High-dose IL-2 Research Articles

A New Humanized Mouse Model for Alopecia Areata

Although alopecia areata (AA) is not life threatening, it may lead to severe psychological disturbances, reducing the quality of life in all ages. Thus, a new animal model is needed for shedding more light onto the pathogenesis of this cell-mediated, organ-specific autoimmune disease to identify more effective therapeutic strategies. Recently, we succeeded in developing a new humanized mouse model of AA, which includes transplantation of healthy human scalp skin obtained from normal volunteers on to severe-combined immunodeficient mice. This is followed by intradermal injection of either autologous or allogeneic peripheral blood mononuclear cells, which had been cultured with high dose of IL-2 and enriched for natural killer group 2D-positive (NKG2D+) and CD56+ cells. This protocol leads to rapid and predictable development of focal hair loss, with all the characteristic clinical, histological, and immunohistochemical features of AA. This humanized mouse AA model underscores the functional importance of NKG2D+ and CD56+ cells in AA pathogenesis and promises to be instrumental for identifying novel AA treatment strategies.

Generation Of Clinical Grade, Highly Cytotoxic NK Cells Using Feeder Cell Free, Particle-Based Technology

NK cell immunotherapy shows exciting promise, but inconsistency and variability remain as a significant challenge. Since NK cells comprise a small fraction (∼5%) of the peripheral blood mononuclear cell fraction, expansion of NK cells in vivo or ex vivo is a critical requirement to attain therapeutically effective dosages and to observe consistent positive clinical outcomes. Most of currently developed ex vivo expansion protocols depend on co-culture with various engineered and/or cancer derived stimulator/feeder cells to induce the proliferation of NK cells. The use of accessory cells poses significant challenges to clinical transfer. Our laboratory has developed a nanoparticle-based expansion technology that utilizes particles, few hundred nanometers in size, derived from the plasma membrane (PM) of K562 feeder cells expressing IL-15 and 41BBL on their surface (PM-mb15-41BBL). These particles in combination with low concentration of IL-2 induce selective and efficient expansion of NK cells within human peripheral blood mononuclear cells (PBMC). When PBMC are stimulated with PM-mb15-41BBL over 21 days the NK cell numbers increase exponentially between days 6 and 18 of culture. The numbers of NK cell increased on average 200 fold (range 104-557, n=11, 4 donors) after 12-13 days of culture in the presence of PM-mb15-41BBL particles (at 200 µg of membrane protein/mL). The expansions with the PM particles are comparable to those in the presence of live feeder cells that gave ∼200 fold (79-895, n=11, 4 donors). The PM-particle based NK expansion is far better in comparison to NK stimulation with soluble purified 41BBL, IL-15 and IL-2, at matching concentrations, that yielded only 3 fold (1-4, n=6, 3 donors) increase in NK cells. Furthermore, the NK cells expand selectively under these conditions where they initially consisted only about 10% of the population of PBMC isolated from fresh peripheral blood, but increased to more than 95% of the cell suspension after 14 days in culture. The extent of expansion and NK cell content on day 12 of culture was dependent on the concentration of PM particles used with 200 µg of PM protein/mL being the optimal dose. Thus, PM nanoparticles can expand NK cells as efficiently and selectively as feeder cells. Furthermore, the PM-particle based expansion is more reproducible between trials and with different donors as compared to NK cell expansion induced with feeder cells (coefficient of variation 63% vs. 88%, respectively). The NK cells expanded in presence of PM-particles were highly cytotoxic against several leukemia cell lines and also against patient derived AML blasts. Expanded NK cells were 4 to 9 times more potent against AML cell lines K562, KG1 and HL-60 as compared to freshly isolated NK cells that were pre-activated with a high dose of IL-2. The PM-particle expanded NK cells also were selectively cytotoxic where they efficiently killed patient derived CD34+ leukemia blasts while sparing healthy CD34- peripheral blood cells. The expanded NK cells were observed to have an increase in the expression of major activating receptors such as NKG2D, NKp44, NKp30 and of the death receptor ligand FasL. This expression difference corresponds well with the activated cytotoxic phenotype and is likely responsible for their increased cytotoxicity against AML cells. Pilot trials in NSG mice are currently ongoing. Disclosures: Solh: Celgene: Speakers Bureau.

IL-2 analogues: novel agents circumventing the expansion of T regulatory cells while promoting NK cell activation during IL-2 therapy

Meeting abstracts High dose (HD) IL-2 treatment is a potent form of immunotherapy that can induce durable complete responses in a fraction of metastatic melanoma patients. Its initial purpose was to activate the expansion and anti-tumor responses of NK cells. However, the efficacy of HD IL-2 in

Clinical benefit of high dose IL-2 (HD IL-2) therapy: evidence for improved overall survival in patients with stable disease

Background HD IL-2 is an approved immunotherapy for advanced melanoma and renal cell carcinoma with a reported objective response rate (ORR) up to 20%. Patients with stable disease (SD) following treatment are not considered objective responders and thus are not included in reported response rates. Since the initial reports of IL-2 our understanding of the delayed pharmacokinetics of immunotherapy has improved. The purpose of this study is to report the clinical outcomes for patients with stable response to HD IL-2. Methods A retrospective chart review of 305 patients diagnosed with metastatic renal cell carcinoma or melanoma and treated with HD IL-2 therapy over a 12-year period was conducted. Age, response to HD IL-2 (based on RECIST criteria) and survival data were available in 215 patients at the time of analysis. Survival analysis was conducted on all patients for whom complete data was available and Kaplan-Meier curves were generated based on overall survival. Results Among the 215 patients with complete data, 62% had progressive disease (PD), 24% had SD and the objective response rate was 14%. Median overall survival was 16.8 months among all 215 patients. Patients with a PR or CR (median survival not reached in the study period) had significantly prolonged survival than the remaining patients (median survival 13.9 months, log-rank p < 0.01). Patients with SD (median survival of 38.2 months) had improved survival compared to patients with progressive disease (median survival of 7.9 months, log-rank p< 0.01). Discussion The IL-2 literature has focused on partial or complete responders when reporting objective response rates. In our data, as well as elsewhere in the literature, there are a large number of patients who have as table response to HD IL-2 that have not been included in the ORR. Our data supports a significant survival benefit associated with stable disease following IL-2, which suggests that HD IL-2 may result in a clinical benefit in a larger number of patients than has previously been reported. Consideration of ad isease control rate (DCR) inclusive of objective responders and stable responses may be more appropriate to better define the clinical benefit of IL-2.

The role of surgery following incomplete response to high-dose IL-2 (HD IL-2)

Meeting abstracts Surgical resection of metastatic cancer is beneficial in select patients with cancer. HD IL-2 is an FDA approved immunotherapy for the treatment of patients with melanoma or renal cell carcinoma. IL-2 induces a complete response (CR) in 4-10% of patients while an additional 10% of

Phenotype and Function of T Cells Infiltrating Visceral Metastases from Gastrointestinal Cancers and Melanoma: Implications for Adoptive Cell Transfer Therapy

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

169 : Direct in vitro generation of antigen specific, memory phenotype, Cd8+ T cells using a cocktail of small molecules and gamma-chain cytokines

Adoptive T cell transfer is an emerging cancer immunotherapy that has yielded complete responses in solid tumors such as B cell lymphoma and melanoma that are incurable by standard therapies. Prior studies have demonstrated that transfer of less differentiated T cells with greater proliferative capacity is associated with better clinical responses. Conventional expansion of antigen specific CD8 T cells uses high dose IL-2 and generates effector cells with high cytolytic and little proliferative capacity. We hypothesized that altering culture conditions by adding known cytokines and small molecules that inhibit acquisition of effector function and encourage memory formation would be associated with greater efficacy of transferred cells. We have used peptide antigen pulsed dendritic cells combined with a cocktail of homeostatic and memory-inducing cytokines that bind to the common gamma chain (IL-7 and IL-21) along with small molecules that inhibit glycolysis and enhance Wnt signaling to rapidly generate tumor specific, less-differentiated CD8 T cells with memory properties. Cells grown by this method are highly antigen specific (>90%) and are ∼50% central memory (CD62L+CD44+) or naive (CD62L+CD44−) phenotype versus virtually undetectable levels of these cells in cultures grown under standard conditions (i.e. with large amounts of IL-2). Compared to highly activated effector cells grown in IL-2, these memory-like cells engraft more efficiently into lymphopenic tumor bearing mice, maintain memory (CD62L+) phenotype for several weeks, and have greater anti-tumor efficacy in vivo. This approach is particularly useful since rapid advances in bioinformatics will soon make practical the identification of CD8 T cell epitopes derived from patient specific mutations. Since this method represents a way to generate more effective T cells that target tumor specific mutations, these results have significant implications for extending the usefulness of adoptive transfer to non-immunogenic (i.e. non-TIL bearing) solid tumors that could be targeted with personalized immunotherapy.

High-dose interleukin-2 (HD IL-2) in the treatment of advanced melanoma: The University of Pittsburgh experience.

9075 Background: IL-2 is a T-cell growth factor tested in a variety of regimens for advanced melanoma (MEL) and renal cell carcinoma (RCC). High-dose IL-2 (600,000-720,000 IU/kg administered intravenously every 8 hours for up to 14 consecutive doses) was approved by FDA for advanced MEL and RCC in 1998 based upon the durability of responses observed. Early studies of HD IL-2 reported overall (OR) and complete response (CR) rates of 16% and 8% respectively. Severe toxicity limited use to specialized centers with standardized protocols, either intensive care (ICU) or oncology specialty settings. The U Pittsburgh has treated 1022 patients with IL-2 at any dosage and we here present outcomes of 550 MEL pts treated with HD IL-2 in an oncology specialty non-ICU setting. Methods: Clinical and radiological data were collected on all pts treated with IL-2 using the UPCI Cancer Registry and Medical Archival System (MARS). Pharmacy records were reviewed for dosing details. The influence of baseline characteristics on treatment outcomes was assessed using Cox proportional hazards analysis. Results: A total of 848 pts received HD IL-2, of which 298 pts had RCC while 550 had MEL. Detailed pharmacy dosing records were reviewed from 176 pts treated over the past 12 years (2000-2012) who received a total of 3738 cycles. Of 165 pts evaluable for response, OR was documented in 24 pts (14.8%) and CR in 5 pts (3.0%). Median overall survival (OS) was 10.0 mos for all patients and 21.5 mos for responders (CR+PR). Median number of doses per cycle was 7. Toxicity was consistent with prior reports. HD IL-2 required ICU transfers in 5% and 1 death was attributed to HD IL-2. Pts with higher baseline lactate dehydrogenase (LDH) had poorer OS (p &lt; 0.05). Conclusions: In this large and uniformly treated series of recent patients treated with IL-2 OR/CR rates with HD IL-2 are 14.8% and 3.0% respectively. Higher LDH is associated with poorer outcome. Biomarkers of response are currently being evaluated in banked clinical specimens collected from patients under the SPORE in Skin Cancer (P50 CA121973).

Salvage-Targeted Kidney Cancer Therapy in Patients Progressing on High-Dose Interleukin-2 Immunotherapy

To analyze the outcomes of patients with metastatic renal cell carcinoma treated with salvage-targeted therapy after progressing on high-dose interleukin (IL)-2 immunotherapy in a tertiary referral center. A retrospective nonrandomized cohort consisting of 286 patients with metastatic renal cell carcinoma treated from 2003 to 2010 was analyzed from the University of California, Los Angeles (UCLA) Kidney Cancer database. All patients underwent cytoreductive nephrectomy, and 21 patients received salvage-targeted therapy after progression on high-dose IL-2, whereas 111 patients received targeted therapy alone. The remaining 154 patients had other treatment combinations or experimental targeted therapy agents only. Since 2003, selection of patients for high-dose IL-2 was increasingly based on clinical, pathologic, and molecular criteria (UCLA CPM criteria). Disease-specific survival was calculated from diagnosis of metastatic renal cell carcinoma. Patients selected according to UCLA CPM criteria and treated with salvage-targeted therapy after progressing on high-dose IL-2 experienced a significantly greater disease-specific survival (median not reached) than those treated with targeted therapy alone (30 months; P = 0.004). Since 2006, all high-dose IL-2 patients met the UCLA CPM criteria and were able to receive salvage-targeted therapy upon progression. Disease-specific survival calculated from initiation of targeted therapy was comparable for patients treated with salvage-targeted therapy after progression on high-dose IL-2 (34 months) versus first-line targeted therapy (26 months; P = 0.175). Patients selected for high-dose IL-2 based on UCLA CPM criteria and treated with salvage-targeted therapy upon progression have achieved outstanding disease-specific survival. Our data suggest a new algorithm for carefully selected patients with metastatic renal cell carcinoma based on UCLA CPM criteria to receive first-line high-dose IL-2 while reserving their option for salvage-targeted therapy with uncompromised efficacy upon progression.

Proliferation promotes functional changes in NK cells during viral infection. (P6054)

Abstract Following mouse cytomegalovirus (MCMV) challenge, NK cells control viral replication by killing infected cells and producing IFNγ in response to IL-12 induced at day 1.5 after infection. Previous work in our laboratory has demonstrated that under conditions of high viral replication, NK cells make significant amounts of IL-10 and deliver immunoregulatory functions at 3 to 4 days of infection (Lee et al., J. Exp. Med. 206:2235, 2009). The condition is accompanied by extensive NK cell proliferation. To test the hypothesis that proliferation is required for the changing function, culture systems were developed examining NK cell responses to IL-12 with and without division supported by high dose IL-2. Proliferating NK cells were induced to express the IL-10-reporter GFP in response to IL-12. One cell division was sufficient as was entrance into cell cycle measured by incorporation of BrdU. In contrast to intracellular IFNγ, proliferation was required for IL-10-GFP expression. Cell transfer studies into MCMV-infected mice demonstrated that IL-10-GFP levels were elevated with increasing divisions. When evaluated either in vitro or in vivo, decreasing intracellular IFNγ expression correlated with increasing IL-10-GFP expression as divisions progressed. Taken together, the studies reveal a previously unappreciated role for proliferation in shaping NK cell responses to infection. (Supported in part by the National Institutes of Health, USA.)

BTLA: new biomarker for a highly proliferative CD8+ TIL subset associated with melanoma regression during adoptive cell therapy (P2189)

Abstract Adoptive T cell therapy using tumor-infiltrating lymphocytes (TIL) expanded ex vivo with high-dose IL-2 is a promising approach for the treatment of metastatic melanoma. Recently, our lab demonstrated the importance of CD8+ T cells expressing B and T lymphocyte attentuator (BTLA) with a positive clinical response. This suggests that functional differences may exist between CD8+BTLA+ and CD8+BTLA- cells resulting in the differential therapeutic potency of TIL in treated patients. Here, we isolated BTLA+ and BTLA- CD8+ TIL from metastatic melanoma patients accrued in a Phase II clinical trial at MD Anderson and performed functional assays measuring proliferation, apoptosis, cytokine production, and CTL activity as well as differences in global gene expression profiles between the subsets using microarray analysis. Overall, BTLA appears to be a new and powerful biomarker distinguishing a less differentiated, more highly active and polyfunctional CD8+ T cell subset with high responsiveness to IL-2. As BTLA ligation by its binding partner HVEM (which is expressed on melanoma cells) results in a decreased proliferative responsiveness to anti-CD3 stimulation, paradoxically, BTLA signaling however may still serve as a negative co-inhibitory molecule. Our results also underscore that increased expression of T cell co-inhibitory molecules may not be necessarily markers of “exhaustion”, but markers of more highly-activated, responsive T cells susceptible to negative regulation.

Enhancing adoptively transferred T cells by stimulating with dendritic cell stimulation: exploring novel therapies (P4405)

Abstract Adoptive Cell Transfer (ACT) of tumor infiltrating lymphocytes (TILs) is a promising strategy for cancer immunotherapy. The current therapy relies on non-myeloablative lymphodepletion and high dose IL-2 therapy, which has serious side effects for patients. Optimization of the current ACT therapy, using animal models, may help reduce or eliminate these additional treatments and enhance the response rate. ACT has been evaluated in the autoimmune model RIP-GP mice, which express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) on the β-islet cells of the pancreas. Our findings showed that LCMV memory T cells stimulated with matured dendritic cells (DCs) and pulsed with peptides derived from LCMV, successfully induced diabetes. These stimulated T cells expanded well in vivo and showed strong killing activity after transfer. However, unstimulated memory tissue specific T cells rarely expanded after transfer and showed limited cytolytic activity. To evaluate this method in the tumor model, RIP-Tag2/GP double transgenic mice were used, which express the large T antigen under the control of RIP and spontaneously develop insulinomas. Islet specific T cells cocultured with DCs and LCMV-GP peptides displayed strong anti-tumor activity after transfer in vivo. Surprisingly, mice bearing insulinomas survived longer after ACT than mice treated with LCMV vaccination. Therefore, stimulating TILs before infusion has high potential for enhancing ACT therapy.

At diagnosis, diffuse large B‐cell lymphoma patients show impaired rituximab‐mediated NK‐cell cytotoxicity

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma in adults. It is generally treated by a combination of chemotherapy and CD20-specific mAbs, such as rituximab, which act, at least partially, by activating antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves NK cells, particularly the CD56(dim) NK-cell subset expressing CD16, the low affinity Fcγ receptor. Here, we show that CD16 expression levels are decreased in a cohort of 36 newly diagnosed DLBCL patients compared with those in 20 healthy controls (HCs). CD137, a co-stimulatory molecule expressed on activated NK cells, was also expressed at lower levels in patients compared with controls. Cells sampled from our cohort also showed severely reduced degranulation activity when challenged with rituximab-coated tumor cells, which could not be corrected by stimulation with high doses of IL-2. These results suggest that rituximab-induced NK-cell ADCC could be defective in some DLBCL patients at diagnosis. These patients should be closely monitored and attempts made to improve their NK-cell function.

The Immunocytokine L19–IL2 Eradicates Cancer When Used in Combination with CTLA-4 Blockade or with L19-TNF

Systemic high-dose IL2 promotes long-term survival in a subset of metastatic melanoma patients, but this treatment is accompanied by severe toxicities. The immunocytokine L19-IL2, in which IL2 is fused to the human L19 antibody capable of selective accumulation on tumor neovasculature, has recently shown encouraging clinical activity in patients with metastatic melanoma. In this study, we have investigated the therapeutic performance of L19-IL2, administered systemically in combination with a murine anti-CTLA-4 antibody or with a second clinical-stage immunocytokine (L19-TNF) in two syngeneic immunocompetent mouse models of cancer. We observed complete tumor eradications when L19-IL2 was used in combination with CTLA-4 blockade. Interestingly, mice cured from F9 tumors developed new lesions when rechallenged with tumor cells after therapy, whereas mice cured from CT26 tumors were resistant to tumor rechallenge. Similarly, L19-IL2 induced complete remissions when administered in a single intratumoral injection in combination with L19-TNF, whereas the two components did not lead to cures when administered as single agents. These findings provide a rationale for combination trials in melanoma, as the individual therapeutic agents have been extensively studied in clinical trials, and the antigen recognized by the L19 antibody has an identical sequence in mouse and man.

Ten-year follow-up of patients (pts) with metastatic renal cell carcinoma (mRCC) treated with interferon alfa-2b (IFN) as first-line therapy: Results from a randomized trial.

365 Background: We previously reported on improved tolerability and maintained efficacy of low-dose IFN in pts with mRCC treated in the cytokine era (Tannir, et al. Cancer 2006). Although VEGFR targeted agents have supplanted cytokines for most pts, IFN, in combination with bevacizumab, is a standard front-line therapy for good-risk and intermediate-risk clear-cell RCC. Methods: Between March 2002 and December 2003, a total of 118 pts (59/arm) were randomized to receive IFN 0.5 million units (MU) twice/day [IFN1] or 5 MU/day [IFN5]. Patients who were progression-free and tolerating therapy well continued IFN for a total of 5 years. The primary endpoint was progression-free survival (PFS). Secondary endpoints were objective response rate (ORR), overall survival (OS), safety, and quality of life (QOL). Results of PFS, ORR, safety, and QOL were previously reported. We provide here updated OS and long-term outcome of complete responders (CR). Results: At 10 years of follow-up, &gt;95% of participants have died. There was no significant difference in OS between the 2 arms (HR=1.32, 95% CI: 0.91-1.91), but favored IFN1 (median OS 2.1 years vs. 1.5 years, log-rank test p=0.14). Compared with IFN5, IFN1 was less toxic and was associated with better QOL. Two nephrectomised pts, 1 from each arm, 1 with multiple hepatic and lung metastases, and 1 with multiple lung and mediastinal metastases, performance status 1 and intermediate-risk clear-cell RCC, remain in unmaintained CR 10 years since starting IFN. Among pts who did not respond to IFN but remain alive, 1 patient received high-dose IL-2 and achieved durable CR; the other patient received multiple approved and investigational targeted agents sequentially. All responders to IFN had tumor regression as early as 8 wks, with PR or CR declared at 16 wks. Conclusions: IFN produces durable CRs and potential cure in about 2% of mRCC pts. Our data have implications for the front-line therapy of mRCC and suggest that IFN, like high-dose IL-2, can be discontinued after 16 wks, if a major response is not achieved. Our updated results are of interest, considering the paucity of durable CRs to VEGFR agents.

Outcomes of patients with metastatic renal cell cancer treated with sunitinib in clinical practice at a reference cancer centre in Manchester, United Kingdom.

477 Background: The development of anti-angiogenic therapy produced a change in the treatment of metastatic renal cancer (mRCC). There are now a range of drugs available with sunitinib being the most widely used first-line anti-angiogenic therapy. In the United Kingdom, until the advent of pazopanib it was the only such drug routinely funded by the NHS. The aim of this study was to audit the clinical outcomes of patients on routine treatment with sunitinib, to compare them with those obtained on trials and to analyze different subsets of patients. Methods: A database of mRCC patients treated at The Christie was set up to record clinical outcomes. Long-term follow-up data is available. Here we present a detailed analysis of 395 patients treated with sunitinib as a first-line anti-angiogenic therapy from 2005 to 2012. 397 patients treated out of a clinical trial. The outcomes were analysed according to various prognostic features. Results: The median OS of all patients audited was 18 months (m) with a PFS of 10.5m. This included clear cell RCC (n=305) and non-clear cell RCC (n=52). OS of clear cell RCC patients who received treatment second-line after cytokine therapy failure was 20.5m, longer than those treated first line 18.6m. This may be because we offer High-Dose IL2 to selected patients and these patients have a longer OS (24m) measured from the start of sunitinib. Baseline performance status was a predictor of outcome with OS being 23.5m for WHO PS 0/1 and 5.9m for WHO PS 2/3. Elderly age was not a predictor of poor outcome as the OS of patients &lt;60, 60-70, and &gt;70 was 15, 23, and 22m respectively. Time from nephrectomy to treatment with sunitinib had an influence on outcome with OS being 15, 24, and 48m for those &lt;1, 1-5, and &gt;5 years since nephrectomy. Further detailed analysis will be presented. Conclusions: Overall, the outcomes of patients treated in routine clinical practice were very similar to comparable groups treated on trials. Interestingly, the outcome post cytokine patients were particularly good but this may reflect particularly, careful choice of patients who receive cytokines since 2008.

Sequential expression of CD39 regulates late developmental T helper type 17 plasticity imparting a regulatory cell phenotype

BackgroundCD39 is an immune cell phenotype marker that exhibits ectonucleotidase activity, converting extracellular nucleotides to nucleosides. Although CD39 has been associated with regulatory T cells (Treg), the ectoenzyme is also expressed by a subpopulation of memory T cells (CD4mem) with effector functions. We postulate that CD39 imparts plasticity to effector T helper type 17 (Th17) as well as co-ordinating Treg cellular programmes of differentiation. MethodsExperiments were performed with peripheral blood mononuclear cells obtained from healthy blood donors. Sorted CD4+CD45RO+ memory cells (CD4mem) were exposed to interleukin (IL) 6 plus IL1β plus recombinant transforming growth factor β1 (rTGF) or IL6 plus IL1β plus IL23, or IL6 plus IL1β plus rTGF β1 plus IL23 to induce Th17 polarisation. Cells at Th17 stage were then treated with high-dose IL2 plus anti-CD3/anti-CD28 to favour Treg differentiation and then re-exposed to Th17 differentiating conditions to induce putative reverse or suppressive Th17 (rev/regTh17) cell. Impacts of purinergic mediators on cell effector phenotype and functions were assessed. FindingsCD4mem could be differentiated sequentially to Th17, Treg, and rev/regTh17. In contrast to the inflammatory properties associated with prototypic Th17 cells, rev/regTh17 exhibited a suppressive phenotype (ie, CD39high, CD73high, FOXP3+) and were able to control CD4+CD25− cell proliferation and pro-inflammatory cytokine (IFNγ, IL17) production. rev/regTh17 did not upregulate CD39, CD73, and FOXP3 and did not undergo increase in their suppressive function after culture with adenosine. InterpretationDifferential levels of expression of CD39 designate early Th17 cells from later Treg/revTh17 cell plasticity. The potential for Treg to revert to the inflammatory Th17 phenotype is mitigated by expression of CD39, as indicated by enhancements of suppressive function in vitro. FundingUK Medical Research Council.

Validation of Stereotyped Immunoglobulin Heavy Chain CDR3 Sequences As Candidate Antigens for Immunotherapy of CLL

AbstractAbstract 1775The immunoglobulin gene repertoire in CLL is remarkably restricted with greater than 30% of cases carrying quasi-identical (stereotyped)heavy complementarity-determining region 3 (VH CDR3) sequences. Indeed, cases can be clustered into different subsets based on shared, subset-biased motifs within the clonotypic VH CDR3s, with, notably, only a handful of subsets accounting for almost 10% of all CLL. VH CDR3 stereotypes are more frequent in cases with unmutated IGHV genes (U-CLL) who are associated with adverse prognosis. In principle, VH CDR3 stereotypy might allow to exploit these IG motifs as candidate Tumor Associated Antigens (TAA) for targeted immunotherapy of CLL. The aim of our study was to validate as potential TAA subset-specific IG motifs from major CLL subsets, focusing especially on subsets #1 and #2 that are the largest overall and both associated with aggressive clinical course.We have so far identified, by in silico analysis, 1–3 long peptides (15-mer) encompassing the VH CDR3 protein regions of subsets #1, 2, 4, 6, 8, 10 with (i) high binding score to MHC class II molecules and (ii) also containing minimal HLA class I-specific epitopes (HLA-A2, -A3, -A24, DR1, DR7, DR13 that are most frequent in the Caucasian population).Blood lymphocytes from 18 CLL patients were collected and phenotyped by flow cytometry with appropriate antibodies to assess the expression of stimulatory, co-stimulatory and negative regulatory molecules on both T and B cells. In addition, HLA typing of CLL patients was performed to select patients expressing the aforementioned HLA molecules. Overall, 13/18 patients matched the defined HLA class I and/or class II molecules.Negatively purified T cells from 11 CLL patients expressing HLA-A2 and/or DR13 have been then stimulated in vitro with the synthesized peptides of the specific stereotype (subset #1 and 2) in the presence of culture medium containing 5% of human serum plus IL-2 (20 IU/ml) and IL-15 (10 ng/ml). These T lymphocytes were then weekly stimulated with autologous irradiated antigen presenting cells (APC; monocytes, B cells, etc.) pulsed with the peptides. Starting from the third week of culture, the specific recognition of CDR3-derived TAAs and of tumor cells (autologous CLL cells) by the T cell cultures has been assessed by in vitro functional assays (ELISPOT assay).We were able to isolate CDR3- (subsets #1 and #2) and tumor-specific T cells from 5/11 CLL patients. In addition, in 4 selected patients the Ag- and tumor specific T lymphocytes have been expanded in vitro by Rapid Expansion Protocol (REP), based on the stimulation of T cells with allogeneic irradiated PBMCs from healthy donors plus OKT3 and high doses of IL-2. Using this protocol we were able to obtain large numbers (2–10 ×109) of anti-CDR3 T cells in all 4 cases tested, thereby, in principle, achieving the potential to use this protocol for expanding sufficient cells for clinical applications. Interestingly, post-REP T cell cultures showed enrichment (85–90%) of CD3+CD8+ T cells and down-modulation of negative regulatory molecules, such as CTLA-4, as compared to pre-REP in vitro stimulated T cells. These cells could be expanded in vitro for up to 6 weeks without any decay in proliferation.Taken together, these results indicate that stereotyped VH CDR3 peptide sequences can represent candidate antigens to elicit T cell-mediated anti-CLL responses, especially in poor prognosis cases, where therapeutic innovation is more urgently needed. After validation of this protocol in a larger series, our results may provide the proof of principle for the design of new immunotherapy protocols for CLL, including both active vaccination and adoptive cell therapy. Disclosures:No relevant conflicts of interest to declare.

Cytokine Activation Induces CD25 Expression and a Signaling-Competent High-Affinity IL-2 Receptor On CD56dim Human NK Cells.

Abstract 2159 Introduction.NK cells are innate immune lymphocytes important for host defense against infection, and also mediate anti-tumor responses. NK cell effector functions are triggered by ligand-mediated engagement of activating receptors and by cytokine stimulation. Human NK cells may be divided into developmental and functional CD56bright and CD56dim subsets with distinct biology. Both NK subsets have constitutive expression of CD122 (IL-2/15Rβ) and CD132 (γc), allowing for responsiveness to IL-15 and high doses (1 nM) of IL-2. The high affinity heterotrimeric IL-2 receptor is formed by CD122, CD132, and CD25 (IL-2Rα), allowing for responses to very low (10 pM) concentrations of IL-2. Previous studies demonstrated that resting CD56bright, but not CD56dim, NK cells constitutively express a functional high-affinity IL-2Rαβγ, as picomolar concentrations of IL-2 result in proliferation and co-stimulate IFN-γ production. We hypothesized that cytokine activation, which occurs at the site of an inflammatory response or interaction with a priming dendritic cell, may induce the expression of CD25 on CD56dimNK cells, allowing for enhanced responsiveness to IL-2. Methods.Purified normal donor NK cells (>95% CD56+CD3-) were cultured for 16h in media containing various cytokines, including IL-15 (100ng/mL) + IL-18 (50ng/mL), IL-15 (100ng/mL) + IL-12 (10ng/mL) or low dose IL-15 (1ng/mL) as a control (to support survival). Following stimulation, cells were washed and analyzed for surface expression of CD25 by antibody staining and flow cytometry. To assess the functional capacity of an induced IL-2Rα chain, NK cells were purified and treated with cytokines as above, washed extensively and replated in media devoid of all cytokines. At 3d after initial stimulation, cultures were briefly (15min) stimulated with IL-2 (10pM, 100pM and 1nM) or IL-15 (100ng/mL) to induce phosphorylation of STAT5. Cells were immediately fixed, permeablized, and assessed for intracellular phosphoSTAT5 by flow cytometry. Results.Pretreatment with IL-15 + IL-18 or IL-12 + IL-18, but no single cytokine, induced marked upregulation of CD25 on both CD56bright as well as CD56dim NK cells, with 93.6 ±1.9% of CD56bright and 97.7 ±1.2% (n=4) of CD56dim NK cells positive for CD25 expression immediately following the initial 16h stimulation with IL-15+18, compared to 20.7 ±6.8 and 3.6 ±1.4% for control treated (low dose IL-15) CD56bright and CD56dim NK. In preliminary experiments, CD25 appears to be markedly upregulated at the mRNA level, following treatment with IL-15+18. While kinetic analyses revealed that the absolute surface receptor expression was maximal at time points early after stimulation, 75.8 ±4.1% of CD56bright and 84.9 ±5.7% (n=4) of CD56dimNK cells pretreated with IL-15+18 retained CD25 surface expression at 7d post-stimulation.Importantly, CD25 induced on both CD56dim and CD56bright NK cells resulted in a signaling-competent high affinity IL-2 receptor, as stimulation with low dose IL-2 at 3d following initial cytokine pre-activation revealed increased production of phosphoSTAT5, versus control treated NK cells. The greatest enhancement was noted in CD56dim NK cells, showing an 8-fold increase in responsiveness to low dose (10pM) IL-2 stimulation (48.2±7.9% pSTAT5+ in IL-15+18 pretreated cells vs. 5.8±2.4% in control treated cells, p<0.02). Cytokine-pretreated CD56bright NK cells demonstrated no enhancement in phosphoSTAT5 following stimulation with low dose IL-2 (10pM) (27.6 ±8.3% in IL-15+18 pretreated vs. 26.0 ±6.4% in control) at this time-point. However, these results are in agreement with published data, which describe expression of CD25 on resting CD56bright but not CD56dimNK cells. Conclusions.Here, we report the induction of CD25, and a signal-competent high-affinity component of the IL-2 receptor, on CD56dim human NK cells, following cytokine pre-activation. These results have implications for the function of both CD56bright and CD56dim NK cells in the context of inflammation and potential for cross-talk with IL-2-producing T cells during an adaptive immune response. In addition, since rhIL-2 is clinically available and used following NK cell adoptive transfer in leukemia patients, these data provides a rationale for low dose IL-2 following allogeneic NK cell pre-activation with combinations of IL-15, IL-12, and IL-18. Disclosures:No relevant conflicts of interest to declare.

Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery

Background. Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. Material and methods. This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. Results and conclusion. Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-β and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.