DATING DRY BURN INJURY IN HUMAN PATIENTS BY FLOW CYTOMETRY OF CD4+ AND CD8+ T-CELLS IN THE BLOOD
OBJECTIVES/SPECIFIC AIMS: Objective: apply checkpoint inhibitors that are specific to the exhaustive markers expressed on tumor CD8+ T-cells ex vivo in order to improve cytokine release and cytotoxic function in comparison to two control groups: (1.) T-cells that receive no antibodies; (2.) T-cells that receive standard inhibition with PD-1 and CTLA-4 antibodies only. Long-term objective: provide personalized medicine in the treatment of HCC by using checkpoint inhibitors that are specific to the receptors expressed by an individual tumor. METHODS/STUDY POPULATION: The study population includes patients undergoing liver transplantation or surgical resection for HCC. Two grams of tumor, two grams of healthy liver tissue at least one centimeter from the tumor margin, and 50 milliliters of blood will be obtained. Solid tissue will be mechanically and enzymatically disrupted and CD8+ T-cells will be isolated from all sites. Using flow cytometry, the expression of surface receptors PD-1, CTLA-4, LAG-3, TIM-3, BTLA, CD244, and CD160 will be categorized in each tissue to identify which receptors are upregulated in the tumor microenvironment. Up to three antibodies specific to the upregulated receptor(s) on the tumor T-cells will be applied per specimen. The experimental arm will receive these antibodies and co-stimulation with CD3/CD28 and will be compared to two controls. One control will receive only CD3/CD28, and the other will receive CD3/CD28 in addition to the standard combination of PD-1 and CTLA-4 inhibitors. From each condition, flow cytometry will be used to assess the mean production of interleukin-2, tumor necrosis factor-α, interferon-γ, granzyme B, and perforin expression as an assessment of T-cell function. RESULTS/ANTICIPATED RESULTS: Preliminary data from the peripheral blood of healthy controls confirms that the developed flow cytometry panels effectively identify the surface receptors and cytokine production of CD8+ T-cells. Two patients have successfully been enrolled in this study. It is predicted that T-cells extracted from the tumor will express more inhibitory receptors than normal liver or peripheral blood and will have increased function after they are targeted with checkpoint inhibitors that are specific to the inhibitory surface receptors they express. DISCUSSION/SIGNIFICANCE OF IMPACT: HCC is the second leading cause of cancer-related death worldwide and therapeutic options are limited for patients who are not surgical candidates. T-cells are a critical component of the anti-tumor response to HCC. However, T-cells can develop an exhausted phenotype characterized by up-regulated inhibitory receptors (PD-1, CTLA-4, LAG-3, TIM-3, CD-244, CD-160, BTLA) and decreased function, allowing for immune escape. Clinical trials using combined checkpoint inhibition with PD-L1 and CTLA-4 antibodies have been considered a breakthrough for patients with advanced HCC, as up to 25% show an objective tumor response. The explanation for the varied susceptibility to checkpoint inhibition remains unknown and is hypothesized to be secondary to inconsistencies in the expression of surface inhibitory receptors. Although inhibitory receptor expression has been shown to be upregulated under conditions of hepatitis and/or HCC, there has been no single study to effectively investigate the expression of all known inhibitors in order to better explore the interplay between them. It will be of great academic interest and clinical purpose to evaluate individual receptor expression and engage the correlating antibodies given the possibility of synergism between receptors and the need for a more profound anti-tumor T-cell response in HCC.
Abstract AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation.
Abstract Background: The severe morbidity and mortality associated with bone marrow transplantation (BMT) is caused by uninhibited immune responses to alloantigen and suppressed immune responses to pathogens. Vasoactive Intestinal Peptide (VIP) is an immunomodulatory neuropeptide produced by T-cells and nerve fibers in peripheral lymphoid organs that suppresses immune responses by induction of tolerogenic dendritic cells. In order to determine the immunoregulatory effects of VIP, we examined T-cell immune responses to allo- and viral-antigens in VIP knockout (KO) mice and mouse BMT recipients of hematopoietic cells from VIP KO donors. Methods: VIP KO mice and VIP WT littermates were infected with lethal or sub-lethal doses (5 × 104− 5 × 105 PFU) of murine cytomegalovirus (mCMV) and the T-cell response to viral antigen was measured by flow cytometry for mCMV peptide-MHC class 1-tetramer+ CD8+ T-cells. We transplanted 5 × 106 BM plus 1 × 106 splenocytes (SP) either from VIP KO or VIP WT donors in an C57BL/6 to F1(BL/6 × Balb/c) allo-BMT model and assessed survival, GvHD, donor T-cell expansion, chimerism, and response to mCMV vaccination and mCMV infection. Results: B-cell, αβ and γδ T-cell, CD8+ T-cell, CD11b+ myeloid cell, and dendritic cell numbers were equivalent between VIP KO and WT mice, while VIP KO mice had higher number of CD4+ and CD4+CD62L+CD25+ T-cells. Non-transplanted VIP KO mice survived mCMV infection better compared to VIP WT, with a brisker anti-viral T-cell response in the blood. In the allogeneic BMT setting, recipients of VIP KO BM plus VIP KO SP had more weight loss and lower (40%) 100 day post-transplant survival compared to the recipients of VIP KO BM plus WT SP (80% survival), recipients of WT BM plus KO SP (100% survival), and recipients of WT BM plus WT SP (80% survival). Recipients of VIP KO grafts had a significantly greater anti-mCMV response that peaked four days earlier than the tetramer response of mice transplanted with WT cells. This increased anti-viral response to vaccination correlated with a greater and more rapid T-cell response to secondary viral challenge. Conclusions: These experiments suggest that the absence of all VIP in the body, or the absence of VIP in a transplanted immune system, enhances anti-viral immunity and allo-immune responses. Modulation of the VIP pathway is a novel method to regulate post-transplant immunity. Figure 1: VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day. Figure 1:. VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day.
Abstract Abstract 3539 Poster Board III-476 Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy. Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures: No relevant conflicts of interest to declare.
Abstract Abstract 2648 Background: Programmed death (PD)-1, a coinhibitory receptor expressed by effector T cells (Teffs) is highly expressed on intratumoral T cells (mean 61%, range 34–86% for CD4+ T cells and mean 44%, range 31–69% for CD8+ T cells) in follicular lymphoma (FL), a finding associated with impaired ability to recognize autologous tumor (Nattamai et al, ASH 2007). Hence, PD-1 expression would be expected to confer an unfavorable prognosis in FL. However, correlation of PD-1 with clinical outcome in FL has been inconsistent with two studies showing favorable (Carreras et al, J Clin Oncol 2009; Wahlin et al, Clin Cancer Res 2010) and one study showing unfavorable (Richendollar et al, Hum Pathol 2011) outcome. While differences in method of analysis and type of treatment may explain the disparate results, a more complex model may be necessary to understand the prognostic impact of PD-1 in FL as PD-1 is expressed not only on antitumor Teffs but also on protumor follicular helper T cells (Tfh) and regulatory T cells (Tregs). Methods: To determine the nature of PD-1+ T cells in FL we performed comprehensive genomic and immunologic studies. By flow cytometry, we observed that the intratumoral CD4+ T cells in FL may be categorized into 3 subsets based on PD-1 expression - PD-1 high (PD-1hi), intermediate (PD-1int), and low (PD-1lo). The intratumoral CD8+ T cells consisted of PD-1int and PD-1lo subsets. The 3 CD4+ T cell subsets were FACSorted from FL tumors (n=3) and whole genome gene expression profiling (GEP) was performed. T cell subsets sorted similarly from tonsils served as controls for reactive follicular hyperplasia (FH) (n=3). Differentially expressed genes in GEP studies were confirmed at the mRNA level by real-time PCR (n=5) and at the protein level by flow cytometry when antibodies were available (n=5–10). Results: Our results suggested that CD4+PD-1hi T cells are Tfh cells (CXCR5hiBcl6hi ICOShiCD40LhiSAPhiPRDM1loIL-4hiIL-21hi); the CD4+PD-1int T cells consisted of a mixture of activated Teffs (CD45RO+CD45RA−) including Th1 (Tbet+IFNg+), Th2 (IL-10+), and Th17 cells (RORc+IL-17+), and Tregs (Foxp3+CD25hiCD127lo); and the CD4+PD-1lo T cells consisted of a mixture of activated Teffs (CD45RO+CD45RA− but IFNg−IL-4−IL-10−IL-17−), Tregs, and naïve T cells (CD45RO−CD45RA+CCR7+). Although these subsets were present in both FL and FH, there were important differences. IL-4 expression was significantly higher in Tfh in FL vs. FH and may play a role in the pathogenesis of FL. IL-17 expression was low and expression of coinhibitory molecules BTLA and CD200 was high in CD4+PD-1int T cells in FL vs. FH. BTLA and CD200 were also increased in CD8+PD-1int T cells in FL vs. FH. However, other coinhibitory molecules (LAG-3, Tim-3, CD160, CTLA-4, CD244, KLRG1) were not significantly different between FL and FH. CD4+PD-1int T cells also had higher expression of BATF, a transcription factor associated with T cell exhaustion in FL vs. FH. Together, these results suggest that the CD4+PD-1int T cells in FL may be in a state of T cell exhaustion whereas the CD4+PD-1int T cells in FH may represent recently activated Teffs. Consistent with this, blocking PD-1 with anti-PD-1 blocking antibody significantly enhanced proliferation and the production of Th1 (IFNg, TNFa) but not Th2 (IL-4, IL-5, IL-10, IL-13) cytokines by intratumoral CD4+ and CD8+ T cells in response to stimulation with autologous FL tumor cells (n=3). As expected, Tregs were increased in number in FL vs. FH and were present in the PD-1int and PD-1lo T cell subsets. We found 74% (range 40–97%) of FL Tregs expressed PD-1. Among the CD4+PD-1lo and CD8+PD-1lo T cells, there were more activated Teffs and fewer naïve T cells in FL vs. FH. Conclusions: Our results suggest that the PD-1+ T cells in FL are comprised of a mixture of antitumor Teffs and protumor Tfh and Tregs. The prognostic impact of PD-1+ T cells in FL may dependent on the relative frequency of these subsets as ligation of PD-1 may produce favorable (inhibition of protumor Tfh and Tregs) or unfavorable (inhibition of antitumor Teffs) outcomes by inhibiting or promoting tumor growth, respectively. Conversely, our results imply that agents that block PD-1/PD-ligand pathway may have the opposite effect on these T cell subsets and enumeration of the intratumoral PD-1+ T cell subsets may serve as biomarker to predict response to these agents in FL and possibly other B-cell malignancies. Disclosures: Dong: GSK: Consultancy; Genentech: Honoraria; Tempero: Consultancy; Ono: Consultancy; AnaptysBio: Consultancy. Neelapu:Cure Tech Ltd: Research Funding.
Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.
AbstractAfrican classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.
Abstract Treatment of glioblastoma (GBM) remains a critical challenge and unmet medical need due to limited treatment options. SL-701 is a novel immunotherapy comprised of synthetic peptides designed to elicit a target-specific anti-tumor immune response against the GBM antigens IL-13Rα2, ephrinA2, and survivin. A multicenter, 2-stage, phase 2 clinical trial (NCT02078648) that evaluated the safety and efficacy of SL-701 in 74 adults with recurrent GBM was previously reported. This report describes preliminary data to suggest a correlation of immunocompetence to clinical outcome. In stage 2 (SL-701 + bevacizumab + poly-ICLC) the overall survival at 12 months was 50%. Two of 28 patients enrolled in stage 2 achieved CR (duration of response: 7.8 and 8.8 months) and 2 achieved PR (duration of response: 7.9 and 8.8 months). In a preliminary analysis to assess CD8+ T-cell responses, long-term survivors were comprised largely of subjects with an SL-701-induced target-specific CD8+ T-cell response, indicating a potential correlation of immunocompetence to clinical outcome. By week 24, SL-701-induced target-specific CD8+ T cells expressing IFNg were detected in 8 of 27 patients (30%) who had sufficient samples, with co-expression of PD-1, TIM3, and LAG3 detected in 4 patients. To further understand the T-cell response to SL-701, deep sequencing of target-specific CD8+ T cells using whole transcriptome-based molecular cytometry and high parameter (25+ color) flow cytometry is currently underway and updated data will be reported.
Abstract CD4+CD25+FOXP3+ regulatory T cells (TR) control proliferative CD4 and CD8 T cell responses to self and foreign antigens such as cytomegalovirus (CMV) and tumor-specific antigens. Thus, depletion of CD25-expressing cells from a resting population of T cells prior to antigen stimulation could boost the generation of antigen-specific T cells for adoptive transfer to treat viral infection or tumors. We depleted peripheral blood mononuclear cells (PBMC) from nine CMV seropositive donors using Miltenyi CD25 microbeads (20 μl/107 cells). CD25-depleted or unmanipulated PBMC were stimulated with CMV pp65-expressing antigen presenting cells for 10–14 days with low dose IL-2, and intracellular interferon-gamma (IFNγ) production by flow cytometry was compared between CD25-depleted and -undepleted cultures. An absolute increase in antigen-specific CD4+ and CD8+ T cells was seen after CD25 depletion in 4/8 and 5/8 cultures respectively. However, in other cultures there was a decrease or no change in IFNγ+ CD4+ T cells in CD25-depleted cultures, suggesting that the pp65-specific precursor cells had also been removed. We then used 4 μl beads per 107 PBMC to selectively remove only the CD25bright (predominantly Treg) population in nine donors and confirmed by flow cytometry that only CD25bright cells had been removed from the starting population. However, real-time quantitative PCR (Q-PCR) showed that even though the CD25+ fraction was enriched in FOXP3-expressing cells, a substantial proportion of the CD25-depleted PBMC still expressed FOXP3. Flow cytometric analysis of FOXP3 expression by CD25+ and CD25-negative CD4+ T cells showed that a substantial proportion of CD25- cells expressed FOXP3, confirming that CD25 is not a suitable single marker for depletion of Tregs. Again no reproducible augmentation of antigen-specific T cell responses was observed: one and five donors showing an increase in CD4 and CD8+ antigen-specific T cells, respectively, while the remainder showed a decrease or no change in CD4+ and CD8+ IFNγ-producing cells. These results suggest that removal of CD25+ cells from PBMC using CD25 microbeads removes both Treg and pp65-specific effector CD4+ and CD8+ T cells. Further, since FOXP3 is induced in responder cells as confirmed by FOXP3 Q-PCR, depletion of Treg at the start of the cultures may only transiently alleviate the negative regulation of the antigen response. Thus, CD25 depletion using microbeads is not a reliable method to boost antigen specific T cell expansions because of the inadvertent removal of a portion of the memory response to the antigen. Since it recently has been demonstrated that Treg act as an IL-2 sink, the addition of this cytokine should functionally silence the Tregs while preserving the inflammatory response.
Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.
