scholarly journals Myeloid-Specific SHP-2 Ablation Induces Robust Anti-Tumor Immunity That Is Not Further Enhanced By PD-1 Blockade

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Anthos Christofides ◽  
Natalia M Tijaro-Ovalle ◽  
Halil-Ibrahim Aksoylar ◽  
Rinku Pal ◽  
Abdelrahman AA Mahmoud ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Immunity ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Tumor Bearing ◽  
Suppressor Function ◽  
Emergency Myelopoiesis ◽  
Myeloid Progenitors

PD-1 is a T cell inhibitor for which blocking agents have achieved success as anti-cancer therapeutics. The current view is that cancer limits host immune responses by upregulating PD-L1 in the tumor microenvironment thereby causing PD-1 ligation and inactivation of CD8+ Teff cells. Recently, we determined that PD-1 alters the differentiation of myeloid progenitors during cancer-mediated emergency myelopoiesis. We found that PD-1 is expressed in granulocyte/macrophage progenitors (GMP), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSC) that promote tumor growth. In tumor-bearing mice with myeloid-specific PD-1 ablation, accumulation of GMP and MDSC was prevented, while output of effector myeloid cells was increased. PD-1-mediated T cell inactivation is attributed to the function of SHP-2 phosphatase, which is activated by recruitment to PD-1 cytoplasmic tail. Temporal activation of SHP-2 is critical for myeloid cell fate. Activating SHP-2 mutations prevent myeloid cell differentiation and lead to the accumulation of immature myelocytes and development of leukemia. To determine whether PD-1-mediated inhibition of anti-tumor immunity relies on SHP-2-mediated effects in T cells or myeloid cells, we generated mice with conditional targeting of the Ptpn11 gene (encoding for Shp-2) and selectively eliminated Shp-2 in T cells (Shp-2fl/flLckCre) or myeloid cells (Shp-2fl/flLysMCre). No significant difference in tumor growth was observed between control Shp2fl/fl and Shp-2fl/flLckCre mice bearing B16-F10 melanoma. Strikingly, Shp-2fl/flLysMCre mice had significantly diminished tumor growth that was not further decreased by anti-PD-1 antibody, in contrast to control Shp-2fl/fl mice in which anti-PD-1 treatment significantly reduced tumor size. To determine how Shp-2 ablation affected the properties of myeloid cells, we examined CD11b+Ly6ChiLy6G- monocytic (M-MDSC), CD11b+Ly6CloLy6G+ polymorphonuclear (PMN-MDSC), CD11b+F4/80+ tumor-associated macrophages (TAM) and CD11c+MHCII+ dendritic cells (DC). No quantitative differences were observed in these myeloid subsets in tumor bearing mice among the different groups. However, M-MDSC from Shp-2fl/flLysMCre mice had elevated expression of CD86 and IFNγ, consistent with effector differentiation. Suppression assays, by measuring antigen-specific responses of OTI transgenic T cells, showed significantly attenuated suppressor function of MDSC isolated from tumor-bearing Shp-2f/fLysMCre mice compared to control or Shp-2f/fLckCre mice. CD38 is a key mediator of MDSC-mediated immunosuppression. It is an ADP-ribosyl cyclase that has ectoenzyme and receptor functions, is induced early during differentiation of myeloid progenitors by retinoic acid receptor alpha (RARα) signaling, and mediates T cell immunosuppression. Because Shp-2 is involved in the differentiation of myeloid progenitors, we examined CD38 expression. We found that expression of CD38 was significantly reduced in MDSC from Shp-2fl/flLysMCre mice compared to control and Shp-2fl/flLckCre-tumor bearing mice. Since the suppressive potency of MDSC is decreased by autophagy, and SHP-2 has been implicated in regulating autophagy in cancer cells, we examined autophagy of MDSC in our system. Assessment of autophagy in ex vivo isolated MDSC, using Cyto-ID that stains the autophagosome membrane and indicates autophagic activity, showed enhanced autophagy in MDSC isolated from tumor bearing Shp-2fl/flLysMCre mice compared to control or Shp-2fl/flLckCre mice. Enhanced autophagy was also detected in bone marrow-derived MDSC from Shp-2fl/flLysMCre mice as determined by accumulation of LC3B-II and p62 during culture under conditions of starvation-induced stress. Consistent with the diminished MDSC suppressor function, myeloid cell-specific Shp-2 ablation in tumor-bearing mice induced an increase of CD8+ T cells showing an effector phenotype with improved functionality, despite preserved expression of PD-1 and Shp-2. Together these results indicate that inhibition of PD-1-mediated SHP-2 activation in myeloid progenitors, thereby preventing the accumulation of immature immunosuppressive MDSC and promoting the differentiation of effector myeloid cells, might be a previously unidentified mechanism by which PD-1 blockade mediates anti-tumor function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Metabolic Reprogramming of Myeloid Cells in Response to Factors of "Emergency" Myelopoiesis By Myeloid-Specific PD-1 Ablation, Regulates Myeloid Lineage Fate Commitment and Anti-Tumor Immunity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 14-14
Author(s):  
Laura Strauss ◽  
Jessica D Weaver ◽  
Rinku Pal ◽  
John Asara ◽  
Nikolaos Patsoukis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Cholesterol Synthesis ◽  
Myeloid Cells ◽  
Metabolic Reprogramming ◽  
Tumor Bearing ◽  
Mtorc1 Signaling ◽  
Emergency Myelopoiesis ◽  
Myeloid Progenitors

Abstract PD-1 is a T cell inhibitor for which blocking agents have achieved success as anti-cancer therapeutics. The current view is that cancer limits host immune responses by upregulating PD-L1 in the tumor microenvironment (TME) thereby causing PD-1 ligation and inactivation of CD8+ Teff cells. However, PD-L1 expression in the TME does not always correlate with therapeutic response. Thus, the mechanism(s) by which PD-1 blockade reverses compromised anti-tumor immunity are poorly understood. The rapid increase in hematopoietic cell output that occurs in response to immunologic stress is known as emergency myelopoiesis. Low-level stimulation by cancer-generated factors induces modest but continuous expansion of myeloid progenitors (MP) (common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP)) albeit with hindered differentiation, leading to output of tumor-promoting myeloid-derived suppressor cells (MDSCs). We determined that myeloid cells expanding during cancer-driven emergency myelopoiesis in tumor-bearing mice express PD-1 and PD-L1. Using PD-1 KO mice we found that PD-1 deletion prevented the accumulation of GMP and stimulated the output of Ly6Chi effector monocytes, macrophages and dendritic cells (DC). To determine whether these outcomes were mediated by a myeloid-intrinsic impact of PD-1 ablation or by the effects of PD-1neg T cells on myeloid cells, we generated mice with conditional targeting of the Pdcd1 gene (PD-1f/f) and selectively eliminated PD-1 in myeloid cells (PD-1f/fLysMcre) or T cells (PD-1f/fCD4cre). Myeloid-specific, but not T cell-specific PD-1 ablation, prevented the accumulation of GMP while promoting the output of effector-like myeloid cells expressing CD80, CD86, CD16/32 (FcRII/III) and CD88 (C5aR). Myeloid cells with PD-1 ablation had elevated expression of IRF8 that drives monocyte and DC differentiation and decreased expression of the MDSC hallmark markers IL-4R, CD206, ARG1 and CD38. Nutrient utilization has a decisive role on the fate of hematopoietic progenitors (HP) and MP. Stemness and pluripotency are regulated by maintenance of glycolysis whereas switch to mitochondrial metabolism is associated with differentiation. To examine whether PD-1 ablation affected these metabolic proceces, bone marrow (BM) from PD-1f/f and PD-1f/fLysMcre mice was cultured with G-CSF/GM-CSF/IL-6, key drivers of emergency myelopoiesis. MP differentiation was documented by decrease of Linneg and increase of Linpos cells, which was more prominent in PD-1f/fLysMcre BM cultures. This coincided with increase of CD45+CD11b+ and dominance of Ly6C+ monocytic cells consistent with a cell-intrinsic mechanism of monocytic lineage commitment. PD-1f/fLysMcre MP had elevated mTORC1, Erk1/2 and Stat1 activation, and enhanced glucose uptake and mitochondrial biogenesis. Bioenergetics studies showed robust development of a mitochondrial-dominant profile, consistent with metabolism-driven enhanced differentiation of MP. Mass spectrometry revealed enhanced intermediates of glycolysis, PPP and TCA cycle, but the most prominent difference was the increased cholesterol. Because mTORC1 signaling, which was enhanced in PD-1f/fLysMcre MP, activates de novo lipid and cholesterol synthesis via SREBP1, we examined the mevalonate pathway of cholesterol synthesis. mRNA for genes mediating cholesterol synthesis and uptake was increased whereas mRNA for genes mediating cholesterol metabolism was decreased. Cholesterol induces a proinflammatory program in myeloid cells, drives differentiation of monocytes, macrophages and DC and promotes antigen-presenting function. We examined how such changes in myeloid cells might affect the function of T cells, which are key anti-tumor mediators. Compared to tumor-bearing PD-1f/f mice, PD-1f/fLysMcre tumor-bearers had no quantitative T cell differences but had an increase in IFNγ- IL-17-, and IL-10-expressing CD8+ Teff-mem and IL-2-expressing Tcentral-mem cells, consistent with superior functionality. These changes correlated with enhanced anti-tumor protection despite preserved PD-1 expression in T cells. Our findings reveal a previously unidentified role of PD-1 in metabolism-driven myeloid cell lineage fate commitment and differentiation and suggest that switch to effector myeloid cells might be a key mechanism by which PD-1 blockade mediates systemic anti-tumor immunity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeted deletion of PD-1 in myeloid cells induces antitumor immunity

2020 ◽  
Vol 5 (43) ◽  
pp. eaay1863 ◽  
Author(s):  
Laura Strauss ◽  
Mohamed A. A. Mahmoud ◽  
Jessica D. Weaver ◽  
Natalia M. Tijaro-Ovalle ◽  
Anthos Christofides ◽  
...  
Keyword(s):  
T Cell ◽  
Antitumor Immunity ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Cell ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Effector Memory ◽  
Emergency Myelopoiesis

PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell–specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1–blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell–specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell–specific PD-1 ablation, myeloid cell–specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell–specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1–deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.

scholarly journals Phenotypic and functional analysis of T-cell precursors in the human fetal liver and thymus: CD7 expression in the early stages of T- and myeloid-cell development

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3401-3414 ◽  
Author(s):  
A Barcena ◽  
MO Muench ◽  
AH Galy ◽  
J Cupp ◽  
MG Roncarolo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fetal Liver ◽  
Cell Lineage ◽  
Myeloid Cell ◽  
Cell Development ◽  
Cell Subpopulation ◽  
Cd34 Antigen ◽  
Fetal Thymic Organ Culture ◽  
Myeloid Progenitors

Abstract It has been proposed that the CD7 molecule is the first antigen expressed on the membrane of cells committed to the T-cell lineage during human fetal T-cell ontogeny. To further identify the pre-T cell subpopulation that migrates to the thymus early in ontogeny, we analyzed the phenotypic and functional characteristics of the fetal liver populations separated on the basis of CD7 expression. Three populations expressing different levels of CD7 were observed: CD7bright, CD7dull, and CD7-. A CD7bright population depleted of mature T, B, and myeloid cells (lineage negative, lin-) and mostly composed of CD56+ CD34- natural killer cells did not mature into T cells in a fetal thymic organ culture (FTOC) assay and was devoid of myeloid progenitors in a clonal colony-forming cell assay. In contrast, the CD7-/dull CD34+ lin- populations were capable of differentiating into phenotypically mature T cells after injection into FTOC and contained early myeloid progenitors. Here we phenotypically compared the fetal liver CD7 populations with the most immature fetal thymic subset that differentiated in the FTOC assay, namely the triple negative (TN, CD3- CD4-CD8-) thymocytes. Fetal TN lin- expressed high levels of CD34 marker and were further subdivided by their expression of CD1 antigen, because CD1- TN thymocytes express higher levels of CD34 antigen compared with CD1+ TN cells. CD1- lin -TN thymocytes are characterized by expressing high levels of CD2, CD7, and CD34 markers and dull levels of CD5, CD10, and CD28 molecules. We could not find fetal liver pre-T cells with a phenotype equivalent to that of TN thymocytes. Our data show that CD7 does not necessarily identify T-cell precursors during fetal T-cell development and strongly support the hypothesis that the acquisition of early T-cell markers as CD2, CD28, and CD5 molecules on the cell surface of T-cell progenitors takes place intrathymically.

scholarly journals T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor.

1980 ◽  
Vol 151 (1) ◽  
pp. 69-80 ◽  
Author(s):  
M J Berendt ◽  
R J North
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Tumor Regression ◽  
Immunocompetent Host ◽  
Suppressor T Cells ◽  
Immunogenic Tumor ◽  
Tumor Bearing ◽  
Concomitant Immunity ◽  
Progressive Growth

The results of this paper are consistent with the hypothesis that progressive growth of the Meth A fibrosarcoma evokes the generation of a T-cell-mediated mechanism of immunosuppression that prevents this highly immunogenic tumor from being rejected by its immunocompetent host. It was shown that it is possible to cause the regression of large, established Meth A tumors by intravenous infusion of tumor-sensitized T cells from immune donors, but only if the tumors are growing in T-cell-deficient recipients. It was also shown that the adoptive T-cell-mediated regression of tumors in such recipients can be prevented by prior infusion of splenic T cells from T-cell-intact, tumor-bearing donors. The results leave little doubt that the presence of suppressor T cells in T-cell-intact, tumor-bearing mice is responsible for the loss of an earlier generated state of concomitant immunity, and for the inability of intravenously infused, sensitized T cells to cause tumor regression. Because the presence of suppressor T cells generated in response to the Meth A did not suppress the capacity of Meth A-bearing mice to generate and express immunity against a tumor allograft, it is obvious that they were not in a state of generalized immunosuppression.

scholarly journals CD4+ T Cells Are Able to Promote Tumor Growth through Inhibition of Tumor-Specific CD8+ T-Cell Responses in Tumor-Bearing Hosts

2005 ◽  
Vol 65 (15) ◽  
pp. 6984-6989 ◽  
Author(s):  
Annemieke Th. den Boer ◽  
Geertje J.D. van Mierlo ◽  
Marieke F. Fransen ◽  
Cornelis J.M. Melief ◽  
Rienk Offringa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Tumor Bearing ◽  
Promote Tumor Growth

scholarly journals Normal Myeloid Cells Are Required for Sustained CAR T Cell Activity Against Myeloid Tumor in a Humanized Mouse Model

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 734-734
Author(s):  
Miriam Y Kim ◽  
Matthew L Cooper ◽  
Julie K Ritchey ◽  
Julia Hollaway ◽  
John F. DiPersio
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Activity ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T cells are effective against B cell malignancies and multiple myeloma, but their efficacy has been limited to date for acute myeloid leukemia (AML). We sought to investigate whether there were fundamental differences in targeting B cell antigens as compared to myeloid antigens with CAR T cells, that may shed light on the mechanism of CAR T cell resistance in patients with AML. For these studies, we utilized human CAR T cells targeting CD19 (CART19) and CD33 (CART33), canonical B cell and myeloid cell antigens, respectively. To ensure that the potency of the two CAR constructs were equivalent, we generated dual CD19 and CD33 expressing cell lines, by adding CD33 to Ramos, a CD19+ B lymphoblastic cell line, and adding CD19 to THP-1, a CD33+ myeloid cell line. We confirmed that CART19 and CART33 were equally potent against CD33+Ramos and CD19+THP-1 cells. To investigate the influence of normal hematopoietic cells on CAR T cell behavior, we incubated CD19+THP-1 cells with CART19 and CART33 in the presence of peripheral blood (PB) or bone marrow (BM) mononuclear cells. We found that both PB and BM enhanced tumor clearance to a similar degree for each CAR construct. Additionally, IL-6 was detected in the supernatant of PB or BM co-cultured with CART19 and CART33, and these levels were markedly increased in the presence of tumor cells. Notably, THP-1 cells by themselves produced high levels of IL-6 upon exposure to CAR T cells, likely reflecting the myeloid origin of this cell line, while Ramos cultured with these same CAR T cells did not produce IL-6. We assessed other myeloid cell lines (U937, KG-1, Kasumi-3, Molm13, HL-60, and K562) and also noted IL-6 production when co-cultured with CART33, although the levels were significantly lower than that produced by THP-1. Of note, IL-6 levels were slightly but consistently higher with CART19 than with CART33 in these in vitro assays, which we attribute to the loss of normal myeloid cells from CART33-mediated killing. To study the effects of normal hematopoiesis on human CAR T cell activity in vivo, we injected NSGS mice with human cord blood CD34+ hematopoietic stem cells (HSCs) to generate a human hematopoietic system in these mice, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from T cells of the same cord blood product as the CD34+ cells. We confirmed the expected loss of human CD19+ B cells and CD33+ myeloid cells in the peripheral blood after CART19 and CART33 treatment, respectively. Surprisingly, we found that only CART33 treatment led to elevated plasma human IL-6 levels in this model. We then injected CD19+THP-1 cells to the mice after HSC engraftment, to assess the anti-tumor activity of the CAR T cells and to increase the potential for toxicity. Consistent with our in vitro data, mice with a human hematopoietic system cleared tumor more efficiently than mice without prior HSC engraftment after treatment with CART19 or CART33. However, while we observed mild weight loss and IL-6 elevation in mice after CART19 treatment, this effect was much more pronounced in mice that received CART33. We hypothesized that the presence of antigen on normal myeloid cells both increased the toxicity and decreased the efficacy of CART33, due to a massive release of inflammatory cytokines from myeloid cells in the immediate aftermath of CART33 treatment, followed by loss of the augmentation of CAR T cell activity mediated by myeloid cells in the long term. To test this hypothesis, we engrafted mice with either control HSCs or CD33 KO HSCs, followed by injection of THP-1 and CART33. Only mice with CD33 KO HSCs maintained myeloid cells after CART33, as expected. CD33 KO HSC-engrafted mice exhibited less toxicity after CART33 treatment than mice with control HSCs, in that they did not lose weight or demonstrate elevated IL-6 levels. Furthermore, absence of CD33 on myeloid cells led to enhanced CAR T cell expansion and persistence, that resulted in better long-term tumor control. In summary, our data suggests that targeting myeloid antigens with CAR T cells may be intrinsically self-defeating due to loss of myeloid cells that are required for sustained CAR T cell activity. These studies illuminate the challenges when extending CAR T cell therapy to myeloid malignancies, and highlight the importance of normal myeloid cells in augmenting T cell-based immunotherapies. Figure 1 Figure 1. Disclosures Kim: Tmunity: Patents & Royalties; NeoImmune Tech: Patents & Royalties. Cooper: RiverVest: Consultancy; Wugen: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties; NeoImmune Tech: Patents & Royalties.

scholarly journals Lactate Increases Stemness of CD8+ T Cells to Augment Anti-Tumor Immunity

2021 ◽  
Author(s):  
Qiang Feng ◽  
Zhida Liu ◽  
Xuexin Yu ◽  
Tongyi Huang ◽  
Jiahui Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Subcutaneous Administration ◽  
Tumor Growth Inhibition ◽  
Mouse Splenocytes

Nutrients and metabolites play important roles in immune functions. Recent studies show lactate instead of glucose can serve as a primary carbon fuel source for most tissues. The role of lactate in tumor immunity is not well understood with immune suppressive functions reported for lactic acid, the conjugate acid form of lactate. In this study, we report lactate increases the stemness of CD8+ T cells and augments anti-tumor immunity. Subcutaneous administration of lactate but not glucose shows CD8+ T cell-dependent tumor growth inhibition. Single cell transcriptomics analysis revealed lactate treatment increased a subpopulation of stem-like TCF-1-expressing CD8+ T cells, which is further validated by ex vivo culture of CD8+ T cells from mouse splenocytes and human peripheral blood mononuclear cells. The inhibition of histone deacetylase activity by lactate increased acetylation in the histone H3K27 site at the Tcf7 super enhancer locus and increased the gene expression of Tcf7. Adoptive transfer of CD8+ T cells pretreated with lactate in vitro showed potent tumor growth inhibition in vivo. Our results elucidate the immune protective role of lactate in anti-tumor immunity without the masking effect of acid. These results may have broad implications for T cell therapy and the understanding of lactate in immune metabolism.

Dipeptidyl Peptidase Inhibition Accelerates Dendritic Cell Cross Priming of Tumor-Reactive T Cells Resulting in Regression of Established Tumors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2579-2579
Author(s):  
Meghaan Walsh ◽  
Aviva C Krauss ◽  
Jessica PE Davis ◽  
Su Young Kim ◽  
Martin Guimond ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Regression ◽  
High Dose ◽  
Complete Regression ◽  
Tumor Bearing ◽  
Dc Vaccine ◽  
Dipeptidyl Peptidases

Abstract BACKGROUND: PT-100 is an aminoboronic dipeptide that competitively inhibits dipeptidyl peptidases. While PT-100 has no direct effect on tumor cells in vitro, it exhibits potent antitumor effects in vivo. We have shown that female C57BL/6 (B6) mice with MB49 tumors, which naturally express the male minor histocompatibility antigen complex (HY), are primed to HY, but the immune response is insufficient to control tumor growth. In this study, we used the well-characterized HY antigen system to examine the immunomodulatory effects of PT-100 during treatment-induced tumor regression. METHODS: B6 female mice were inoculated subcutaneously with MB49 (106 cells) on day 0 and treated daily with PT-100 by gavage. For re-challenge experiments mice received high dose MB49 (3×106 cells) three weeks after complete regression of primary tumors. IFN-g ELISPOT was used to measure HY antigen specific T cell responses in the spleen and lymph nodes (LNs) during tumor growth. For adoptive transfer experiments, T cells were magnetic-bead purified from LNs and spleens of tumor-bearing PT-100 treated, tumor-bearing sham treated, or naïve mice and injected intravenously into Rag1−/− recipients (1.2×106 cells) which were then inoculated with high dose MB49. T cells were depleted with monoclonal antibodies to CD4 and CD8. Dendritic cells (DCs) were depleted with diphtheria toxin (DT) in bone marrow chimeras expressing the DT receptor under the CD11c promoter. DC activation examined by flow cytometry. For vaccine experiments, HY-expressing DCs were cultured from male B6 bone marrow and injected intraperitoneally (1×105 cells). RESULTS: PT-100 treatment resulted in complete regression of MB49, even when limited to the first week (days 3–7) during tumor progression. Treatment started later than week 1 was insufficient to establish consistent, complete tumor regression. High-dose re-challenge of PT-100 treated mice resulted in initial growth followed by regression without additional PT-100. IFN-gELISPOT revealed a robust response against HY in spleens of controls on day 17. Interestingly, PT-100 treated mice had quantitatively similar priming, but the response peaked earlier (day 10), just prior to tumor regression. Purified T cells from PT-100 treated donors collected on day 17 mediated markedly enhanced tumor protection compared to recipients of T cells from sham treated tumor-bearing mice despite significantly more HY-reactive cells in the spleen and LNs of sham treated-tumor bearing mice by that time. T cell or DC depletion independently abrogated the anti-tumor effect of PT-100 and treatment with PT-100 increased CD80 and CD86 expression on LN DC populations in vivo. Although HY DC vaccination does not affect tumor growth, supplementation of the DC vaccine with PT-100 mediated a therapeutic effect resulting in regression of well-established tumors. CONCLUSIONS: PT-100 establishes a consistent and potent antitumor effect against MB49 dependent on T cells and DCs. Treatment results in a memory response that is protective against high dose MB49 re-challenge. PT-100-induced tumor regression is associated with enhanced early tumor priming, associated with increases in activated DCs. T cells from PT-100 treated mice elicit superior protection upon adoptive transfer compared to shams, despite quantitatively less tumor-primed T cells, suggesting the PT-100 antitumor effect may involve a qualitative difference in T cell function. PT-100 given as an adjuvant to a DC vaccine results in increased potency and regression of established tumors. Inhibition of dipeptidyl peptidases modulate naturally occurring anti-tumor immune responses and contribute to the generation of a therapeutic anti-cancer vaccine.

Agonist anti-GITR antibody induces CD8 T cell-mediated tumor rejection

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3058-3058
Author(s):  
A. D. Cohen ◽  
A. Diab ◽  
M. A. Perales ◽  
F. Duan ◽  
R. Jenq ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Tumor Antigens ◽  
Cell Function ◽  
Cd8 T Cell ◽  
Tumor Rejection ◽  
Cd8 Cells

3058 Background: Signaling through GITR (glucocorticoid-induced tumor necrosis factor receptor) can abrogate the suppressive effects of CD4+foxp3+ regulatory T cells and co-stimulate activated effector CD4+ and CD8+ T cells. We have previously shown that in vivo GITR ligation using the agonist anti-GITR mAb DTA-1 augments concomitant immunity and immunity generated by active immunization with self- tumor antigens. In the present study, we assessed the activity of anti-GITR mAb used alone, focusing on the effects of GITR ligation on CD8+ T cells during tumor growth. Methods: C57BL/6 mice were injected intradermally with B16 melanoma and received 1mg of DTA-1 or control rat IgG intraperitoneally on various days after tumor injection. In some experiments, naïve, CFSE-labeled pmel-1 CD8+ transgenic T cells (specific for the melanoma antigen gp10025–33 epitope) were transferred into naïve recipients 1 day prior to B16 inoculation. Results: DTA-1 treatment on days 0 and 4 led to tumor rejection in 20–30% and 50–60% of mice, respectively, compared with rejection in 0–5% of mice treated with control IgG (p<0.05 for both). Treatment at day 7 or later had no significant impact on tumor-free survival. The importance of CD8+ T cells in mediating DTA-1-induced tumor immunity was demonstrated by 4 findings: 1) in untreated mice, tumor-infiltrating CD8+ lymphocytes significantly upregulated GITR expression during tumor growth; 2) DTA-1-treated mice had greater CD8+ T cell infiltration into tumors than IgG-treated mice; 3) depletion of CD8+ cells completely abrogated the tumor protection provided by DTA-1; and 4) tumor-specific CD8+ cells proliferated more extensively, became more activated, and exhibited greater effector function following DTA-1 administration compared with control IgG. This was most dramatically seen within the tumor (compared with spleen or draining lymph node), suggesting that a major mechanism of tumor immunity induced by anti-GITR mAb may be overcoming impaired CD8+ T cell function within the tumor microenvironment. Conclusions: Ligating GITR using an agonist mAb can by itself augment tumor-specific CD8+ T cell responses and induce rejection of an aggressive, poorly immunogenic tumor. This strategy merits further consideration as an immune-modulating therapy for cancer. No significant financial relationships to disclose.

scholarly journals 2363 Inducing anti-tumor immunity in colorectal cancer

2018 ◽  
Vol 2 (S1) ◽  
pp. 15-16
Author(s):  
Jonathan B. Mitchem ◽  
Yue Guan ◽  
Mark Daniels ◽  
Emma Teixeiro
Keyword(s):  
Colorectal Cancer ◽  
Statistical Analysis ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Immunity ◽  
Suppressor Cells ◽  
The United States ◽  
Cytotoxic T Cell ◽  
Myeloid Derived Suppressor Cells ◽  
Tumor Bearing

OBJECTIVES/SPECIFIC AIMS: Despite significant advances in screening and treatment, colorectal cancer is the second leading cancer killer in the United States today. Some of the most promising recent developments in cancer therapy have come from immune-based therapy. Immune-based therapy, however, has shown limited utility in patients with colorectal cancer. Studies have previously shown that certain chemotherapy regimens may be more effective in combination with immune-based therapy due to induction of inflammation in the tumor microenvironment. In this study, we sought to determine how standard chemotherapy (FOLFOX) affects the generation of antigen-specific anti-tumor immunity in colorectal cancer. METHODS/STUDY POPULATION: To determine the how antigen-specific immunity and T cell responses are affected by FOLFOX, we utilized a model antigen expressing murine colon cancer cell line syngeneic to C57BL/6 (MC38-CEA). Treatment was initiated when tumor size reached 50 mm2. Mice were treated with either vehicle (PBS), 5-Fluorouracil (5-FU), Oxaliplatin, or combination (FOLFOX). Antigen-specific cytotoxic T cell (tet+Tc) were detected using Db-CEA-tetramer obtained from the NIH-tetramer core facility. Flow cytometry was performed for phenotypic analysis and tetramer positivity. Tumor growth was measured using standard caliper measurements. Statistical analysis was performed using t-test for continuous variables and ANOVA was used when comparing multiple groups. Statistical analysis was performed using SPSS. All arms were completed with n=3–7. RESULTS/ANTICIPATED RESULTS: To determine how systemic treatment with chemotherapy affects cytotoxic T cell development (Tc), we established that we could detect antigen-specific Tc (tet+Tc) in the spleen, tumor, and draining lymph nodes of tumor-bearing mice. After establishing that the system worked appropriately, tumor-bearing mice were treated with different chemotherapy regimens and tumor growth was monitored. As expected, the combination of FOLFOX was significantly better than either drug individually (2-way ANOVA, p<0.01). FOLFOX therapy also showed a significant (p<0.05) increase in the number of tumor-associated tet+Tc, and tet+Tc expressing phenotypic markers of effector (Te) and resident memory (Trm) subsets. Tumor-associated tet+Tc highly expressed PD-1 (>50%); however, this was not significantly different between treatment or vehicle arms. Since 5-FU, one component of FOLFOX has previously shown a selective reduction of myeloid-derived suppressor cells, we also investigated the myeloid compartment. There were no significant differences in conventional or plasmacytoid dendritic cells, myeloid-derived suppressor cells, or tumor-associated macrophages. DISCUSSION/SIGNIFICANCE OF IMPACT: The future of cancer care involves multi-modality care tailored to patients. To more effectively combine therapy it is critical that we understand how currently utilized therapy works. In this study, we show that the primary chemotherapy regimen utilized in colorectal cancer increases tumor-associated antigen-specific cytotoxic T cells and the majority of these cells are PD-1 positive. This suggests that FOLFOX may work in concert with immune-based therapy when selected appropriately. Further study is warranted to determine optimal combination therapy and ways to maximize anti-tumor immunity in order to improve the treatment of patients with this deadly disease.

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