CD49d (high) T cells in the ovarian cancer microenvironment are a potential target for the optimization of immune checkpoint therapy in ovarian cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e17075-e17075 ◽  
Author(s):  
Henning De May ◽  
Sharina Palencia Desai ◽  
Ichiko Kinjyo ◽  
Jaryse Harris ◽  
Sarah Foster Adams
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
T Cell Subsets ◽  
Antigen Specificity ◽  
Cd8 Cells ◽  
Tumor Bearing

e17075 Background: Despite the correlation between tumor infiltrating lymphocytes and long-term survival, immune-based therapies have underperformed for the treatment of ovarian cancer. This is attributed to an immune suppressive intraperitoneal microenvironment. With evidence that T cell dysfunction in the ovarian tumor environment is not reflected peripherally, we hypothesized that anatomically restricted T cell subsets play a role in local disease regulation. High expression of integrin α4 (CD49d) is selectively seen on peritoneal T cells in patients and healthy mice. Here we tested whether CD49d(high) CD8 T cells also contribute to anti-tumor immunity in ovarian cancer models. Methods: Using a syngeneic immune competent model of high grade serous ovarian cancer (ID8ova), we evaluated the phenotype of CD49d(high) T cells at varying stages of intraperitoneal disease by flow cytometry. Antigen specificity was tested using a SIINFEKL/H-2Kb NIH tetramer assay. Results: CD49d is highly expressed by peritoneal CD8 T cells but not by splenocytes in tumor-bearing mice (29.8% vs. 3.3% of CD8 cells respectively). Supporting a role in anti-tumor immunity, 92% of tumor antigen-specific CD8 T cells in the peritoneal cavity expressed high CD49d. While the proportion of peritoneal CD8 cells that express high CD49d is similar in healthy and tumor-bearing mice, CD49d(high)CD8 cells upregulate the expression of co-inhibitory receptors with tumor progression. At late stages of the disease, PD-1, TIM3, and LAG3 are exclusively expressed by peritoneal CD49d(high) cells (range 94.7 +/- 3.05%). Consistent with our prior data, PD1+TIM3+LAG3+ CD8 T cells were not present in the spleen, confirming the anatomic specificity of this lymphocyte subset. Conclusions: These findings add to the accumulating evidence that tumor immunity is locally regulated and identify an IP specific subset of CD8 T cells that could be selectively targeted with immune checkpoint blockade. We predict that strategies directing immune therapy to the peritoneal tumor microenvironment will enhance treatment efficacy and limit off-target toxicities in women with ovarian cancer.

scholarly journals Engineered Exosomes Expressing HLA and Co-Stimulatory Molecules to Generate Antigen-Specific CD8+ T Cells for Adoptive Cell Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1338-1338
Author(s):  
Sueon Kim ◽  
Hyun-Jung Sohn ◽  
Hyun-Joo Lee ◽  
Dae-Hee Sohn ◽  
Seung-Joo Hyun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Adoptive Cell Therapy ◽  
K562 Cells ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Antigen Specificity

Abstract S.K and H.-J.S. contributed equally for this works Dendritic cell-derived exosome (DEX) has been known as an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to broad utility for immunotherapy. In this study, we engineered K562 cells expressing triple-co-stimulatory signals (CD80, 4-1BBL, and CD83) with HLA-A2 as an AAPC. Specifically, CD137L (4-1BBL) is an ideal signaling molecule for long-term propagation of CD8+ T cells, and the addition of other co-stimulatory molecules, such as CD80 and CD83, is used to support the expansion of naive T cell subsets. DC-derived exosomes display immunologically important molecules such as HLA and co-stimulatory molecules. Likewise, CoEX-A2 expressed high levels of HLA-A2, CD80, CD83, and CD137L (41BBL) and mediate strong, antigen-specific CD8+ T lymphocyte activation. The stimulation of freshly isolated peripheral CD8+ T cells with the appropriate antigen specificity observed here was likely made possible by the use of the sensitive ELISPOT assay. Viral or tumor protein-pulsed exosomes can directly stimulate CD8+ T cell proliferation and differentiation into CTLs in vitro. In addition, exosomes can be taken up by both CD8+ T cells and K562 cells. Meanwhile, K562 cells that have taken up exosomes can also stimulate CD8+ T cells, which may be due to the higher levels of HLA-A2, CD80, CD83, and 41BBL expression observed on exosomes. Therefore, the CD8+ T cell antigen-specific expansion observed in our cultures is likely the result of coated CoEX-A2s working directly or in a cross-dressed manner. The results suggest that these novel exosomes may provide a crucial source to generate antigen-specific CD8+ T cells for adoptive cell therapy against viral infection and tumors. Disclosures No relevant conflicts of interest to declare.

Age-related differences in T cell subsets in a nationally representative sample of people over age 55: Findings from the Health and Retirement Study

2021 ◽  
Author(s):  
Bharat Thyagarajan ◽  
Jessica Faul ◽  
Sithara Vivek ◽  
Jung Ki Kim ◽  
Janko Nikolich-Žugich ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Subsets ◽  
Health And Retirement Study ◽  
Cd8 Cells ◽  
Age Related ◽  
Nationally Representative ◽  
Retirement Study ◽  
Age And Sex

Abstract Though T cell immunosenescence is a major risk factor for age-related diseases, susceptibility to infections, and responses to vaccines, differences in T cells subset counts and representation by age and sex have not been determined for a large sample representative of the national population of the US. We evaluated the counts of T cell subsets including total, CD4+ and CD8+ T cells, and their naïve (Tn), effector memory (Tem) and effector subsets, in the context of age, sex and exposure to cytomegalovirus (CMV) infection among 8,848 Health and Retirement Study (HRS) participants, a nationally representative study of adults over 55 years. Total T cells (CD3+) and CD4+ cells declined markedly with age; CD8+ T cells declined somewhat less. While CD4+ T cell declines with age occurred for both CMV seropositive and CMV seronegative groups, total T cells and CD8+ cells were both substantially higher among the CMV seropositive group. Numbers of Tn CD4+ and CD8+ cells were strongly and inversely related to age, were better conserved among women, and were independent of CMV seropositivity. By contrast, accumulation of the CD8+ and CD4+ Tem and effector subsets was CMV-associated. This is the first study to provide counts of T cell subsets by age and sex in a national sample of older US adults over the age of 55 years. Understanding T cell changes with age and sex is an important first step in determining strategies to reduce its impact on age-related diseases and susceptibility to infection.

scholarly journals The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules [corrected]

1993 ◽  
Vol 178 (5) ◽  
pp. 1837-1842 ◽  
Author(s):  
J M Penninger ◽  
N Neu ◽  
E Timms ◽  
V A Wallace ◽  
D R Koh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Severe Disease ◽  
Heart Tissue ◽  
T Cell Subsets ◽  
Autoimmune Myocarditis ◽  
Cd8 Cells ◽  
Organ Specific

Experimental induction of most autoimmune diseases appears to depend on the activation of CD4+ T helper cells, while CD8+ lymphocytes may have a role in disease progression. To study the role of CD4+ and CD8+ T cell subsets in T cell-dependent autoimmunity, mice lacking CD4 or CD8 molecules after gene targeting were injected with cardiac myosin to induce organ specific autoimmune myocarditis. Mice homozygous for the CD8 mutation (CD8-/-) developed significantly more severe disease as compared to CD4+/-CD8+/- controls. Surprisingly, CD4-/- mice developed autoimmune myocarditis with infiltration of TCR alpha beta +CD4-CD8- T cells in the heart tissue and appearance of autoantibodies. These data demonstrate that the lack of CD4+ or CD8+ T cells has no significant influence on the initiation of autoimmune myocarditis. CD4+ and CD8+ cells regulate disease severity and these results may explain the occurrence of autoimmunity in CD4 immunodeficiencies.

scholarly journals Clinical Observation of the Number of Lymphocytes and T Cell Subsets in Patients with COVID-19 at Different Stages

2020 ◽  
Author(s):  
Jing Bai ◽  
Hui Zhou ◽  
Bao-sheng Dai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
White Blood Cells ◽  
Peripheral Blood Lymphocytes ◽  
T Cell Subsets ◽  
Control Group ◽  
Cd8 Cells ◽  
Significant Difference ◽  
Severe Group

Abstract To explore the changes of lymphocytes and T cell subsets at different stages in patients with COVID-19. 86 patients with COVID-19 were enrolled, and the dynamic changes of peripheral blood lymphocytes and T cell subsets of CD3+, CD4+, and CD8+ were measured on admission, after treatment for1 week, 2 weeks, and before discharge. There were no significant differences in the number of white blood cells and lymphocytes between admission and 2 weeks after treatment or before discharge in severe patients. The counts of CD3+, CD4+, and CD8+ T cells decreased significantly on admission. After 2 weeks of treatment, the CD3+ counts were significantly higher than that on admission. The CD4+ and CD8+ counts increased significantly after 1 week of treatment, and went up remarkably before discharge compared with that on admission. There was no significant difference in the number of CD3+ cells between the mild group and the control group on admission, but it was significantly lower in the severe group than that in the control group and the mild group. The CD4+ and CD8+ counts decreased significantly in both mild and severe patients on admission, and increased significantly before discharge. At the time of discharge, the CD4+ counts in the severe and mild groups were still significantly lower than in the control group, but there was no significant difference in CD8+ counts among the three groups. The counts of CD3+,CD4+,and CD8+ T cells in the patients with COVID-19 is significantly correlated with the short-term prognosis, and is more sensitive than lymphocytes. In the earliest stage, the numbers of CD4+ and CD8+ cells are more sensitive to early reduction and faster to late recovery.

Expression and Function of Tigit in B-Cell Non-Hodgkin Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4138-4138 ◽  
Author(s):  
Zhi-Zhang Yang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
T Cell Subsets ◽  
Non Hodgkin Lymphoma ◽  
And Function

Background:T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and a novel member of CD28 family. TIGIT is expressed on NK cells, effector and memory T cells, and Treg cells. Upon ligation with CD155, TIGIT delivers an inhibitory signal and negatively regulates anti-tumor responses. While important in normal T-cell biology, the expression and function of TIGIT in the blood and tumor microenvironment of patients with B-cell non-Hodgkin lymphoma (NHL) is completely unknown. Goal: To phenotypically and functionally characterize TIGIT+T cell subsets in B-cell NHL and compare expression of TIGIT on intratumoral T cells to normal controls. Results: In peripheral blood, TIGIT expression was not detected on resting T cells. In contrast, when we analyzed biopsy specimens from B-cell NHL patients, we observed that TIGIT is variably but highly expressed on a subset of intratumoral T cells. A median of 41.1% (range: 24.9-56.1, n=5) of CD4+ or 22.2% (range: 13.4-33.3, n=5) of CD8+ T cells from tumor specimens expressed TIGIT on the cell surface. In normal tonsil tissue, expression level of TIGIT was modest or negligible. A median of 12.0% (range: 9.1-15.8, n=5) of CD4+ or 3.9% (range: 1.3-5.7, n=5) of CD8+ T cells express TIGIT on the cell surface, which is significantly lower than that in B-cell NHL (p=0.005 and p=0.003 for CD4+ and CD8+, respectively). We observed that both CD45RA+ and CD45RA- T cells express TIGIT with the greatest expression on the CD45RA- population in B-cell NHL specimens. Furthermore, we found that TIGIT+ T cells coexpressed other immune checkpoint molecules including PD-1 and TIM-3. Functionally, TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and IL-2-producing cells was lower in the TIGIT+ population than in TIGIT-T cells. Unlike TIM-3 that is coexpressed with PD-1 and whose expression is upregulated by IL-12, TIGIT expression is not upregulated by IL-12, suggesting that a different mechanism is involved in TIGIT upregulation in B-cell NHL. Conclusion: Taken together, these results indicated that TIGIT is highly expressed on intratumoral T cells in B-cell NHL and is expressed on a population of T-cells with suppressed immune function. TIGIT, while co-expressed with PD-1 and TIM-3, is not upregulated by cytokines such as IL-12, suggesting that it is regulated by an alternate pathway. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression and exhaustion in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

Effects of rhIL-7 administration in humans on in vivo expansion of naïve, memory and effector subsets of CD4+ & CD8+ T-cells

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2504-2504
Author(s):  
C. Sportes ◽  
F. Hakim ◽  
M. Krumlauf ◽  
R. Babb ◽  
T. Fleisher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Down Regulation ◽  
Cd8 Cells ◽  
Interleukin 7 ◽  
Circulating Cells

2504 Background: IL-7 has a critical and non-redundant role in T-cell lymphopoiesis and peripheral T-cell homeostasis. IL-7 administration may prove clinically valuable in conditions of disease induced (HIV) or iatrogenic T-cell depletion and for modulation of vaccine immune responses. In the first phase I study in humans, recombinant human interleukin-7 (“CYT 99–007”, Cytheris Inc., Rockville, MD) was administered subcutaneously every other day for two weeks in adults with refractory malignancies at 3, 10, 30 and 60 μg/kg/dose. Biologic activity, defined as a 50% increase over baseline of peripheral blood CD3+ T-cells, was seen at and above the 10μg/kg/dose in all patients. The kinetics of proliferation and expansion of peripheral blood T-cell subsets were analyzed. Methods: Multicolor flow cytometry was performed at baseline, 1, 2 and 3 weeks. Among CD4+ cells, the most naïve were defined as CD45RA+ /CD31+. Among CD4+ & CD8+ cells, the main naïve, memory and effector populations were defined respectively as CD45RA+/CD27+, CD45RA-/CD27+ and CD45RA-/CD27-. Within each subset, the number of cells in cycle was defined by Ki67 staining. Results: Following IL-7 therapy, there was marked proliferation of all T-cells subsets, peaking at week 1, most striking for the naive subsets with 30–70% of circulating cells induced to cycle. Proliferation rates were halved by week 2 despite continuation of treatment, coincident with the observed down-regulation of the IL-7 receptor. Cycling returned to baseline by week 3. Significant proliferation was also induced in effector and memory CD4+ and CD8+ T-cells but to a lesser magnitude, resulting in a greater net expansion of the naïve subsets, still ongoing one week after the end of treatment. Conclusions: IL-7 administration induces marked expansion of naïve, memory and effector CD4+ & CD8+ T-cells in humans. Consistent with the known down-regulation of the IL-7 receptor upon IL-7 exposure, proliferation rates decrease during the second week of treatment. rhIL-7 induced T-cell expansion may prove clinically valuable in adoptive immunotherapy as an adjunct to tumor vaccination and / or immunorestorative agent. [Table: see text]

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.

Defining the Immune Checkpoint Landscape in the Bone Marrow and Peripheral Blood of Patients with Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2012-2012
Author(s):  
Nitin Jain ◽  
Sreyashi Basu ◽  
Beenu Thakral ◽  
Jan Burger ◽  
Philip A Thompson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Checkpoint Blockade ◽  
T Cell Subsets ◽  
Cd8 Cells ◽  
Checkpoint Receptors

Abstract Background: Limited data is available on expression levels of checkpoint receptors, respective ligands, and other immune markers in patients with CLL (Ramsay et al. Blood 2012). Checkpoint blockade has been a successful therapy of many cancers including melanoma, and more recently, Hodgkin's lymphoma. Understanding expression patterns of checkpoint receptors and ligands may help in the clinical development of checkpoint blockade as a therapy for patients with CLL. Methods: Between September 2015 and April 2016, we performed 17-color multi-parameter flow-cytometry (MFC) in paired peripheral blood (PB) and bone marrow (BM) samples from 30 patients with CLL who presented as new patients for evaluation at MDACC. Patients may have received prior CLL therapy. We evaluated expression of immune receptors (inhibitory receptors: PD1, CTLA4, LAG3, TIM3; activating receptors: GITR, OX40, 41BB, ICOS) on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. CLL cells were assessed for both immune receptors (as above), and ligands (4-1BBL, B7-1, B7-2, ICOSL, PDL-1, PDL-2, OX40L). These analyses were performed on freshly collected PB and BM samples by the M. D. Anderson Cancer Center Immunotherapy Platform. Results: A total of 30 patients with CLL were enrolled. The median age was 66 years (range, 35-83). Nine were women. Nineteen were treatment-naive. Prognostic markers included FISH [del(17p) = 6; del(13q) = 9, del(11q) = 4, trisomy 12 = 3, negative = 8]. IGHV mutation status was available for 19 patients (13 unmutated IGHV, 6 mutated IGHV). B2M was ≥3.5 in 11 pts. Baseline expression of costimulatory receptors in CD8 T cells in the marrow, and of the ligands in CLL cells in the marrow is shown in Figure 1. In paired PB and BM sample analysis, there was a high correlation between expression level of PD1 on Treg (Pearson correlation, r = 0.90, p<0.00001), Teff (r = 0.87, p<0.00001), CD8+ cells (r = 0.80, p<0.00001), and CLL cells (r = 0.75, p<0.00001). PD-L1 expression on CLL cells was moderately correlated between PB and BM (r = 0.57, p<0.001). Patients with prior therapy had significantly higher expression of PDL1 on the CLL cells in both PB and BM (p=0.01 and p=0.002, respectively) compared to previously untreated patients. OX40 expression on CD8 cells was significantly higher in both PB and BM in previously treated patients (compared to previously untreated patients). Patients with unmutated IGHV (p = 0.003) and del17p (p = .03) had higher PDL1 expression on CLL cells in the marrow. Conclusions: There is a strong correlation in the expression levels of PD1 on various T cell subsets between PB and BM. Clinically targetable checkpoint receptors including PD1, OX40, CTLA4, and ICOS are consistently expressed across patients with CLL, and present on cells in both PB and BM. Disclosures Jain: BMS: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novimmune: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Infinity: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding. Burger:Roche: Other: Travel, Accommodations, Expenses; Pharmacyclics, LLC, an AbbVie Company: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Portola: Consultancy; Gilead: Research Funding. Thompson:Pharmacyclics: Consultancy, Honoraria. Daver:Otsuka: Consultancy, Honoraria; Ariad: Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Research Funding; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Kiromic: Research Funding. Wierda:Acerta: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Novartis: Research Funding; Abbvie: Research Funding.

T cell receptor activation status as biological context for interpreting PD1/PDL1 biomarker measurements.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 54-54
Author(s):  
Ralph E. Parchment ◽  
Tony Navas ◽  
Kristin Fino ◽  
Andy Fung ◽  
Facundo Cutuli ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Immunofluorescence Microscopy ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Tcr Activation

54 Background: Direct cytolysis of tumor cells by CD8+ T cells results from the net effect of at least two biochemical pathways: (1) stimulatory signaling from the activated T cell receptor (TCR) complex in response to its recognition of a tumor neoantigen presented in the context of autologous MHC class I, and (2) suppressive signaling from immune checkpoints, such as the response of PD1 to binding its ligand, PDL1. Because the PD1:PDL1 immune checkpoint is significant for therapy only when there is tumor cell-specific TCR activation and signaling, it is not surprising that simple measurements of either PD1 or PDL1 in tumor biopsies are, at best, imperfect predictive biomarkers. Instead, a more precise test that quantifies PD1 signaling due to PDL1 binding only in the subset of CD8+ T cells exhibiting activated TCR signaling should provide a more accurate assessment of the extent of immune checkpoint suppression of tumor immunity and therefore be a more predictive biomarker of response to PD1/PDL1-targeted immunotherapy. Methods: We have developed a multiplexed immunofluorescence microscopy test capable of simultaneous quantitation of TCR activation (phospho-CD3zeta), immune checkpoint signaling via PD1 (phospho-SHP1 and -SHP2), and the net stimulation or inhibition resulting from the integration of these two pathways (phospho-ZAP70). Results: Specific antibodies to these biomarkers have been qualified, including peptide inhibition studies to establish antibody specificity, and their performance established by fit-for-purpose studies of in vitro models of CD8+ T cell activation. This multiplex biomarker panel is suitable for clinical use with formalin-fixed, paraffin embedded core needle biopsies of tumor and quantitative immunofluorescence microscopy (qIFA). Conclusions: The additional biomarkers of tumor immunity are expected to add an important context for interpreting PD1/PDL1 measurements. Funded by NCI Contract No. HHSN261200800001E.

T Cells from Chronic Lymphocytic Leukemia (CLL) Patients Display Dysregulated Expression of Immune Checkpoint Molecules and Activation Markers Mainly Restricted to CD4+ Cells and Correlated with Disease Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4132-4132
Author(s):  
Marzia Palma ◽  
Giusy Gentilcore ◽  
Fariba Mozaffari ◽  
Kia Heimersson ◽  
Barbro Näsman-Glaser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Immune Checkpoints ◽  
Activated T Cells ◽  
Activation Markers ◽  
Cd8 Cells

Abstract Background CLL patients (pts) have impaired humoral and cellular immune functions, which is largely due to profound defects of T-cells. Regulation and activation of T lymphocytes depend not only on T cell receptor signaling but also on co-signaling receptors delivering either inhibitory or stimulatory signals, known as immune checkpoints. CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) is transiently expressed on activated T cells, binding the same ligands as CD28, inhibiting T-cell activation. Similarly, programmed cell death protein 1 (PD-1) is expressed on activated CD4+ and CD8+ T cells inhibiting T-cell functions upon binding to the ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). CD137 is an inducible costimulatory receptor expressed by activated T cells. Dysregulated expression of immune checkpoint receptors on T cells of CLL pts may have an impact on T-cell responsiveness and might be a mechanism for the immune deficiency in the disease. Aim To evaluate the expression of the immune checkpoint molecules CTLA-4, PD-1 and CD137 as well as of the cell proliferation marker Ki67, the activation marker CD69 and of CD103, a marker expressed on regulatory T cells, in T cells from CLL pts in different disease phases. Methods Peripheral blood samples were obtained from 69 CLL pts and 13 healthy control donors (HD). Pts were sub-grouped according to disease phase: indolent vs progressive (i.e. fulfilling criteria for active disease). The expression of CTLA-4, PD-1, PD-L1, CD69, CD103, CD137 and Ki-67 was assessed by flow-cytometry on CD4+ and CD8+ T cells. We also analysed the change in expression of these markers on T cells after 72 hours of PHA stimulation. Results CLL pts (n=17) had a significanty higher percentage of proliferating (Ki67+) CD3+ cells compared to HD (n=7) (median 3.7% in progressive vs 1.7% in indolent CLL vs 0.9% in HD, p=0.004 and p=0.04, respectively) (Fig.1). Progressive CLL pts had a significantly higher percentage Ki67+ CD4+ compared to indolent pts as well as HD (p=0.007 and p=0.001, respectively). Both indolent and progressive pts had higher percentage of Ki67+ CD8+ T cells compared to HD (p=0.01 and p=0.03, respectively). The percentage of CTLA-4+ CD4+ and CTLA-4+ CD8+ cells was low in CLL pts as well as in HD. However, the percentage of PD-1+ CD4+ T cells was significantly higher in progressive (n=32) as compared to indolent (n=35) CLL pts (median 40.3% vs 23.3%, p<0.0001) and HD (n=13) (median 21.5%, p<0.0001) (Fig.2) and correlated positively to the white blood cell counts (WBC) at the time of testing (r=0.29, p=0.03), while no difference was found with regard to the percentage of PD-1+ CD8+ T cells. No difference was observed between CLL pts and HD regarding the expression of PD-L1 on T cells. Both the percentage of CD69+ CD4+ and CD137+ CD4+ T cells were significantly higher in progressive as compared to indolent disease and correlated positively to WBC while no difference was found seen in CD8+ T cells. The percentage of CD103+ T cells was significantly lower in progressive compared to and HD within both the CD4+ (p=0.02) and the CD8+ subpopulations (p=0.02). After 72-hrs of PHA stimulation, PD-1 and CTLA-4 expression increased in CD4+ and CD8+ cells to a similar extent in CLL pts and HD, while PD-L1 increased in HD but not in progressive CLL pts (p=0.03 and p=0.007 for CD4+ and CD8+ cells, respectively). CD69 expression increased to a similar extent in CLL pts and HD, while CD137 expression increased more in T cells from progressive pts compared to HD (p=0.03 and 0.01 for the CD4+ and CD8+ cells, respectively). No increase in CD103 on CD8+ T-cells was observed in CLL pts compared to HD (p=0.04 and p=0.01 for the indolent and progressive pts, respectively). Conclusions Progressive CLL pts have more proliferating (Ki67+) T cells in both the CD4+ and CD8+ compartments compared to HD. CD4+ T-cells in progressive CLL pts display an activated phenotype (CD69+) and express the immune co-stimulatory molecule CD137 at a significantly higher level compared to indolent pts and HD. Nevertheless, the expression of the inhibitory immune checkpoint molecule PD-1 is so high that it is reasonable to assume that these cells are heavily impaired in their immune functions. The differences observed in the expression of immune checkpoints and activation markers between CLL pts in different phases of the disease suggest that major changes occur in the CD4+ T-cell compartment during disease progression. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Hansson: Jansse Cilag: Research Funding. Österborg:Janssen, Pharmacyclics, Gilead: Consultancy, Research Funding; Novartis: Research Funding.

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