CCR4 – a potential marker for effector-type regulatory T cells

CCR4 – a potential marker for the This note exemplifies the use of the gentleMACS™ Dissociator and the autoMACS® Pro Separator for an automated selective removal of effector-type experimental setup, providing reproducible results from + + FOXP3 CD4 regulatory T cells in sample preparation to cell separation, in our search for more cancer immunotherapies specific cell markers in cancer immunotherapies.

Daisuke Sugiyama and Hiroyoshi Nishikawa Department of Immunology, Nagoya University Graduate Material School of Medicine, Nagoya, Japan • gentleMACS Dissociator • gentleMACS C Tubes Background • autoMACS Pro Separator • Biotin-anti-CD25 (BC96) CD4+CD25+ regulatory T (Treg) cells expressing the • Biotin-anti-CCR4 monoclonal antibody (1G1) transcription factor forkhead box P3 (FOXP3) play • Anti-Biotin MicroBeads (Miltenyi Biotec) an important role in suppressing antitumor immune responses. Some clinical studies have shown the potential of depleting CD25-expressing lymphocytes to augment Methods antitumor immune responses1,2. Yet other similar studies did not support this claim3-5. The depletion of CD25+ • Peripheral blood mononuclear cells (PBMCs) were cells is debatable, because activated effector T cells prepared from peripheral blood of healthy donors also express CD25 and promote the expansion of CD8+ and melanoma patients. cytotoxic lymphocytes, for example. Their depletion may • Primary human melanomas were resected and the abrogate the effect of Treg cell depletion, i.e., counteract surrounding healthy tissue was removed. Single-cell the augmentation of antitumor immunity3. Moreover, suspensions were prepared using the gentleMACS based on studies with animal models it has been suggested Dissociator and C Tubes. that depletion of Treg cells can result in autoimmunity6-8. • CD25 – or CCR4– cells were prepared from PBMCs and Therefore, a current challenge is to determine more specific melanoma-derived single-cell suspensions using markers to control the immunosuppressive effect of Treg biotin-anti-CD25 or biotin-anti-CCR4 antibodies cells without compromising effector T cells or eliciting (0.01 mg/mL), respectively. Cells were incubated with deleterious autoimmunity. antibodies for 15 min at 4 °C. Subsequently, Anti-Biotin Compared to peripheral blood T cells, tumor-infiltrating MicroBeads were added as described in the T cells contain a larger percentage of effector Treg (eTreg) manufacturer’s protocol. Then cells were washed using cells, which are defined as FOXP3hi and CD45RA–, terminally PBS containing 2% (v/v) FCS. CD25– or CCR4– cells were differentiating, and most suppressive. We could show that separated using an autoMACS Pro Separator. eTreg cells, but not FOXP3lo and CD45RA+ naive Treg cells, • Original fractions before separation and fractions predominantly express C-C receptor 4 (CCR4) in depleted of CD25+ or CCR4+ cells were analyzed by both melanoma tissues and peripheral blood. In vivo and flow cytometry using CD4, CD25, CD45RA, CD194 in vitro anti-CCR4 mAb treatment selectively depleted eTreg (CCR4), and Anti-FOXP3 antibodies. cells and efficiently induced tumor-antigen-specific CD4+ and CD8+ T cells.9

CCR4 – a potential marker for effector-type regulatory T cells | January 2018 1/3 Results Figure.1Figure.1 A A + Depletion of CCR4 cells removes 105 105 105 105 V: 14.0 V:I :14.0 0.67 I: 0.67 22.8 2.022.8 3.52.0 3.5 eTreg cells from PBMCs selectively 4 4 10 10 104 104 When PBMCs from melanoma patients were stained with 3 3 II: 3.7 II: 3.7 + 10 10 103 103 CCR4 various antibodies and analyzed by gating on CD4 100100 100100 CD45RA

8080 8080 102 102 II IIII T cells, CCR4 expression was found to be particularly high 6060 6060 0 0 4040 4040

IV: 76.6 IV:III: 76.6 4.8 III: 4.8 Count Count CD45RA CD45RA 0 0 CCR4 CCR4 in fractions of eTreg cells and almost non-existent in naive 2020 2020 3 43 5 4 5 69.5 32.169.5 432.1 5 4 5 0 10 0 1010 1010 10 0 00 10 0 1010 00 1010 10 Treg (nTreg) cell fractions (fig. 1A). This tendency was similar FOXP3 00 101033 101044 10FOXP31055 00 101033 101044 101055 CCR4 in PBMCs from healthy individuals⁹. Depletion of CCR4+ cells FOXP3FOXP3 FOXP3FOXP3

100100 100100 100100 100100 led to a remarkable reduction of eTreg cells whereas nTreg 8080 8080 8080 8080 I II II IIII 6060 6060 6060 6060 cells remained unaffected (fig. 1B). However, both eTreg 4040 4040 4040 4040 Count Count Count Count

2020 2020 2020 2020

+ Relative Relative cells and nTreg cells were greatly reduced after CD25 cell 00 00 00 00 cell number cell number cell 00 101033 101044 101055 00 101033 101044 101055 00 101022 101033 101044 101055 00 101022 101033 101044 101055 depletion (fig. 1C). CCR4 CCR4 CD25 CD25

+ 100100 100100 CCR4 eTreg cells infiltrate into melanoma tissue 8080 8080 I II predominantly and can be efficiently depleted Figure.1Figure.1B B and6060 B Cand C6060 C 4040 4040 Count Count It is known that FOXP3+ T cells invade tumors locally, but 1052020 105 2020 105 105 00 00 22 V: 33 32.444 V:55 I: 32.41.3 22 I33: 1.344 55 V: 37.0 V:I: 37.00.39 I: 0.39 00 1010 1010 1010 1010 00 1010 1010 1010 1010 their detailed phenotype is not well defined. Therefore, 104 104 CD25 104 104 tumor-infiltrating lymphocytes (TILs) from melanoma 3 3 II: 0.05 II: 0.05 3 3 II: 0.02 II: 0.02 10 10 10 10

tissue were prepared using the gentleMACS Dissociator CD45RA CD45RA and the same flow cytometric analysis was performed as in 102 102 102 102

CD45RA IV:CD45RA 62.8 IV:III: 62.8 3.2 III: 3.2 CD45RA CD45RA IV: 59.7 IV:III: 59.7 2.4 III: 2.4 figure 1A. A high percentage of eTreg cells infiltrated into 0 0 0 0 3 43 5 4 5 3 43 5 4 5 0 FOXP310 0 1010 1010 10 0 10 0 10FOXP310 1010 10 the melanoma tissue (fig. 2A) resulting in higher eTreg cell FOXP3FOXP3 FOXP3FOXP3 frequencies compared to the PBMC samples. Nevertheless, we could significantly reduce the number of melanoma Fraction I Fraction I 2 1.5 tissue-infiltrating eTreg cells by selective depletion of P < 0.005 + 1.5 CCR4 T cells (fig. 2B). 1

T cells 1 T cells + + 0.5 CD4 Conclusions 0.5 CD4 Percentage among Percentage Screening new marker candidates for effective cancer 0 among Percentage 0 immunotherapies is challenging and labor intensive. The PBMCs CCR4 depl. PBMCs CD25 depl. combination of gentleMACS Dissociator and autoMACS Fraction II Fraction II Pro Separator enabled us to automate our workflow and to 4 4 use CCR4 as a selective marker to remove eTreg cells – the P < 0.005 P < 0.005 most immunosuppressive Treg cell type – in a standardized 3 3

manner. Depletion of eTreg cells based on CCR4 did not T cells T cells

+ 2 + 2 affect other Treg cell subpopulations. In contrast, depletion based on CD25 resulted in the removal of various Treg CD4 1 CD4 1 Percentage among Percentage among Percentage cell subsets. 0 0 PBMCs CCR4 depl. PBMCs CD25 depl.

Figure 1: In vitro CCR4+ cell depletion reduces effector Treg cells effectively. Expression of FOXP3, CD45RA, and CCR4 in CD4+ T cells from PBMCs obtained from a melanoma patient was analyzed by flow cytometry (A). Numbers in the dot plots show the percentage of cells in each fraction with CD4+ T cells representing 100%. The various fractions indicate nTreg cells (I), eTreg cells (II), FOXP3lo non-Treg cells (III), memory and activated Th (IV), and naive Th cells (V). Expression of CCR4 and CD25 in fractions I and II is shown in the histograms (A; lower panel). CCR4+ cells (B) or CD25+ cells (C) were depleted from PBMCs using the autoMACS Pro Separator and the CCR4– and CD25– cells were analyzed for FOXP3 and CD45RA expression (B, C; upper panel). The percentage of nTreg cells (fraction I) and eTreg cells (fraction II) in PBMCs prior to depletion and after CCR4+ (B) or CD25+ (C) cell depletion is shown for six different donors. CD4+ cells represent 100%.

CCR4 – a potential marker for effector-type regulatory T cells | January 2018 2/3 References A B Melanoma TILs 1. Dannull, J. et al. (2005) Enhancement of vaccine-mediated antitumor Melanoma TILs MelanomaafterMelanoma TILs CCR4 depletion TILs immunity in cancer patients after depletion of regulatory T cells. J. Clin. MelanomaMelanoma TILs TILsAfter CCR4After depletion CCR4 depletion Invest. 115: 3623–3633.

105 105 105 105 2. Rech, A.J. et al. (2012) CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients. Sci.

104 104 104 104 Transl. Med. 4: 134ra162. 3. Nishikawa, H. and Sakaguchi, S. (2010) Regulatory T cells in tumor 3 3 3 3 immunity. Int. J. Cancer 127: 759–767. 10 10 12.2 10 10 5.0 12.2 12.2 5.0 5.0 4. Attia, P. et al. (2005) Inability of a fusion of IL-2 and diphtheria CD45RA CD45RA

2 2 102 102 10 10 toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma. J. Immunother. 28: 582–592. 0 0 CD45RA CD45RA 0 0 5. Litzinger, M.T. et al. (2007) IL-2 immunotoxin denileukin diftitox reduces 3 4 3 5 4 5 3 4 3 5 4 5 0 FOXP310 0 10 1010 10 10 0 10 0 10 FOXP310 10 10 10 regulatory T cells and enhances vaccine-mediated T-cell immunity. FOXP3 FOXP3 Blood 110: 3192–3201. 6. Sakaguchi, S. (2004) Naturally arising CD4+ regulatory T cells for

5 5 10 10 105 105 immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531–562. 4 4 12.2 4 4 10 10 12.2 12.210 10 7. Sakaguchi, S. et al. (1995) Immunologic self-tolerance maintained by 5.0 5.0 activated T cells expressing IL-2 receptor α-chains (CD25). Breakdown 3 3 10 10 103 103 of a single mechanism of self-tolerance causes various autoimmune CCR4 CCR4 diseases. J. Immunol. 155: 1151–1164. 102 102 8. Kim, J.M. et al. (2007) Regulatory T cells prevent catastrophic 0 0 autoimmunity throughout the lifespan of mice. Nat. Immunol. 8: 191–197. CCR4 CCR4 0 0 9. Sugiyama, D. et al. (2013) Anti-CCR4 mAb selectively depletes effector- 3 4 3 5 4 5 3 4 3 5 4 5 0 FOXP310 0 10 10 10 10 10 0 10 0 10 FOXP310 10 10 10 type FoxP3+CD4+ regulatory T cells, evoking antitumor immune FOXP3 FOXP3 responses in humans. Proc. Natl. Acad. Sci. USA 110: 17945–17950.

Figure 2: CCR4+ eTreg cells invade into melanoma tissue predominantly. (A) Melanoma tissue-infiltrating lymphocytes were prepared with the gentleMACS Dissociator and analyzed by flow MACS Product Order no. cytometry. (B) After depletion of CCR4+ cells using the autoMACS Pro gentleMACS Dissociator 130-093-235 Separator the frequency of FOXP3hi eTreg cells was analyzed. Numbers indicate the percentage of FOXP3hi CCR4+ cells among CD4+ T cells. gentleMACS C Tubes 130-096-334 autoMACS Pro Separator – Starter Kit 130-092-545 Anti-Biotin MicroBeads 130-090-485

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CCR4 – a potential marker for effector-type regulatory T cells | January 2018 3/3