臺灣博碩士論文加值系統

調節性T細胞為一特化的細胞群，他們透過抑制其他免疫細胞的功能，以達成調節個體免疫反應之效果。近來有研究指出，調節性T細胞會展現適應性，以因應環境中的刺激。在老鼠的實驗模型中，調節性T細胞因發炎細胞激素的影響，甚至能發展出增進免疫反應之功效。然而，目前對於人類調節性T細胞是否具有相同的促進免疫特性還不甚了解。此篇研究的目的為鑑定出人類表現CD40L的調節性T細胞，並分析其表型特徵與功能特性。
來自健康成人血液中的調節性T細胞，在強烈的CD3/TCR刺激下，得以表現CD40L。我們也從口腔癌患者的細胞中觀察到，相較於來自周邊血的調節性T細胞，腫瘤浸潤型淋巴球內的調節性T細胞面對刺激，有較高的比例會表現CD40L。藉由分析DNA的甲基化程度，發現CD40L+和CD40L-的調節性T細胞在FOXP3啟動子和TSDR的位置，皆展現低程度的甲基修飾，表示這兩群細胞均屬穩定的調節性T細胞。根據調節性T細胞常見的標記表現模式，顯示CD40L+和CD40L-調節性T細胞具有相似的表型特徵。在傳統的調節性T細胞功能分析中，CD40L+調節性T細胞展現較強的免疫抑制活性；而當單核球存在於實驗系統內，兩種調節性T細胞的免疫抑制活性則相當。此外，CD40L+與CD40L-調節性T細胞相比，具有較強的細胞激素生產率。與抑制功能相反的是，CD40L+調節性T細胞能夠促進樹突細胞表現成熟的細胞表徵，CD83。並且，與前述的樹突細胞表型特徵相符，經CD40L+調節性T細胞刺激後的樹突細胞，更能增進異體CD8+ T細胞的增生與活化。綜上所述，本篇研究指出，人類CD40L+調節性T細胞除了免疫抑制功能外，也可能具有促進免疫反應的效力。

Regulatory T cells are first discovered to regulate immune response through suppression to other immune cells. Upon facing environmental stimuli, it has been suggested that Treg cells show adaptive properties, even reported to provide a helper function in a mouse model. However, little is unknown about the helper activity of human Treg cells. The aims of this study are to identify human CD40L-expressing Treg cells along with their phenotypic and functional characteristics.
In this study, we isolated human Treg cells from healthy donors and identified CD40L-expressing Treg cells after strong CD3/TCR stimulation. We found Treg cells from tumor-infiltrating lymphocytes (TILs) of oral cancer showed stronger tendency to express CD40L under restimulation. Through DNA methylation analyses, both CD40L+ and CD40L- Treg cells were hypomethylated at FOXP3 promoter and Treg-specific demethylated region (TSDR), suggesting that both subsets are stable Treg cells. According to the expression patterns of Treg-associated markers, CD40L+ Treg cells exhibited similar phenotype to CD40L- counterpart. As for the suppression function, CD40L+ Treg cells showed superior suppressive activity on proliferation and cytokine production of Tconv cells. Nonetheless, the suppressive activities of both Treg subsets were equivalent in suppression assay with monocytes. CD40L+ Treg cells displayed consistently higher cytokine productivity than CD40L- Treg cells in both suppression systems. Furthermore, CD40L+ Treg cells promoted CD83 expression on dendritic cells, a marker for dendritic cell maturation. Consistent with the phenotype, CD40L+ Treg-treated dendritic cells possessed better capacity to promote allogeneic CD8+ T cell activation and proliferation. In summary, these results indicated that human CD40L-expressing Treg cells might have positive effects on immune response apart from their suppressive function.

誌謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS iv
LIST OF TABLES vii
LIST OF FIGURES viii
Chapter 1 Introduction 1
1.1 Regulatory T cells 1
1.1.1 Identification of human Treg cells 1
1.1.2 Development of human Treg cells 2
1.1.3 Suppressive activity of human Treg cells 3
1.1.4 Heterogeneity and plasticity of human Treg cells 5
1.2 CD40 ligand 6
1.2.1 Expression of CD40L on CD4+ T cells 7
1.2.2 Function of CD40-CD40L engagement in immune system 8
1.3 Expression of CD40L on human Treg cells 9
Chapter 2 Purposes and Aims 11
Chapter 3 Materials and Methods 12
3.1 Materials 12
3.2 Methods 12
3.2.1 Isolation of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes 12
3.2.2 Enrichment of CD4+, CD8+ T cells and CD14+ monocytes 13
3.2.3 Generation of human monocyte-derived dendritic cells 13
3.2.4 Fluorescent staining of surface, intracellular and intranuclear proteins 14
3.2.5 Fluorescence activated cell sorting (FACS) 14
3.2.6 Labeling cells with various proliferation dyes 15
3.2.7 CD40L induction 15
3.2.8 DNA bisulfite conversion and methylation analysis 16
3.2.9 In vitro Treg suppression assay 16
3.2.10 Allogeneic CD8+ T cell activation by dendritic cells 17
3.2.11 Statistical analysis 18
Chapter 4 Results 19
4.1 Phenotypic analyses of CD40L+ Treg cells 19
4.1.1 CD40L expression on Treg cells was induced by strong CD3/TCR stimulation 19
4.1.2 TIL Treg cells were prone to express CD40L upon αCD3/CD28 bead restimulation 20
4.1.3 CD40L+ Treg cells exhibited hypomethylation on FOXP3 promoter and TSDR 20
4.1.4 The expression patterns of Treg-associated markers were similar between CD40L+ and CD40L- Treg cells 21
4.1.5 Naïve and memory CD40L+ Treg cells resembled its CD40L- counterpart regardless of differential CD40L expression 21
4.2 Functional analyses of CD40L+ Treg cells 22
4.2.1 CD40L+ Treg cells possessed stronger suppressive activity and cytokine productivity than CD40L- Treg cells under αCD3/CD28 beads stimulation 22
4.2.2 CD40L+ Treg cells showed equivalent suppressive activity to CD40L- Treg cells under monocyte stimulation 23
4.2.3 CD40L+ Treg cells promoted dendritic cell maturation 23
4.2.4 CD40L+ Treg-treated dendritic cells had the capacity to induce allogeneic CD8+ T cell activation and proliferation 24
Chapter 5 Discussion 26
5.1 Induction of CD40L on Treg cells 26
5.2 CD40L expression on Treg cells in TILs from oral cancer patients 27
5.3 The phenotype of CD40L+ and CD40L- Treg cells 28
5.4 The suppressive activities of CD40L+ and CD40L- Treg cells 28
5.5 Phenotype of dendritic cells after coculture with CD40L+/- Treg cells 29
REFERENCES 30
TABLES 37
FIGURES 45

1.DuPage, M. and J.A. Bluestone, Harnessing the plasticity of CD4(+) T cells to treat immune-mediated disease. Nat Rev Immunol, 2016. 16(3): p. 149-63.

2.Shevach, E.M., Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity, 2009. 30(5): p. 636-45.

3.Sakaguchi, S., et al., FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol, 2010. 10(7): p. 490-500.

4.Cools, N., et al., Regulatory T cells and human disease. Clin Dev Immunol, 2007. 2007: p. 89195.

5.Bennett, C.L., et al., The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet, 2001. 27(1): p. 20-1.

6.Gershon, R.K. and K. Kondo, Infectious immunological tolerance. Immunology, 1971. 21(6): p. 903-14.

7.Sakaguchi, S., et al., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol, 1995. 155(3): p. 1151-64.

8.Fontenot, J.D., M.A. Gavin, and A.Y. Rudensky, Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol, 2003. 4(4): p. 330-6.

9.Hori, S., T. Nomura, and S. Sakaguchi, Control of regulatory T cell development by the transcription factor Foxp3. Science, 2003. 299(5609): p. 1057-61.

10.Roncador, G., et al., Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol, 2005. 35(6): p. 1681-91.

11.Liu, W., et al., CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med, 2006. 203(7): p. 1701-11.

12.Seddiki, N., et al., Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med, 2006. 203(7): p. 1693-700.

13.Gavin, M.A., et al., Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc Natl Acad Sci U S A, 2006. 103(17): p. 6659-64.

14.Mazzucchelli, R. and S.K. Durum, Interleukin-7 receptor expression: intelligent design. Nat Rev Immunol, 2007. 7(2): p. 144-54.

15.Hamann, A., et al., Activation induces rapid and profound alterations in the trafficking of T cells. Eur J Immunol, 2000. 30(11): p. 3207-18.

16.Abbas, A.K., et al., Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol, 2013. 14(4): p. 307-8.

17.Shevach, E.M. and A.M. Thornton, tTregs, pTregs, and iTregs: similarities and differences. Immunol Rev, 2014. 259(1): p. 88-102.

18.Nishizuka, Y. and T. Sakakura, Thymus and reproduction: sex-linked dysgenesia of the gonad after neonatal thymectomy in mice. Science, 1969. 166(3906): p. 753-5.

19.Itoh, M., et al., Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol, 1999. 162(9): p. 5317-26.

20.Hsieh, C.S., H.M. Lee, and C.W. Lio, Selection of regulatory T cells in the thymus. Nat Rev Immunol, 2012. 12(3): p. 157-67.

21.Ohkura, N., et al., T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity, 2012. 37(5): p. 785-99.

22.Spits, H., Development of alphabeta T cells in the human thymus. Nat Rev Immunol, 2002. 2(10): p. 760-72.

23.Davidson, T.S., et al., Cutting Edge: IL-2 is essential for TGF-beta-mediated induction of Foxp3+ T regulatory cells. J Immunol, 2007. 178(7): p. 4022-6.

24.Tran, D.Q., H. Ramsey, and E.M. Shevach, Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood, 2007. 110(8): p. 2983-90.

25.Lu, L., et al., Characterization of protective human CD4CD25 FOXP3 regulatory T cells generated with IL-2, TGF-beta and retinoic acid. PLoS One, 2010. 5(12): p. e15150.

26.Hippen, K.L., et al., Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease. Am J Transplant, 2011. 11(6): p. 1148-57.

27.Vignali, D.A., L.W. Collison, and C.J. Workman, How regulatory T cells work. Nat Rev Immunol, 2008. 8(7): p. 523-32.

28.Schmidt, A., N. Oberle, and P.H. Krammer, Molecular mechanisms of treg-mediated T cell suppression. Front Immunol, 2012. 3: p. 51.

29.Ito, T., et al., Two functional subsets of FOXP3+ regulatory T cells in human thymus and periphery. Immunity, 2008. 28(6): p. 870-80.
30.Jonuleit, H., et al., Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J Exp Med, 2001. 193(11): p. 1285-94.

31.Hawrylowicz, C.M. and A. O''Garra, Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma. Nat Rev Immunol, 2005. 5(4): p. 271-83.

32.Annacker, O., et al., Interleukin-10 in the regulation of T cell-induced colitis. J Autoimmun, 2003. 20(4): p. 277-9.

33.Levings, M.K., et al., Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J Exp Med, 2002. 196(10): p. 1335-46.

34.Fahlen, L., et al., T cells that cannot respond to TGF-beta escape control by CD4(+)CD25(+) regulatory T cells. J Exp Med, 2005. 201(5): p. 737-46.

35.Baecher-Allan, C., V. Viglietta, and D.A. Hafler, Inhibition of human CD4(+)CD25(+high) regulatory T cell function. J Immunol, 2002. 169(11): p. 6210-7.

36.Godfrey, W.R., et al., Cord blood CD4(+)CD25(+)-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function. Blood, 2005. 105(2): p. 750-8.

37.Oberle, N., et al., Rapid suppression of cytokine transcription in human CD4+CD25 T cells by CD4+Foxp3+ regulatory T cells: independence of IL-2 consumption, TGF-beta, and various inhibitors of TCR signaling. J Immunol, 2007. 179(6): p. 3578-87.

38.Collison, L.W., et al., The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature, 2007. 450(7169): p. 566-9.

39.Bardel, E., et al., Human CD4+ CD25+ Foxp3+ regulatory T cells do not constitutively express IL-35. J Immunol, 2008. 181(10): p. 6898-905.

40.Zhao, D.M., et al., Activated CD4+CD25+ T cells selectively kill B lymphocytes. Blood, 2006. 107(10): p. 3925-32.

41.Grossman, W.J., et al., Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood, 2004. 104(9): p. 2840-8.

42.Garin, M.I., et al., Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells. Blood, 2007. 109(5): p. 2058-65.

43.Strauss, L., C. Bergmann, and T.L. Whiteside, Human circulating CD4+CD25highFoxp3+ regulatory T cells kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis. J Immunol, 2009. 182(3): p. 1469-80.

44.Thornton, A.M. and E.M. Shevach, CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med, 1998. 188(2): p. 287-96.

45.de la Rosa, M., et al., Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur J Immunol, 2004. 34(9): p. 2480-8.

46.Tran, D.Q., et al., Analysis of adhesion molecules, target cells, and role of IL-2 in human FOXP3+ regulatory T cell suppressor function. J Immunol, 2009. 182(5): p. 2929-38.

47.Borsellino, G., et al., Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood, 2007. 110(4): p. 1225-32.

48.Mandapathil, M., et al., Isolation of functional human regulatory T cells (Treg) from the peripheral blood based on the CD39 expression. J Immunol Methods, 2009. 346(1-2): p. 55-63.

49.Sakaguchi, S., et al., Regulatory T cells and immune tolerance. Cell, 2008. 133(5): p. 775-87.

50.Ernst, P.B., J.C. Garrison, and L.F. Thompson, Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology. J Immunol, 2010. 185(4): p. 1993-8.

51.Bopp, T., et al., Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression. J Exp Med, 2007. 204(6): p. 1303-10.

52.Klein, M., et al., Repression of cyclic adenosine monophosphate upregulation disarms and expands human regulatory T cells. J Immunol, 2012. 188(3): p. 1091-7.

53.Cederbom, L., H. Hall, and F. Ivars, CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur J Immunol, 2000. 30(6): p. 1538-43.

54.Fallarino, F., et al., Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol, 2003. 4(12): p. 1206-12.

55.Liang, B., et al., Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II. J Immunol, 2008. 180(9): p. 5916-26.

56.Mohr, A., et al., Human FOXP3(+) T regulatory cell heterogeneity. Clin Transl Immunology, 2018. 7(1): p. e1005.

57.Miyara, M., et al., Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity, 2009. 30(6): p. 899-911.

58.Baecher-Allan, C., E. Wolf, and D.A. Hafler, MHC class II expression identifies functionally distinct human regulatory T cells. J Immunol, 2006. 176(8): p. 4622-31.

59.Fuhrman, C.A., et al., Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226. J Immunol, 2015. 195(1): p. 145-55.

60.Gu, J., et al., Human CD39(hi) regulatory T cells present stronger stability and function under inflammatory conditions. Cell Mol Immunol, 2017. 14(6): p. 521-528.

61.Campbell, D.J. and M.A. Koch, Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol, 2011. 11(2): p. 119-30.

62.Rubtsov, Y.P., et al., Stability of the regulatory T cell lineage in vivo. Science, 2010. 329(5999): p. 1667-71.

63.Dominguez-Villar, M., C.M. Baecher-Allan, and D.A. Hafler, Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease. Nat Med, 2011. 17(6): p. 673-5.

64.Cretney, E., A. Kallies, and S.L. Nutt, Differentiation and function of Foxp3(+) effector regulatory T cells. Trends Immunol, 2013. 34(2): p. 74-80.

65.Komatsu, N., et al., Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc Natl Acad Sci U S A, 2009. 106(6): p. 1903-8.

66.Hori, S., Developmental plasticity of Foxp3+ regulatory T cells. Curr Opin Immunol, 2010. 22(5): p. 575-82.

67.Sakaguchi, S., et al., The plasticity and stability of regulatory T cells. Nat Rev Immunol, 2013. 13(6): p. 461-7.

68.Hori, S., Lineage stability and phenotypic plasticity of Foxp3(+) regulatory T cells. Immunol Rev, 2014. 259(1): p. 159-72.

69.van Kooten, C. and J. Banchereau, CD40-CD40 ligand. J Leukoc Biol, 2000. 67(1): p. 2-17.

70.Karpusas, M., et al., 2 A crystal structure of an extracellular fragment of human CD40 ligand. Structure, 1995. 3(10): p. 1031-9.

71.Grewal, I.S. and R.A. Flavell, CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol, 1998. 16: p. 111-35.

72.Elgueta, R., et al., Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev, 2009. 229(1): p. 152-72.

73.Koguchi, Y., et al., Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner. Blood, 2007. 110(7): p. 2520-7.
74.Schubert, L.A., et al., The human gp39 promoter. Two distinct nuclear factors of activated T cell protein-binding elements contribute independently to transcriptional activation. J Biol Chem, 1995. 270(50): p. 29624-7.

75.Lee, B.O., et al., The biological outcome of CD40 signaling is dependent on the duration of CD40 ligand expression: reciprocal regulation by interleukin (IL)-4 and IL-12. J Exp Med, 2002. 196(5): p. 693-704.

76.Peng, X., et al., IL-12 up-regulates CD40 ligand (CD154) expression on human T cells. J Immunol, 1998. 160(3): p. 1166-72.

77.Fayen, J.D., Multiple cytokines sharing the common receptor gamma chain can induce CD154/CD40 ligand expression by human CD4+ T lymphocytes via a cyclosporin A-resistant pathway. Immunology, 2001. 104(3): p. 299-306.

78.Ma, D.Y. and E.A. Clark, The role of CD40 and CD154/CD40L in dendritic cells. Semin Immunol, 2009. 21(5): p. 265-72.

79.Mackey, M.F., R.J. Barth, Jr., and R.J. Noelle, The role of CD40/CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells. J Leukoc Biol, 1998. 63(4): p. 418-28.

80.Quezada, S.A., et al., CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol, 2004. 22: p. 307-28.

81.Summers deLuca, L. and J.L. Gommerman, Fine-tuning of dendritic cell biology by the TNF superfamily. Nat Rev Immunol, 2012. 12(5): p. 339-51.

82.Lozano, T., et al., Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation. J Immunol, 2015. 195(7): p. 3180-9.

83.Koguchi, Y., et al., Preformed CD40L is stored in Th1, Th2, Th17, and T follicular helper cells as well as CD4+ 8- thymocytes and invariant NKT cells but not in Treg cells. PLoS One, 2012. 7(2): p. e31296.

84.Litjens, N.H., K. Boer, and M.G. Betjes, Identification of circulating human antigen-reactive CD4+ FOXP3+ natural regulatory T cells. J Immunol, 2012. 188(3): p. 1083-90.

85.Schoenbrunn, A., et al., A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg. J Immunol, 2012. 189(12): p. 5985-94.

86.Noyan, F., et al., Isolation of human antigen-specific regulatory T cells with high suppressive function. Eur J Immunol, 2014. 44(9): p. 2592-602.

87.Nowak, A., et al., CD137+CD154- Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures. Front Immunol, 2018. 9: p. 199.

88.Sharma, M.D., et al., An inherently bifunctional subset of Foxp3+ T helper cells is controlled by the transcription factor eos. Immunity, 2013. 38(5): p. 998-1012.

89.Lee, J.J., et al., Increased prevalence of interleukin-17-producing CD4(+) tumor infiltrating lymphocytes in human oral squamous cell carcinoma. Head Neck, 2011. 33(9): p. 1301-8.

90.Sallusto, F., et al., Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med, 1995. 182(2): p. 389-400.

91.Li, D.Y., et al., Maturation induction of human peripheral blood mononuclear cell-derived dendritic cells. Exp Ther Med, 2012. 4(1): p. 131-134.

92.Morelli, A.E. and A.W. Thomson, Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol, 2007. 7(8): p. 610-21.

93.Gordon, J.R., et al., Regulatory dendritic cells for immunotherapy in immunologic diseases. Front Immunol, 2014. 5: p. 7.

94.Yoo, S. and S.J. Ha, Generation of Tolerogenic Dendritic Cells and Their Therapeutic Applications. Immune Netw, 2016. 16(1): p. 52-60.

95.Hubo, M., et al., Costimulatory molecules on immunogenic versus tolerogenic human dendritic cells. Front Immunol, 2013. 4: p. 82.

96.Aerts-Toegaert, C., et al., CD83 expression on dendritic cells and T cells: correlation with effective immune responses. Eur J Immunol, 2007. 37(3): p. 686-95.