Differential phenotypes of memory CD4 and CD8 T cells in the spleen and peripheral tissues following immunostimulatory therapy

Gail D Sckisel, Annie Mirsoian, Christine M Minnar, Marka Crittenden, Brendan Curti, Jane Q Chen, Bruce R Blazar, Alexander D Borowsky, Arta M Monjazeb, William J Murphy, Gail D Sckisel, Annie Mirsoian, Christine M Minnar, Marka Crittenden, Brendan Curti, Jane Q Chen, Bruce R Blazar, Alexander D Borowsky, Arta M Monjazeb, William J Murphy

Abstract

Background: Studies assessing immune parameters typically utilize human PBMCs or murine splenocytes to generate data that is interpreted as representative of immune status. Using splenocytes, we have shown memory CD4-T cells that expand following systemic immunostimulatory therapies undergo rapid IFNg-mediated activation induced cell death (AICD) resulting in a net loss of total CD4-T cells which correlates with elevated PD-1 expression. This is in contrast to CD8-T cells which expand with minimal PD-1 upregulation and apoptosis. In this study we expand upon our previous work by evaluating CD4 and CD8-T cell phenotype and distribution in peripheral organs which are more representative of immune responses occurring at metastatic sites following immunotherapy.

Methods: Phenotypic assessment of T cells in both lymphoid (spleen and LN) as well as peripheral organs (liver and lungs) in control and immunotherapy treated mice was performed to survey the impact of location on memory phenotype and activation marker status. Peripheral blood from patients undergoing systemic high dose IL-2 was also assessed for expression of PD-1 and memory phenotype.

Results: Here we reveal that, similar to what occurs in the spleen and lymph nodes, CD4-T cell numbers decreased while CD8-T cells expanded at these peripheral sites. In contrast to having differential expression of PD-1 as occurs in the spleen, both CD4 and CD8-T cells had significantly elevated levels of PD-1 in both the liver and lungs. Further analysis correlated PD-1 expression to CD62Llow (T effector/effector memory,TE/EM) expression which are more prevalent in CD4-T cells in general as well as CD8-T cells in peripheral organs. Similar elevated PD-1 expression on TE/EM cells was observed in patients undergoing systemic high-dose IL-2 therapy.

Conclusions: These data highlight PD-1 expressing and/or TE/EM subsets of T cells in circulation as more representative of cells at immune sites and underscore the importance of valuation both in lymphoid as well as target organs when making determinations about immune status.

Trial registration: ClinicalTrials.gov NCT01416831. Registered August 12, 2011.

Keywords: Bystander Activation; CD8; Cancer; Immunotherapy; NKG2D; PD-1.

Figures

Fig. 1
Fig. 1
CD4 and CD8 T cells have differential proliferative and apoptotic responses to immunostimulatory therapies in lymphoid organs. Mice were treated with anti-CD40/IL-2 immunotherapy and assessed for various immune parameters on day 12 of treatment in lymphoid (spleen or LN) organs. Percentage (a) and total numbers (b) of CD4 (Foxp3-ve) and CD8 T cells in lymphoid organs. Percentage of proliferating (c), as assessed by BrdU, and apoptotic (d), as assessed by surface Annexin V expression, of CD4 (Foxp3-ve) and CD8 T cells in lymphoid organs. These data are representative of 2-5 independent experiments with 3 mice per group. Data are presented as mean ± SEM. Statistics were derived using ANOVA with Bonferroni’s post-test, *P < 0.05, **P < 0.01, ***P < 0.001, ns: P > 0.05
Fig. 2
Fig. 2
CD4 and CD8 T cells have differential proliferative and apoptotic responses to immunostimulatory therapies in peripheral organs. Mice were treated with anti-CD40/IL-2 immunotherapy and assessed for various immune parameters on day 12 of treatment in peripheral (lungs or liver) organs. Percentage (a) and total numbers (b) of CD4 (Foxp3-ve) and CD8 T cells in peripheral organs. Percentage of proliferating (c), as assessed by BrdU, and apoptotic (d), as assessed by surface Annexin V expression, of CD4 (Foxp3-ve) and CD8 T cells in peripheral organs. These data are representative of 2-3 independent experiments with 3 mice per group. Data are presented as mean ± SEM. Statistics were derived using ANOVA with Bonferroni’s post-test, *P < 0.05, **P < 0.01, ***P < 0.001, ns: P > 0.05
Fig. 3
Fig. 3
T cell memory phenotype differs in lymphoid and peripheral organs following immunotherapy. Mice were treated with anti-CD40/IL-2 immunotherapy and assessed for various immune parameters on day 12 of treatment in lymphoid (spleen or LN) or peripheral (lungs or liver) organs. a-b Representative dot plots of CD44 vs CD62L expression in CD8 (a) and CD4 (Foxp3-ve) (b) T cells in control and IT-treated mice. c-d Pie charts depicting central memory (white) vs effector/effector memory (black) frequency in the CD44high sub-population in CD8 (c) T cells and CD4 (d) T cells; frequencies of CD44high depicted within pie slices for given population. (e-f) Frequency of effector/effector memory (e) and central memory (f) CD8 (left panels) and CD4 (Foxp3-ve) (right panels) T cells in various organs from control or anti-CD40/IL2-treated mice. These data are representative of 4-5 independent experiments with 3 mice per group. Data are presented as mean ± SEM
Fig. 4
Fig. 4
Differential expression of activation and inhibitory markers in CD8 T cells dependent upon location. Mice were treated with anti-CD40/IL-2 immunotherapy and assessed for various immune parameters on day 12 of treatment in lymphoid (spleen or LN) or peripheral (lungs or liver) organs. Percentage NKG2D+ (a-b) and PD-1+ (c-d) of CD8 (a, c) and CD4 (Foxp3-ve) (b, d) T cells across various organs. Pie graphs depicting CD8 EM/CM of each organ under given treatment conditions/organ. These data are representative of 2-4 independent experiments with 3 mice per group. Data are presented as mean ± SEM. Statistics were derived using ANOVA with Bonferroni’s post-test, *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
Differential phenotypes of CD8 T cells by location correlates with enhanced expansion and activation marker upregulation on the effector/effector memory T cell phenotype. Mice were treated with anti-CD40/IL-2 immunotherapy and assessed for various immune parameters on day 12 of treatment in lymphoid (spleen or LN) or peripheral (lungs or liver) organs. Frequencies of NKG2D+ (a-b) and PD-1+ (c-d) in CD25negCD44highCD8+ T cells in control (a, c) and anti-CD40/IL-2 (b, d) treated mice as stratified by TCM (CD62L+, white) and TE/EM (CD62L-, black). These data are representative of 2-3 independent experiments with 3 mice per group. Data are presented as mean ± SEM. Statistics were derived using ANOVA with Bonferroni’s post-test, *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
Effector/effector memory T cells from human T cells undergoing IL-2 therapy express upregulated PD-1. PBMCs were isolated prior to therapy and on day 8 of treatment from patients undergoing high dose systemic IL-2 therapy for melanoma. PBMCs were assessed for T cell subset expression of PD-1 by flow cytometry. a Representative gating strategy for staining of human PBMCs. b-c Frequency of PD-1 expression on memory CD4 (b) and CD8 (c) T cells. (d-e) Frequency of PD-1 expression on central (CD45RO + CD62L+) and effector memory (CD45RO + CD62L-) subsets in CD4 (d) and CD8 (e) T cells. Six patient samples were included in this data set. Data are presented as mean ± SEM. Statistics were derived using Student’s T test, *P < 0.05, **P < 0.01, ***P < 0.001

References

    1. Weiss JM, Back TC, Scarzello AJ, Subleski JJ, Hall VL, Stauffer JK, Chen X, Micic D, Alderson K, Murphy WJ, et al. Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment. Proc Natl Acad Sci U S A. 2009;106(46):19455–19460. doi: 10.1073/pnas.0909474106.
    1. Monjazeb AM, Tietze JK, Grossenbacher SK, Hsiao HH, Zamora AE, Mirsoian A, Koehn B, Blazar BR, Weiss JM, Wiltrout RH, et al. Bystander activation and anti-tumor effects of CD8+ T cells following Interleukin-2 based immunotherapy is independent of CD4+ T cell help. PLoS ONE. 2014;9(8):e102709. doi: 10.1371/journal.pone.0102709.
    1. Sckisel GD, Mirsoian A, Bouchlaka MN, Tietze JK, Chen M, Blazar BR, Murphy WJ. Late administration of murine CTLA-4 blockade prolongs CD8-mediated anti-tumor effects following stimulatory cancer immunotherapy. Cancer Immunol Immunother. 2015;64(12):1541–1552. doi: 10.1007/s00262-015-1759-4.
    1. Sckisel GD, Tietze JK, Zamora AE, Hsiao HH, Priest SO, Wilkins DE, Lanier LL, Blazar BR, Baumgarth N, Murphy WJ. Influenza infection results in local expansion of memory CD8(+) T cells with antigen non-specific phenotype and function. Clin Exp Immunol. 2014;175(1):79–91. doi: 10.1111/cei.12186.
    1. Tietze JK, Sckisel GD, Hsiao HH, Murphy WJ. Antigen-specific versus antigen-nonspecific immunotherapeutic approaches for human melanoma: the need for integration for optimal efficacy? Int Rev Immunol. 2011;30(5-6):238–293. doi: 10.3109/08830185.2011.598977.
    1. Tietze JK, Wilkins DE, Sckisel GD, Bouchlaka MN, Alderson KL, Weiss JM, Ames E, Bruhn KW, Craft N, Wiltrout RH, et al. Delineation of antigen-specific and antigen-nonspecific CD8(+) memory T-cell responses after cytokine-based cancer immunotherapy. Blood. 2012;119(13):3073–3083. doi: 10.1182/blood-2011-07-369736.
    1. Berner V, Liu H, Zhou Q, Alderson KL, Sun K, Weiss JM, Back TC, Longo DL, Blazar BR, Wiltrout RH, et al. IFN-gamma mediates CD4+ T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy. Nat Med. 2007;13(3):354–360. doi: 10.1038/nm1554.
    1. Alderson KL, Zhou Q, Berner V, Wilkins DE, Weiss JM, Blazar BR, Welniak LA, Wiltrout RH, Redelman D, Murphy WJ. Regulatory and conventional CD4+ T cells show differential effects correlating with PD-1 and B7-H1 expression after immunotherapy. J Immunol. 2008;180(5):2981–2988. doi: 10.4049/jimmunol.180.5.2981.
    1. Damonte P, Hodgson JG, Chen JQ, Young LJ, Cardiff RD, Borowsky AD. Mammary carcinoma behavior is programmed in the precancer stem cell. Breast Cancer Res. 2008;10(3):R50. doi: 10.1186/bcr2104.
    1. Murphy WJ, Welniak L, Back T, Hixon J, Subleski J, Seki N, Wigginton JM, Wilson SE, Blazar BR, Malyguine AM, et al. Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses. J Immunol. 2003;170(5):2727–2733. doi: 10.4049/jimmunol.170.5.2727.
    1. Seung SK, Curti BD, Crittenden M, Walker E, Coffey T, Siebert JC, Miller W, Payne R, Glenn L, Bageac A, et al. Phase 1 study of stereotactic body radiotherapy and interleukin-2--tumor and immunological responses. Sci Transl Med. 2012;4(137):137ra174. doi: 10.1126/scitranslmed.3003649.
    1. Dutton RW, Bradley LM, Swain SL. T cell memory. Annu Rev Immunol. 1998;16:201–223. doi: 10.1146/annurev.immunol.16.1.201.
    1. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol. 2004;22:745–763. doi: 10.1146/annurev.immunol.22.012703.104702.
    1. Shin H, Iwasaki A. Tissue-resident memory T cells. Immunol Rev. 2013;255(1):165–181. doi: 10.1111/imr.12087.
    1. Duraiswamy J, Ibegbu CC, Masopust D, Miller JD, Araki K, Doho GH, Tata P, Gupta S, Zilliox MJ, Nakaya HI, et al. Phenotype, function, and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy human adults. J Immunol. 2011;186(7):4200–4212. doi: 10.4049/jimmunol.1001783.
    1. Sckisel GD, Bouchlaka MN, Monjazeb AM, Crittenden M, Curti BD, Wilkins DE, Alderson KA, Sungur CM, Ames E, Mirsoian A, et al. Out-of-Sequence Signal 3 Paralyzes Primary CD4(+) T-Cell-Dependent Immunity. Immunity. 2015;43(2):240–250. doi: 10.1016/j.immuni.2015.06.023.
    1. Hokey DA, Johnson FB, Smith J, Weber JL, Yan J, Hirao L, Boyer JD, Lewis MG, Makedonas G, Betts MR, et al. Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaques. Eur J Immunol. 2008;38(5):1435–1445. doi: 10.1002/eji.200737857.
    1. Kinter AL, Godbout EJ, McNally JP, Sereti I, Roby GA, O'Shea MA, Fauci AS. The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J Immunol. 2008;181(10):6738–6746. doi: 10.4049/jimmunol.181.10.6738.
    1. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, Zhu G, Tamada K, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8(8):793–800.
    1. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992;11(11):3887–3895.
    1. Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, Adams WC, Precopio ML, Schacker T, Roederer M, Douek DC, et al. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med. 2006;203(10):2281–2292. doi: 10.1084/jem.20061496.
    1. Gros A, Robbins PF, Yao X, Li YF, Turcotte S, Tran E, Wunderlich JR, Mixon A, Farid S, Dudley ME, et al. PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest. 2014;124(5):2246–2259. doi: 10.1172/JCI73639.
    1. Lages CS, Lewkowich I, Sproles A, Wills-Karp M, Chougnet C. Partial restoration of T-cell function in aged mice by in vitro blockade of the PD-1/PD-L1 pathway. Aging Cell. 2010;9(5):785–798. doi: 10.1111/j.1474-9726.2010.00611.x.
    1. Shifrut E, Baruch K, Gal H, Ndifon W, Deczkowska A, Schwartz M, Friedman N. CD4(+) T Cell-Receptor Repertoire Diversity is Compromised in the Spleen but Not in the Bone Marrow of Aged Mice Due to Private and Sporadic Clonal Expansions. Front Immunol. 2013;4:379. doi: 10.3389/fimmu.2013.00379.
    1. Yang H, Youm YH, Vandanmagsar B, Ravussin A, Gimble JM, Greenway F, Stephens JM, Mynatt RL, Dixit VD. Obesity increases the production of proinflammatory mediators from adipose tissue T cells and compromises TCR repertoire diversity: implications for systemic inflammation and insulin resistance. J Immunol. 2010;185(3):1836–1845. doi: 10.4049/jimmunol.1000021.

Source: PubMed

Sponsorzy i współpracownicy

Warunki medyczne

Interwencje lekowe

3
Subskrybuj