Moreover, in naive Th cells activated with anti-CD3 antibodies, ICOS stimulation induced IL-17 secretion in the presence of exogenous IL-1b. In this work we showed that B7h-mediated reverse signaling induced IL-1b by DCs which in turn favors IL-17 secretion by Th cells. On the other side, we demonstrated that ICOS costimulation directly favors Th17 differentiation.

A Novel Approach to Prevent GvHD: Donor Cells Engineered to Display on Their Surface a Recombinant Form of FasL Protein Effectively Prevent Lethal GvHD in a Mouse Model

Allogeneic bone marrow transplantation (BMT) has the potential to cure a series of inherited and acquired hematological disorders and malignancies. BMT can also be used as a cell-based immunomodulatory approach to induce tolerance to foreign and auto-antigens for the prevention and/or treatment of foreign graft rejection and autoimmune disorders. The routine application of allogeneic BMT as a therapeutic intervention in the clinic, however, is complicated by graft-versus-host (GVH) reaction, which is the major cause of graft-versus-host disease (GVHD) with potential life-threatening complications. T cells specific for alloantigens are the primary culprit of GVHD. Although elimination of T cells from the donor bone marrow inoculum can curtail GVH reaction, it results in compromised engraftment. Thus, strategies targeting specific and effective elimination of only the pathogenic T cells may have important implications for routine application of BMT to the clinic for the treatment of a variety of diseases. The main objective of this study was to use a novel and practical approach, designated as ProtEx™, to engineer bone marrow cells to display on their surface a modified form of FasL protein with potent apoptotic activity as an immunomodulatory agent and test the capacity of the engineered cells to engraft in allogeneic recipients without complications of GVHD. ProtEx™ technology involves generation of chimeric molecules with a core streptavidin (SA), modification of cell membrane with biotin, and the display of chimeric molecules on the cell surface taking advantage of strong noncovalent interaction (10−15 M) between biotin and SA. This technology allows for rapid (~ 2 hr) and efficient (100% of the targeted cells) display of exogenous proteins of interest on any cell without compromising the function of the cell or the proteins. In this study, ProtEx™ was used to display SA-FasL protein on C57BL/6 T cells and BMCs and these cells were tested for elimination of alloreactive T cells as well as prevention of acute GVHD following transplantation into lethally (1000 cGy) irradiated F1 (C57BL/6xBALB/c) mice. We hypothesized that mature T cells in the BM inoculum displaying FasL will respond to the host alloantigens, upregulate the death receptor Fas, and undergo apoptosis following the engagement of FasL with Fas on the same or a different cell, resulting in the prevention of GVHD. In support of this hypothesis, we demonstrated specific elimination of C57BL/6 T cells engineered to display SA-FasL on their surface in response to BALB/c antigen presenting cells in mixed lymphocyte cultures. Transplantation of unmodified 10×106 BMCs comixed with 20×106 splenocytes of C57BL/6 mice into lethally irradiated F1 recipients (n=12) resulted in lethal GVHD in all recipients within 34 days. In marked contrast, transplantation of cells engineered to display SA-FasL on splenocytes only (n=9) or splenocytes and BMCs (n=9) effectively prevented acute GVHD in all recipients that survived over 80 days of an observation period. The importance of FasL-mediated apoptosis in immune homeostasis and tolerance combined with our ability to use a practical approach, ProtEx™, to display SA-FasL with potent apoptotic activity on the surface of BMC or mature T cells at the protein level to prevent acute GVHD is significant and may have immediate clinical application.

Delayed rejection of MHC class II-disparate skin allografts in mice treated with farnesyltransferase inhibitors

Farnesyltransferase inhibitors (FTIs), developed as anti-cancer drugs, have the potential to modulate immune responses without causing nonspecific immune suppression. We have investigated the possibility that FTIs, by affecting T cell cytokine secretion, can attenuate alloreactive immune responses. The effects of FTIs on murine alloreactive T cells were determined both in vitro, by measuring cytokine secretion or cell proliferation in mixed lymphocyte cultures, and in vivo, by performing skin allografts from H-2 bm12 mice to MHC class II-disparate B6 mice. We found that two different FTIs, ABT-100 and L-744,832, blocked secretion of IFN-γ, IL-2, IL-4, and TNF-α from naïve T cells in vitro. ABT-100 and L-744,832 blocked cytokine production from both CD4 + and CD8 + naïve T cells stimulated with CD3 and CD28 antibodies, but only if the cells were pretreated with the FTIs for 48 h. Proliferation of alloreactive T cells in mixed lymphocyte cultures was blocked by either FTI. We also found that the proliferation of enriched T cells stimulated with IL-2 was blocked by ABT-100 treatment. In mice with an MHC class II-disparate skin graft, rejection of primary allografts was significantly delayed by treatment with either ABT-100 or L-744,832. Secondary rejection in mice previously primed to the alloantigen was found to be unaffected by L-744,832 treatment. We have shown that FTIs can block T cell cytokine secretion and attenuate alloreactive immune responses. The ability of FTIs to block secretion of cytokines, including IFN-γ and IL-4, from naïve T cells provides a likely biological mechanism for the specific suppression of class II MHC-mediated allorejection. These results demonstrate that FTIs may have useful immunomodulatory activity due to their ability to delay priming to alloantigens.

Cord Blood Nucleated Cells Induce Delayed T Cell Alloreactivity

Cord blood (CB) mononuclear cells (MNCs) can be transplanted in HLA mismatched recipients with limited graft rejection or graft-versus-host disease (GVHD). Previous studies have shown that naïve T cells and hyporesponsive dendritic cells are largely represented in CB. Data presented here demonstrate that CB MNCs are unable to stimulate allogeneic T cell proliferative or cytotoxic responses in standard in vitro assays. However, a suppressive effect of CB MNCs was ruled out because purified CD34 + cells or CD14 + monocytes stimulated T cell responses that were not inhibited by add-back of CB MNCs. The lack of antigen-presenting cell (APC) activity of CB MNCs in primary mixed lymphocyte culture (MLC) did not induce allogeneic T cell anergy. In fact, rechallenge of T cells with CB CD34 + cells, or immature monocyte-derived dendritic cells (iMo-DCs) in secondary MLC induced potent T cell proliferative responses. A delayed APC activity of CB MNCs was observed after stimulation with irradiated allogeneic T cells for 6 days, likely because of the upregulation of CD86 and HLA-DR on CB cells. Cytotoxic lymphocytes (CTL) were generated after stimulation of blood T cells with CB MNCs for 4 weeks or CB-derived iMo-DCs for 1 week. Concomitant stimulation of T cells with CB iMo-DC obtained from 2 CB units resulted in the generation of CTLs specific for each CB, independently of the CB:CB cell ratio. These data suggest that the APC activity of CB cells possibly increases posttransplant, and may contribute to delayed graft rejection and/or acute and chronic GVHD.

Enhancement of T cell activation by immobilized hu5C8 (anti‐CD40L) monoclonal antibody

Soluble monoclonal antibodies (MoAb) targeting CD40L on T cells can partially block T cell alloreactivity by preventing the costimulatory signal of antigen presenting cells through CD40. However, it is not known if these MoAbs can also deliver inhibiting or stimulating signals through the CD40L receptor. Blood mononuclear cells were stimulated by mitogens or allogeneic stimulator cells in the presence of hu5C8 MoAb, either in soluble form, or immobilized to the culture wells. T cell responses were evaluated by means of primary and secondary mixed lymphocyte culture (MLC), cytotoxic T lymphocyte (CTL) generation, immunophenotype, apoptosis assay and cytokine release. Also, the effect of hu5C8 on cells inhibited by CTLA4-Ig was tested. While the soluble hu5C8 inhibited T cell proliferation, the immobilized hu5C8 enhanced both mitogen and alloantigen-induced proliferative and cytotoxic T cell responses, without inducing further apoptotic T cell death. In the presence of CTLA4-Ig, immobilized hu5C8 increased the residual CD28-independent proliferation of alloantigen-specific T cells both in primary and secondary MLC, and prevented the inhibiting effect of CTLA4-Ig on the generation of CTL. Immobilized hu5C8 MoAb-stimulated T cells also showed a limited capacity of producing interleukin (IL)-10, even in the presence of CTLA4-Ig. We show that the hu5C8 MoAb has a strong mitogenic activity when immobilized, likely due to higher crosslinking capacity as compared to the soluble antibody. Strategies to induce ex-vivo T cell responses against tumor or viral antigens by means of hu5C8 MoAb antibody will be exploited based on these findings.

Studies on naïve CD4+CD25+T cells inhibition of naïve CD4+CD25−T cells in mixed lymphocyte cultures

BackgroundNaïve CD4+CD25+T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4+CD25+T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood. MethodThe alloreactivity of naïve CD4+CD25+, CD4+CD25− and unfractionated CD4+T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (Lewis×PVG)F1 cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4+CD25+T cells. ResultsProliferation of CD4+CD25−T cells was significantly greater than unfractionated CD4+T cells to both allogeneic and syngeneic stimulator cells. CD4+CD25+T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3− cells. Admixing CD4+CD25+T cells with CD4+CD25−T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4+T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-γ, IL-10, TGF-β and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4+CD25+T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4+CD25−T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-γ, IL-10, TGF-β, IL-5 or iNOS did not prevent CD4+CD25+T cell's inhibition of CD4+CD25−T cell proliferation. Blocking IFN-γ or iNOS enhanced CD4+CD25−T cell proliferation only in the absence of CD4+CD25+T cells. Depletion of CD4+CD25+T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators. ConclusionsTwo independent mechanisms that control proliferation of CD4+CD25−T cells in MLC were identified that naive CD4+CD25+T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4+CD25−T cells producing IFN-γ to induce iNOS.

Rapamycin Enhances the Number of Alloantigen-Induced Human CD103+CD8+ Regulatory T Cells In Vitro

Regulatory T cells (T(reg) cells) may be operational in both the induction and maintenance of transplantation tolerance. We recently showed that alloantigen-induced CD103+ CD8+ T cells strongly suppressed T-cell proliferation in mixed lymphocyte culture (MLC) via a contact-dependent mechanism. CD103 directs T lymphocytes to their ligand E-cadherin, which is expressed on renal tubular epithelial cells, and CD103+ CD8+ T cells have been described to be present in late renal allograft rejection. We studied the influence of prednisolone, cyclosporin, tacrolimus, CD25 monoclonal antibodies, rapamycin, and mycophenolate mofetil (MMF) on the development and functional activity of alloantigen-activated CD103+ CD8+ T cells in MLC. Calcineurin inhibitors, MMF, and CD25mAb did not influence the number of CD103 expressing CD8+ T cells. In contrast, corticosteroids diminished CD103 expression on alloactivated CD8+ T cells, which appeared to be caused by their inhibitory action on myeloid dendritic cells. Addition of rapamycin to allocultures led to an increased percentage of CD103+ CD8+ alloreactive T cells. Moreover, in the presence of rapamycin, these cells tended to show higher suppressive capacity. Alloreactive CD103+ CD8+ T(reg) cells may expand and exert their suppressive function during immunosuppressive treatment with rapamycin. These data are relevant in the design of immunosuppressive drug regimens intended to induce and/or maintain transplantation tolerance.

Rapamycin Does Not Induce Anergy but Inhibits Expansion and Differentiation of Alloreactive Human T Cells

Studies in mice have shown that rapamycin inhibits cell cycle progression and promotes the development of clonal anergy. We here addressed the question if rapamycin can induce anergy of human T cells and studied the effects of rapamycin on activation, proliferation and expression of cytotoxic effector molecules of alloresponsive T cells in mixed lymphocyte cultures. Peripheral blood mononuclear cells from healthy individuals were labeled with CFSE to monitor subsequent cell divisions. Cells were cocultured with allogeneic irradiated cells in the presence or absence of rapamycin. Flowcytometric analysis was performed after staining for surface CD4, CD8, and CD25 and for intracellular perforin, granzyme B, active caspase-3, and TGF-beta. Bio-Plex cytokine assay was done to measure the secretion of IL-2, IL-4, IL-10, and IFN-gamma. Addition of rapamycin at a final concentration of 10 ng/ml strongly decreased precursor frequencies of alloreactive CD4+ and CD8+ T cells. However, when these cells were washed and subsequently specifically restimulated in the absence of rapamycin, the proliferative capacity appeared normal. Next to lowering precursor frequencies, rapamycin also inhibited T cell expansion by inducing apoptosis in divided alloreactive CD4+ and CD8+ T cells. Rapamycin did not interfere with the formation of CD25brightCD4+ T cells during allogeneic stimulation and did not inhibit their suppressive function. Furthermore, the drug decreased production of effector molecules perforin and granzyme B by alloreactive T cells and diminished alloreactive cytotoxicity. Our data show that rapamycin strongly inhibits proliferation and effector functions of alloreactive T cells in vitro, but does not induce alloantigen specific nonresponsiveness.