Primary CLL Xenograft: A Model System to Study the Role of T-Cells in CLL Biology and Therapeutic Response

Abstract The interaction between chronic lymphocytic leukaemia (CLL) cells and T-cells is an important aspect of CLL biology. CLL cells require T-cell support for their proliferation and in addition induce proliferation of regulatory and cytotoxic (CD8+) T-cells. T-cell number and repertoire are both markedly affected by CLL therapy and there is considerable interest in how current treatments modulate the interaction between T-cells and the tumour clone. In this study we investigated whether this relationship was maintained in a xenotransplantation model. CLL engraftment in NOG mice was facilitated by humanisation of the murine microenvironment by allogeneic CD34+ umbilical cord cells or CD14+ monocytes. Accelerated engraftment of both CLL and T-cell compartments was observed in xenografts derived from patients with progressive CLL, suggesting that the biological properties of both subsets are maintained in the murine model. Furthermore, the distribution of helper (CD4+), cytotoxic (CD8+) and regulatory (CD4+CD25+FoxP3+) T-cells was maintained within the xenografts, including retention of the CD4:CD8 ratio. Interestingly, the anergic PD-1+CD160+CD244+TIM3+ T-cell phenotype reported in CLL patients was also evident in T-cells expanded in xenograft models. Consistent with an anergic T-cell phenotype, T-cells from CLL xenografts lacked anti-tumour activity in vitro. Importantly, such anergic cells were observed when T-cells were reconstituted from allogeneic cord blood cells as well as autologous cells, suggesting that CLL cells have the ability to shape T-cell populations of different origin in diverse microenvironments. Finally, to investigate the interaction between specific T-cell subsets and engrafted CLL cells, CD4+, CD8+, and CD25+ T-cells were depleted prior to generation of xenografts. CD8+ T-cell depletion significantly prolonged CLL engraftment (p≤0.01) whereas neither depletion of CD4+ nor CD25+cells had a significant impact. In summary, our results demonstrate that the relationship between CLL tumour cells and reactive T-cells is accurately maintained in a murine xenograft model. Such models will be of great value for investigation of aspects of T-cell function in CLL biology. Disclosures No relevant conflicts of interest to declare.

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Free radicals and redox signalling in T-cells during chronic inflammation and ageing

Biochemical Society Transactions ◽

10.1042/bst0391273 ◽

2011 ◽

Vol 39 (5) ◽

pp. 1273-1278 ◽

Cited By ~ 26

Author(s):

Helen R. Griffiths ◽

Christopher R. Dunston ◽

Stuart J. Bennett ◽

Melissa M. Grant ◽

Darren C. Phillips ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Inflammation ◽

Cell Function ◽

Cell Phenotype ◽

Cell Functions ◽

Cytoplasmic Proteins ◽

Local Cell ◽

Species Production

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2′,7′-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

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Increased Regulatory T Cell Function in a Mouse Model of Beta-Thalassemia.

Blood ◽

10.1182/blood.v114.22.638.638 ◽

2009 ◽

Vol 114 (22) ◽

pp. 638-638

Author(s):

Weili Bao ◽

Susanne Heck ◽

Wu He ◽

Karina Yazdanbakhsh

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Proinflammatory Cytokine ◽

Cell Function ◽

Conflicts Of Interest ◽

Therapeutic Interventions ◽

Beta Thalassemia ◽

Transfusion Therapy

Abstract Abstract 638 The development of red blood cell (RBC) alloantibodies complicates transfusion therapy for chronically transfused patients with thalassemias. Understanding the regulation of immune responses to transfused RBCs may help in future design of therapeutic interventions to prevent RBC alloimmunization in this patient population. We have previously reported in a mouse model that key immune response regulators, CD4+CD25− regulatory T cells (Tregs) expressing Foxp3, control the rate and frequency of RBC alloimmunization in wildtype recipient mice. As an initial step to study and characterize immune regulation in thalassemias, we studied the Treg status of Hbb(th1/th1) mouse model of beta-thalassemia intermedia, known to exhibit mild anemia, reticulocytosis and splenomegaly. Compared to wildtype littermate controls (WT) (n=11), 3 month old thalassemic (THAL) mice (n=12) had significantly higher frequency of CD25+Foxp3+ Tregs in CD4+ population in peripheral blood (5.5±0.5% versus 4.1±0.3%, p=0.03) but not in the spleen (11.4%±0.9% versus 9.5±0.5%, p=0.1). Sorted splenic Tregs from THAL and WT mice were equally anergic to stimulation through the T cell receptor and Tregs from THAL mice secreted the expected low background levels of IL-2 and IFN-gamma in stimulated culture supernatants, similar to Tregs from WT mice. Surprisingly, however, the THAL stimulated Tregs, but not the WT counterparts, secreted the proinflammatory cytokine IL-17. To determine whether THAL Tregs were functionally active, we performed in vitro Treg suppressive assay. We found that thalassemic Tregs were in fact more suppressive than Tregs from their WT counterparts in their ability to suppress proliferation of CD4+CD25–T cells (at 1:4 ratio of Tregs : CD4+CD25–cells 91±3% thalassemic versus 66±3% WT , p=0.03; at 1:8 ratio, 84±4% % versus 63±7%, p=0.02 and at 1:16 ratio, 76±4% % versus 28±14%, p=0.03). We next performed red cell transfusion studies to determine if the increased in vitro THAL Treg suppressive activity is associated with decrease in frequency of alloimmunization in THAL mice. A cohort of age- and sex-matched THAL and WT mice were transfused on a weekly basis for 4 weeks with red cells from transgenic mice expressing human glycophorin A. We found that considerably lower (2/9) alloimmunization rates in THAL compared to WT mice (8/11). Altogether, our data indicate that qualitative differences exist between Tregs from THAL mice and their littermate controls and raise the interesting possibility that THAL Tregs may represent a novel population of Tregs. Specifically, only THAL Tregs secrete the proinflammatory cytokine IL-17 normally associated with Th17 subset and yet they are functionally more suppressive. The mechanisms responsible for these differences are under further study. Disclosures: No relevant conflicts of interest to declare.

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Primary CLL Xenograft: T-Cells Friend or Foe? the Role of T-Cells in the Primary CLL Xenograft Model.

Blood ◽

10.1182/blood.v120.21.2888.2888 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2888-2888

Author(s):

Ceri Oldreive ◽

Anna Skowronska ◽

Paul Moss ◽

Alexander Taylor ◽

Tatjana Stankovic

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Human Disease ◽

Xenograft Model ◽

Rapid Expansion ◽

T Cell Subsets ◽

Xenograft Models ◽

Kinetics Of

Abstract Abstract 2888 Chronic lymphocytic leukaemia (CLL) is a malignancy characterised by the gradual accumulation of mature B cells in peripheral lymphoid organs. However, limited access to this proliferating tumour population in CLL patients and difficulties in modelling this proliferation in vitro necessitates the establishment of human xenograft models that can recapitulate the human disease. Several CLL xenograft models have already been established, however due to their inability to fully recapitulate human tissue distribution and their restricted period of engraftment, these models are not suitable for testing novel chemotherapeutics. To address this, modifications were made to a recent xenograft model established by Bagnara et al, utilising immuno-compromised NOG mice on a human haematopoietic microenvironment provided by prior propagation of human CD34+ umbilical cord blood cells. This was reported to reliably replicate the human disease but was of limited duration purportedly due to T-cell outgrowth. Our aim therefore was to determine the role various T-cells subsets play in engraftment and its demise in order to enhance the duration of the CLL engraftment in this primary CLL xenograft model. This was accomplished, by a) sequential cull of animals engrafted by primary CLL cells to monitor kinetics of T-cell engraftment over time, b) by addressing clonality and the potential anti-CLL effect of expanded T-cells and c) by depletion of implicated autologous T-cell subsets prior to their co-administration into mice with CLL tumour cells. The engraftment kinetics of patient PBMC in murine spleen; encompassing both T-cells and tumour CLL; were tracked and we observed that the relative proportions of patient T-cell subsets altered within 8 weeks following injection. Initially, during the first 6 weeks, there was rapid expansion of helper (CD4+) and regulatory (CD4+/CD25+) T-cells. Subsequently, their proportion decreased as the cytotoxic (CD8+) T-cells expanded becoming the dominant T-cell subtype. Interestingly, by using PCR amplification of TCR rearrangements we noted that these T-cells were not clonally expanded and that their co-incubation with autologous CLL cells in vitro failed to exert a cytotoxic effect. We have previously demonstrated that CLL engraftment could occur even if patient T-cells were depleted to a minimal level of 0.6–5×104 cells or 2% of initial T-cell numbers. Here, we demonstrate that depletion of either helper (CD4+) or regulatory (CD25+) T-cells resulted in a reduced level of engraftment in murine peripheral blood and spleen, extended graft duration and prolonged survival of the animals. In contrast, depletion of either cytotoxic (CD8+) or natural killer (CD56+) T-cells had limited effect on both engraftment initiation and duration of the graft (none<CD8<CD56<CD4<CD25<CD3, 101 vs 96 vs 115 vs 127 vs 130 vs 151 days). Taken together our results indicate a crucial role for helper (CD4+) and regulatory (CD25+) T-cell subsets in both initiation and termination of CLL engraftment. The expansion of CD8+ T-cells at the end of engraftment may be co-incidental and not a reflection of an anti-tumour effect. Further studies are necessary to address the exact interaction between patient CLL cells and regulatory T-cells. Finally, manipulation of the regulatory T-cell subset in this xenograft model can provide robust CLL proliferation amenable for drug testing. Disclosures: No relevant conflicts of interest to declare.

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Vitamin D Inhibits Pro-Inflammatory T Cell Function in Patients With Inflammatory Bowel Disease

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz090 ◽

2019 ◽

Vol 13 (12) ◽

pp. 1546-1557 ◽

Cited By ~ 8

Author(s):

Josefine Schardey ◽

Anna-Maria Globig ◽

Christine Janssen ◽

Maike Hofmann ◽

Philipp Manegold ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Vitamin D ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Bowel Disease ◽

T Cell Subsets ◽

Cell Phenotype ◽

Inflammatory Bowel

Abstract Background and Aims Dysregulated T cell responses contribute to the pathogenesis of inflammatory bowel disease [IBD]. Because vitamin D [vitD] deficiency is a risk factor for adverse disease outcomes, we aimed to characterize the impact of vitD on intestinal and peripheral T cell profiles. Methods T cells were isolated from peripheral blood and intestinal biopsies of IBD patients, incubated with vitD and characterized by flow cytometry. To translate these in vitro findings to the clinic, serum vitD concentrations and clinical outcomes were correlated with T cell phenotype and function in a prospective patient cohort. Results Incubation of peripheral and intestinal T cells with 1,25(OH)2-vitD resulted in strongly reduced frequencies of pro-inflammatory CD4+ and CD8+ T cells producing interferon γ [IFNγ], interleukin-17 [IL-17], IL-22, IL-9 and tumour necrosis factor [TNF]. Univariable analysis of 200 IBD patients revealed associations of vitD deficiency with non-compliant vitD intake, season of the year and anaemia in Crohn’s disease [CD] as well as disease activity in ulcerative colitis [UC]. Ex vivo immunophenotyping revealed that CD4+ and CD8+ T cell subsets were not substantially altered in vitD-deficient vs vitD-sufficient patients while regulatory T cell frequencies were reduced in UC and non-smoking CD patients with vitD deficiency. However, normalization of serum vitD concentrations in previously deficient CD patients resulted in significantly reduced frequencies of CD4+ T cells producing IFNγ, IL-17 and IL-22. Conclusion vitD exerts profound anti-inflammatory effects on peripheral and intestinal CD4+ and CD8+ T cells of IBD patients in vitro and inhibits TH1 and TH17 cytokine production in CD patients in vivo.

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Reversal of the CD8+ T-Cell Exhaustion Induced by Chronic HIV-1 Infection Through Combined Blockade of the Adenosine and PD-1 Pathways

Frontiers in Immunology ◽

10.3389/fimmu.2021.687296 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jing Li ◽

Hui-Huang Huang ◽

Bo Tu ◽

Ming-Ju Zhou ◽

Wei Hu ◽

...

Keyword(s):

T Cells ◽

Viral Load ◽

T Cell ◽

Cell Function ◽

T Cell Subsets ◽

Cell Counts ◽

T Cell Function ◽

Treatment Naïve ◽

Hiv 1

BackgroundTargeting immune checkpoints for HIV treatment potentially provides a double benefit resulting from the ability to restore viral-specific CD8+ T-cell functions and enhance HIV production from reservoir cells. Despite promising pre-clinical data, PD-1 blockade alone in HIV-1-infected patients with advanced cancer has shown limited benefits in controlling HIV, suggesting the need for additional targets beyond PD-1. CD39 and PD-1 are highly co-expressed on CD8+ T cells in HIV-1 infection. However, the characteristics of CD39 and PD-1 dual-positive CD8+ T-cell subsets in chronic HIV-1 infection remain poorly understood.MethodsThis study enrolled 72 HIV-1-infected patients, including 40 treatment naïve and 32 ART patients. A total of 11 healthy individuals were included as controls. Different subsets of CD8+ T cells defined by CD39 and/or PD-1 expression were studied by flow cytometry. The relationships between the frequencies of the different subsets and parameters indicating HIV-1 disease progression were analyzed. Functional (i.e., cytokine secretion, viral inhibition) assays were performed to evaluate the impact of the blockade of adenosine and/or PD-1 signaling on CD8+ T cells.ResultsThe proportions of PD-1+, CD39+, and PD-1+CD39+ CD8+ T cells were significantly increased in treatment naïve patients but were partially lowered in patients on antiretroviral therapy. In treatment naïve patients, the proportions of PD-1+CD39+ CD8+ T cells were negatively correlated with CD4+ T-cell counts and the CD4/CD8 ratio, and were positively correlated with viral load. CD39+CD8+ T cells expressed high levels of the A2A adenosine receptor and were more sensitive to 2-chloroadenosine-mediated functional inhibition than their CD39- counterparts. In vitro, a combination of blocking CD39/adenosine and PD-1 signaling showed a synergic effect in restoring CD8+ T-cell function, as evidenced by enhanced abilities to secrete functional cytokines and to kill autologous reservoir cells.ConclusionIn patients with chronic HIV-1 infection there are increased frequencies of PD-1+, CD39+, and PD-1+CD39+ CD8+ T cells. In treatment naïve patients, the frequencies of PD-1+CD39+ CD8+ T cells are negatively correlated with CD4+ T-cell counts and the CD4/CD8 ratio and positively correlated with viral load. Combined blockade of CD39/adenosine and PD-1 signaling in vitro may exert a synergistic effect in restoring CD8+ T-cell function in HIV-1-infected patients.

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A population of CD4+ T cells with a naïve phenotype stably polarized to the TH1 lineage

10.1101/2020.07.14.202168 ◽

2020 ◽

Author(s):

Jonathan W. Lo ◽

Maria Vila de Mucha ◽

Luke B. Roberts ◽

Natividad Garrido-Mesa ◽

Arnulf Hertweck ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cell Function ◽

T Cell Subsets ◽

T Helper

AbstractT-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH17 cells and TREG, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells with naïve cell surface markers and that this novel cell population is phenotypically and functionally distinct from conventional naïve CD4+ T cells. These cells are also distinct from previously described populations of memory phenotype and stem cell-like T cells. Naïve-like T-bet-experienced cells are polarised to the TH1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can function to polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response.

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PD-1 Blockade Modulates Functional Activities of Exhausted-Like T Cell in Patients With Cutaneous Leishmaniasis

Frontiers in Immunology ◽

10.3389/fimmu.2021.632667 ◽

2021 ◽

Vol 12 ◽

Author(s):

Renan Garcia de Moura ◽

Luciana Polaco Covre ◽

Carlos Henrique Fantecelle ◽

Vitor Alejandro Torres Gajardo ◽

Carla Baroni Cunha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cutaneous Leishmaniasis ◽

Cell Function ◽

Parasite Clearance ◽

Skin Lesions ◽

T Cell Subsets ◽

Leishmania Braziliensis ◽

T Cell Function

Patients infected by Leishmania braziliensis develop debilitating skin lesions. The role of inhibitory checkpoint receptors (ICRs) that induce T cell exhaustion during this disease is not known. Transcriptional profiling identified increased expression of ICRs including PD-1, PDL-1, PDL-2, TIM-3, and CTLA-4 in skin lesions of patients that was confirmed by immunohistology where there was increased expression of PD-1, TIM-3, and CTLA-4 in both CD4+ and CD8+ T cell subsets. Moreover, PDL-1/PDL-2 ligands were increased on skin macrophages compared to healthy controls. The proportions PD1+, but not TIM-3 or CTLA-4 expressing T cells in the circulation were positively correlated with those in the lesions of the same patients, suggesting that PD-1 may regulate T cell function equally in both compartments. Blocking PD-1 signaling in circulating T cells enhanced their proliferative capacity and IFN-γ production, but not TNF-α secretion in response to L. braziliensis recall antigen challenge in vitro. While we previously showed a significant correlation between the accumulation of senescent CD8+CD45RA+CD27- T cells in the circulation and skin lesion size in the patients, there was no such correlation between the extent of PD-1 expression by circulating on T cells and the magnitude of skin lesions suggesting that exhausted-like T cells may not contribute to the cutaneous immunopathology. Nevertheless, we identified exhausted-like T cells in both skin lesions and in the blood. Targeting this population by PD-1 blockade may improve T cell function and thus accelerate parasite clearance that would reduce the cutaneous pathology in cutaneous leishmaniasis.

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Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection

Journal of Experimental Medicine ◽

10.1084/jem.20141520 ◽

2015 ◽

Vol 212 (9) ◽

pp. 1449-1463 ◽

Cited By ~ 42

Author(s):

Egidio Torrado ◽

Jeffrey J. Fountain ◽

Mingfeng Liao ◽

Michael Tighe ◽

William W. Reiley ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Cd4 T Cells ◽

Cell Function ◽

Killer Cell ◽

Cell Number ◽

Cd4 T Cell ◽

Cell Phenotype ◽

Mycobacterium Tuberculosis Infection

CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra−/− mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R–deficient T cells is not associated with increased proliferation but with decreased expression of cell death–associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R–deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB.

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Hirsutella Sinensis Fungus Regulates CD8+ T Cell Exhaustion Through Involvement of T-Bet/Eomes in the Tumor Microenvironment

Frontiers in Pharmacology ◽

10.3389/fphar.2020.612620 ◽

2021 ◽

Vol 11 ◽

Author(s):

Lu Jin ◽

Lushuai Jin ◽

Renjie Wu ◽

Xia Liu ◽

Xinhai Zhu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Tumor Growth ◽

Lung Metastasis ◽

Cell Function ◽

T Cell Subsets ◽

Effector Memory

Background: Targeting exhausted T (Tex) cells is a promising strategy for anti-tumour treatment. Previously, we demonstrated that Hirsutella sinensis fungus (HSF) could significantly increase T cell infiltration and the effector T cell ratio in the tumor microenvironment, activating systemic immune responses. However, we do not know how HSF regulates Tex cells in the tumor microenvironment. Here, we explored the mechanism underlying HSF inhibition of Tex cells and tumor growth and metastasis in breast cancer.Methods: We examined the effects of HSF on various tumor mouse models using in vivo imaging technology. Lung metastasis was detected by H&E staining and the T cell subsets in the tumor microenvironment were assayed with flow cytometry. The in vitro proliferation, function and apoptosis of CD8+ T cells were measured, as well as the T-bet and PD-1 mRNA expressions.Results: HSF inhibited tumor growth and lung metastasis in the mice, and had significantly higher CD44LowCD62LHi and CD44HiCD62LLowpopulations in the tumour-infiltrating CD8+ T cells. However, HSF significantly reduced levels of inhibitory receptors, such as PD-1, TIGIT, CTLA-4, and regulatory T cells. In vitro, HSF inhibited the CD8+ T cell apoptosis rate, and promoted CD8+ T cell proliferation and secretion of interferon (IFN)-γ and granzyme B. Furthermore, HSF treatment both in vivo and in vitro significantly increased Eomes expression, while decreasing T-bet expression.Conclusion: HSF exerted anti-tumour effects mainly through the immune system, by promoting effector/memory T cells and reducing Tex cell production in the tumor microenvironment. The specific mechanisms involved inhibiting T-bet and promoting Eomes to decrease the expression of immune inhibitor receptors and enhance the T cell function, respectively.

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696 Brentuximab vedotin, a CD30-directed antibody-drug conjugate, selectively depletes activated Tregs in vitro and in vivo

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0696 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A738-A738

Author(s):

Bryan Grogan ◽

Reice James ◽

Michelle Ulrich ◽

Shyra Gardai ◽

Ryan Heiser ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Efflux Pump ◽

Apoptotic Cell ◽

T Cell Subsets ◽

Memory Cells ◽

Antibody Drug Conjugate ◽

Selective Depletion

BackgroundRegulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs.MethodsHere we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo.ResultsTreatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression.ConclusionsTogether, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs.AcknowledgementsThe authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation.Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

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