scholarly journals 255 Investigating VISTA’s role intrinsic to T cells in the tumor microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A276-A276
Author(s):  
Cassandra Gilmour ◽  
Li Wang ◽  
Juan Dong ◽  
Sarah Stone ◽  
Keman Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Immunity ◽  
Patient Survival ◽  
Cytotoxic T Cells ◽  
Peripheral Tolerance ◽  
Checkpoint Blockade

BackgroundCancer immunotherapies, specifically checkpoint blockade therapies, have demonstrated clinical importance for long term patient survival. One of the major limitations to checkpoint blockade therapies, is the low response rate: ~30% with anti-CTLA4 and anti-PD1 treatment. This may be due to heterogeneity of the patients‘ immune system and the tumor microenvironment including T cell inhibitions. There is a clear need to study this phenomenon and develop additional therapies for long term survival to include a broad range of patients. V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) is a suppressive protein expressed on many cell types in the tumor microenvironment including cytotoxic T cells. VISTA’s role on T cells has been described as maintaining quiescence and peripheral tolerance in a graft vs host disease model, but is not fully understood in context of the tumor microenvironment.MethodsWe use a series of invivo experiments, including T cell specific VISTA knock outs, to understand the role of VISTA on T cells in the tumor microenvironment.ResultsHere we show a series of in vivo experiments that suggest VISTA has a potent intrinsic role on T cells and therefore anti-tumor immunity. Using a T cell specific VISTA knock out, our results suggest that the absence of VISTA on T cells in combination with anti-CTLA4 and vaccine is a very powerful tumor suppressor compared to vaccine and anti-CTLA4 treatment alone. These results also indicate that the absence of VISTA alters the phenotype of cytotoxic T cells in several ways including the production of inflammatory cytokines.ConclusionsOur preliminary data provides foundation to study VISTA’s role intrinsic to T cells in the tumor microenvironment and how disrupting VISTA’s influence intrinsic to T cells may be advantageous for anti-tumor immunity and long term patient survival.Ethics ApprovalAll in vivo studies were reviewed and approved by Institutional Animal Care and Use Committee (Approval number 2019–2142).

502 The co-expression of two immune complex molecules, VISTA and TIGIT, define a dysfunctional cytotoxic T cell subset

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A537-A537
Author(s):  
Cassandra Gilmour ◽  
Li Wang ◽  
Juan Dong ◽  
Hieu Ta
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Nk Cells ◽  
Tumor Immunity ◽  
Immune Checkpoint ◽  
Cytotoxic T Cell ◽  
List Type ◽  
Clinical Models

BackgroundCancer immunotherapies have proven, over the last decade, to be of extreme importance for long term survival of patients. Specifically, immune checkpoint inhibitors such as anti-CTLA-4 and anti-PD-1/PD-L1 have had tremendous clinical success treating many cancers including melanoma, lung, breast, colon, and bladder cancer. The low response rate (~30%) of these drugs suggest a mechanism of resistance within the tumor microenvironment, and it demonstrates the immense need to study and develop alternative routes to long-term anti-tumor immunity.1V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) has been shown to be a suppressive molecule in the tumor microenvironment in preclinical models and VISTA’s expression is correlated with poor patient outcome across several cancers.2T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) is a functional receptor expressed on NK cells and T cells that contributes to a suppressive tumor microenvironment by acting on T cells, NK cells, and antigen presenting cells.3MethodsWe use flow cytometry to visualize single cell expression of VISTA and TIGIT on CD8+ tumor infiltrating lymphocytes in pre-clinical models. Functional studies include cytotoxic assays as well as intracellular cytokine staining after cell sorting.ResultsHere we show the expression of these two immune checkpoint molecules, VISTA and TIGIT, across several pre-clinical models, and how their co-expression subsets a distinctly dysfunctional population of cytotoxic T cells.ConclusionsOur data provides foundation to study the rejuvenation of this subset of T cells to restore cytotoxic function and therefore, anti-tumor immunity.Trial RegistrationNAEthics ApprovalAll in vivo studies were reviewed and approved by Institutional Animal Care and Use Committee (Approval number 2019-2142).ConsentNAReferencesFares CM, Allen EMV, Drake CG, Allison JP & Hu-Lieskovan S. Mechanisms of resistance to immune checkpoint blockade: why does checkpoint inhibitor immunotherapy not work for all patients?American Society of Clinical Oncology Educational Book 2019;147–164.Xu W, Hieu T, Malarkannan S. & Wang L. The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti-tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol 2018;15:438–446.Manieri NA, Chiang EY & Grogan JL. TIGIT: A Key Inhibitor of the Cancer Immunity Cycle. Trends Immunol 2017;38:20–28.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

scholarly journals 323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A348-A348
Author(s):  
Jessie Wang ◽  
Kaixia Lian ◽  
Jia Zheng ◽  
Chenpan Nie ◽  
Annie An ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Immune Evasion ◽  
Syngeneic Mouse ◽  
Ifn Γ ◽  
Draining Lymph Nodes ◽  
Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

scholarly journals New Insights into Mechanisms of Long-term Protective Anti-tumor Immunity Induced by Cancer Vaccines Modified by Virus Infection

Biomedicines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 55 ◽  
Author(s):  
Volker Schirrmacher
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Infection ◽  
Tumor Immunity ◽  
Cancer Vaccines ◽  
Cell Mediated Immunity ◽  
Anti Cancer ◽  
Antigen Presenting ◽  
Molecular Patterns

The topic is how to achieve long-term protective anti-tumor immunity by anti-cancer vaccination and what are its mechanisms. Cancer vaccines should instruct the immune system regarding relevant cancer targets and contain signals for innate immunity activation. Of central importance is T-cell mediated immunity and thus a detailed understanding of cognate interactions between tumor antigen (TA)-specific T cells and TA-presenting dendritic cells. Microbes and their associated molecular patterns initiate early inflammatory defense reactions that can contribute to the activation of antigen-presenting cells (APCs) and to costimulation of T cells. The concommitant stimulation of naive TA-specific CD4+ and CD8+ T cells with TAs and costimulatory signals occurs in T-APC clusters that generate effectors, such as cytotoxic T lymphocytes and T cell mediated immunological memory. Information about how such memory can be maintained over long times is updated. The role that the bone marrow with its specialized niches plays for the survival of memory T cells is emphasized. Examples are presented that demonstrate long-term protective anti-tumor immunity can be achieved by post-operative vaccination with autologous cancer vaccines that are modified by virus infection.

scholarly journals IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell–mediated rejection of systemic lymphoma in immunodeficient mice

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3508-3519 ◽  
Author(s):  
John C. Markley ◽  
Michel Sadelain
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Tumor Rejection ◽  
Effector Memory ◽  
Transfer Model ◽  
Human T Cell

Abstract The γc-cytokines are critical regulators of immunity and possess both overlapping and distinctive functions. However, comparative studies of their pleiotropic effects on human T cell–mediated tumor rejection are lacking. In a xenogeneic adoptive transfer model, we have compared the therapeutic potency of CD19-specific human primary T cells that constitutively express interleukin-2 (IL-2), IL-7, IL-15, or IL-21. We demonstrate that each cytokine enhanced the eradication of systemic CD19+ B-cell malignancies in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice with markedly different efficacies and through singularly distinct mechanisms. IL-7– and IL-21–transduced T cells were most efficacious in vivo, although their effector functions were not as enhanced as IL-2– and IL-15–transduced T cells. IL-7 best sustained in vitro T-cell accumulation in response to repeated antigenic stimulation, but did not promote long-term T-cell persistence in vivo. Both IL-15 and IL-21 overexpression supported long-term T-cell persistence in treated mice, however, the memory T cells found 100 days after adoptive transfer were phenotypically dissimilar, resembling central memory and effector memory T cells, respectively. These results support the use of γc-cytokines in cancer immunotherapy, and establish that there exists more than 1 human T-cell memory phenotype associated with long-term tumor immunity.

Leukemia-Reactive Virus-Specific T Cells Generated by T Cell Receptor (TCR) Transfer Preserve Their Dual Specificity upon Repetitive Stimulation of the Endogenous TCR.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 136-136
Author(s):  
M.M. van Loenen ◽  
R.S. Hagedoorn ◽  
M. Hoogeboom ◽  
M.G.D. Kester ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Repetitive Stimulation ◽  
Cell Surface Expression ◽  
Viral Antigens ◽  
Low Expression ◽  
Stimulation Of

Abstract TCR-transfer to engineer tumor-specific T cells may be a strategy for adoptive immunotherapy. For complete eradication of leukemic cells and to achieve long-term protection, potent effector T cell function and long-term T cell persistence are necessary. Therefore, we propose to use virus specific T cells for TCR transfer since such engineered dual specific T cells can be triggered via their endogenous TCR by latent presence of viral antigens, improving their long-term persistence. We have previously shown that virus specific T cells can be redirected towards anti-leukemic reactivity by transfer of the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). The TCR-transferred virus specific T cells showed differences in TCR cell surface make up, which was stable for months after repetitive non-specific TCR triggering. The T cells expressed either both TCRs intermediately at the cell surface, or the endogenous TCR was highly expressed with a low expression of the introduced TCR, or the introduced TCR was highly expressed with a low expression of the endogenous TCR. It may be anticipated that frequent encounter with viral antigens in vivo leads to selective outgrowth of TCR-transferred dual specific T cells with high expression of the endogenous viral specific TCR but low expression of the introduced tumor specific TCR, resulting in reduced anti-leukemic reactivity. To address this issue, we generated CMVA2-specific T cells transduced with the HA-2-TCR. This resulted in dual specific cells with different TCR cell surface make up. The dual specific T cells were repetitively stimulated specifically either via their endogenous virus specific TCR or via the introduced HA-2 specific TCR. In time, the cell surface expression of the endogenous and introduced TCRs as measured with CMVA2 and HA-2A2 tetramers diverged. Repetitive stimulation of the endogenous TCR skewed the dual specific T cells towards a cell population that predominantly expressed the endogenous TCR. In contrast, repetitive stimulation of the introduced TCR skewed the cells towards T cells that predominantly expressed the introduced TCR. However, this divergence in tetramer stainings was shown to quickly revert after a single stimulation via the other TCR. To study whether this divergence was the result of a difference in TCR cell surface distribution or of selective outgrowth of different T cells, T cells were sorted that predominantly expressed either the endogenous or the introduced TCR. These cells were subsequently stimulated on the endogenous or introduced TCR, and compared regarding TCR cell surface expression and functional activity. Directly after sorting dual specific T cells preferentially expressing the endogenous TCR were still reactive against HA-2+ target cells, although the reactivity was reduced compared to cells preferentially expressing the introduced TCR. However, when restimulated on the introduced HA-2-TCR, the dual specific T cells expanded antigen specifically, and reverted within several days into cells with high expression of the introduced TCR that exerted potent HA-2 specific anti-leukemic effector functions. In conclusion, we demonstrate that these dual specific T cells are likely to persist in vivo due to repetitive encounter with viral antigens with preservation of anti-leukemic effector function. Moreover, in vivo exposure to the tumor associated antigen will further enhance the relevant specificity.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 866-866
Author(s):  
Carolina Berger ◽  
Michael C. Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Annexin V ◽  
T Cell Memory ◽  
T Cell Clones ◽  
Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for &gt;30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

Synergistic Tumor Immunity Induced by Chemotherapy and Agonist Anti-GITR Antibody.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1788-1788
Author(s):  
Adam D. Cohen ◽  
Daniel Hirschhorn-Cymmerman ◽  
Adi Diab ◽  
Miguel A. Perales ◽  
Taha Merghoub ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Tumor Antigens ◽  
Tumor Necrosis Factor Receptor ◽  
Cd4 Cells ◽  
Tumor Cell Death ◽  
Free Survival

Abstract Ligation of GITR (glucocorticoid-induced tumor necrosis factor receptor) can both co-stimulate effector CD4 and CD8 T cells (Teff) and abrogate suppression by CD4+foxp3+ regulatory T cells (Tregs). This may be beneficial for the purposes of tumor immunotherapy, and we and others have demonstrated that the agonist anti-GITR antibody DTA1 can enhance both vaccine-induced and naturally-arising tumor immunity in murine models (Turk et al, JEM 2004; Cohen et al, Cancer Res 2006). In this study, we assessed the efficacy of combining GITR ligation with cyclophosphamide (CTX), a cytotoxic chemotherapeutic with immunomodulatory properties, to treat established, poorly immunogenic tumors. C57BL/6 mice received 50,000 B16 melanoma intradermally and were treated on day 6 with CTX 250 mg/kg intraperitoneally (ip), followed 1 day later by 1mg DTA1 or control rat IgG ip. In repeated experiments, 0–20% of mice treated with DTA1 alone or CTX + IgG had long-term tumor-free survival, compared with 60–80% long-term tumor-free survival in mice treated with CTX + DTA1. Starting CTX + DTA1 treatment on day 10 led to 40% tumor-free survival, with no survivors seen with either treatment alone. Synergy was lost with lower doses of CTX, or when CTX was given prior to tumor inoculation, indicating a likely requirement of tumor cell death and cross-presentation of tumor antigens to T cells. Consistent with this, the proliferation and activation of naïve pmel-1 CD8+ T cells (specific for the melanoma antigen gp100) was significantly enhanced when transferred into B16-bearing mice treated 1 day earlier with CTX, compared with untreated mice. This was not simply due to homeostatic proliferation in a lymphopenic state, as no differences were seen when cells were transferred into mice without tumors. In addition, increased GITR expression on both Teff and Tregs was observed for up to 4 days after in vivo CTX treatment, particularly on the proliferating (Ki67+) fraction, providing a greater target for GITR ligation by DTA1. Analysis of T cell populations in spleen, tumor-draining lymph node (DLN), and tumor following CTX + IgG treatment showed a relative decrement in Treg frequency in the first week, followed by a strong rebound, such that by day 14 after CTX roughly 50–60% of the tumor-infiltrating CD4+ cells were foxp3+. This rebound was largely abrogated, however, in the CTX + DTA1-treated mice, with only 5–15% of tumor-infiltrating CD4+ cells expressing foxp3. This led to a dramatic increase in the ratio of CD8+ to CD4+foxp3+ in the tumor (40:1 vs. 5:1 for CTX + IgG-treated mice), with lesser increases seen in the spleen and DLN as well, without significant changes in overall cellularity. Granzyme B expression was also increased in CD8+ and, to a lesser extent, CD4+foxp3- T cells from CTX + DTA1-treated mice, demonstrating increased cytolytic potential by Teff. In sum, CTX + agonist anti-GITR antibody can induce rejection of an aggressive syngeneic tumor, at a stage when either agent alone is ineffective. This synergy likely involves enhanced cross-priming and co-stimulation of Teff, with concomitant decrease in tumor-infiltrating Tregs, leading to a more effective anti-tumor immune response. This combination warrants further evaluation as an immunotherapeutic strategy for cancer.

Alemtuzumab Treatment Can Affect CD52 Positive As Well As CD52 Negative GPI-Deficient, Sezary Cells Allowing Long-Term Disease Control

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2491-2491
Author(s):  
C.J.M. Halkes ◽  
I Jedema ◽  
H.M. van Egmond ◽  
L van der Fits ◽  
J.H.F. Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fold Increase ◽  
Cell Populations ◽  
Proliferative Potential ◽  
Small Populations ◽  
Collateral Damage ◽  
Sézary Cells

Abstract Abstract 2491 Alemtuzumab (ALT) is a monoclonal anti CD52 antibody used for the treatment of CD52 positive lymphoid malignancies and to deplete T cells in vivo and in vitro to prevent graft rejection or GVHD after allogeneic stem cell transplantation (alloSCT). Membrane CD52 expression depends on the presence of a glycosylphosphatidylinositol (GPI) anchor. GPI deficiency is frequently found in small populations of normal and malignant hematopoietic cells, including T and B cells (frequencies from <0.01 to 2%). These cells lack expression of GPI-linked proteins like CD52 as can be detected by absence of staining of FLAER, which is an aerolysin that specifically binds to mammalian GPI anchors. After alloSCT using ALT for T cell depletion, reconstitution of FLAER and CD52 double negative cells is seen, and outgrowth of CD52 negative malignant cell populations has been found after single agent treatment with ALT in malignant diseases. However, GPI deficient cells have been suggested to have a lower proliferative potential and a decreased survival due to their increased susceptibility to spontaneous complement mediated cell lysis, possibly explaining the infrequent dominant outgrowth of GPI deficient clones in healthy individuals. Sézary Syndrome (SS) is an aggressive cutaneous T cell lymphoma characterized by the presence of high numbers of neoplastic T cells expressing CD4 and CD52 in peripheral blood, lymph nodes and skin. Clinical responses in SS patients after single drug treatment with low dosed ALT have been described by several investigators. However, in 6 out of 6 patients analyzed, we found a small population of CD52 and FLAER negative Sézary cells, illustrating that a GPI negative subpopulation is frequently observed which may lead to outgrowth of CD52 negative Sézary cells. We treated 3 patients with successive courses of low dose ALT (10 mg subcutaneously once weekly until circulating malignant cells were < 1,000/mm3) and followed the kinetics of CD52- and CD52+ Sézary cells. Before ALT treatment, a CD4+CD52-FLAER- T cell population was found in all three patients (0.01–0.06% of all circulating CD4+ T cells). As expected, a rapid decrease in absolute numbers of CD4+CD52+FLAER+ cells was observed after ALT treatment (decrease 94 to 100%). Surprisingly, despite the absence of the CD52 target molecule, the absolute number of CD4+CD52-FLAER- T cells also decreased after the first and second treatment cycles in all three patients (decreases between 22 and 96%), indicating that the massive in vivo ALT mediated lysis of CD52+ Sézary cells coincided with collateral damage of CD52- Sézary cells. Between successive treatment courses in the absence of circulating ALT, the absolute numbers of CD4+CD52+FLAER+ T cells showed a more rapid increase compared to CD4+CD52-FLAER- T cells in all patients (median 193 fold increase (range 17–896) versus 9 fold increase (range 2–144) respectively), illustrating a decreased in vivo proliferative potential of these GPI negative cells. After repeated doses of ALT, one of the patients developed resistance to ALT treatment. Phenotype analysis revealed that 97% of the 23,000/mm3 circulating Sézary cells were CD4+CD52-FLAER-. Clonality analysis showed that CD4+CD52+FLAER+ and CD4+CD52-FLAER–Sézary cell populations expressed identical T cell receptor V-beta chains demonstrating that both cell populations are part of the same initial clone of Sézary cells. At present, one year after the start of ALT treatment, reponses are still observed in both other patients without overgrowth of a CD4+CD52-FLAER–Sézary cells. We conclude that despite presence of small populations of CD52 and GPI negative cells in patients with Sézary Syndrome, all patients respond to treatment with alemtuzumab. CD52 negative, GPI deficient Sézary cells showed high susceptibility to collateral damage during antibody treatment. During treatment intervals, CD52+ cells showed a faster recovery compared to CD52- cells, indicating a lower proliferative potential of the GPI deficient Sézary cells. Although, as shown in one patient, ultimate outgrowth of GPI deficient CD52- sezary cells can occur, the capacity to achieve long term control of both CD52+ and CD52- Sézary cells in several patients offers a rationale for treatment of SS with alemtuzumab, possibly in combination with a low dosed cytotoxic drug Disclosures: Off Label Use: Alemtuzumab for treatment of Sezary Syndrome.

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