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Originally published online as doi:10.1189/jlb.1105640 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:663-666.)
© 2006 by Society for Leukocyte Biology

Sex-specific phenotypical and functional differences in peripheral human V{gamma}9/V{delta}2 T cells

Nadia Caccamo*, Francesco Dieli*, Daniela Wesch{dagger}, Hassan Jomaa{ddagger} and Matthias Eberl{ddagger},1

* Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Italy;
{dagger} Institut für Immunologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Germany; and
{ddagger} Biochemisches Institut, Infektiologie, Justus-Liebig-Universität Giessen, Germany

1Correspondence at current address: Institute of Cell Biology, Baltzerstrasse 4, University of Bern, 3012 Bern, Switzerland. E-mail: meberl{at}izb.unibe.ch

ABSTRACT

V{gamma}9/V{delta}2 T cells constitute a minor proportion of human peripheral blood T cells that can expand rapidly upon infection with microbial pathogens. V{gamma}9/V{delta}2 T cell numbers change characteristically with age, rising from birth to puberty and gradually decreasing again beyond 30 years of age. In adults, female blood donors have significantly higher levels than males, implying that circulating V{gamma}9/V{delta}2 T cells in women remain elevated for a longer period in life and drop less strikingly than in men. This loss in men is accompanied by a substantial depletion of CD27–CD45RA– and CD27–CD45RA+ effector T cells and a parallel increase in CD27+CD45RA– central memory T cells while in women, the distribution of V{gamma}9/V{delta}2 T cell subsets remains virtually unchanged. The phenotypical conversion in men older than 30 years is mirrored by an increased proliferative response of V{gamma}9/V{delta}2 T cells and a reduced interferon-{gamma} secretion upon stimulation with isopentenyl pyrophosphate in vitro.

Key Words: {gamma}{delta} T lymphocytes • memory subsets • male immune system • gender-related bias • aging • IPP

V{gamma}9/V{delta}2 T cells constitute a minor proportion of human peripheral blood T cells yet expand rapidly upon infection with microbial pathogens, which produce (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a highly immunogenic intermediate of the nonmevalonate pathway of isoprenoid biosynthesis [1 ]. In addition, viruses and tumors may activate V{gamma}9/V{delta}2 T cells through surface expression of the mitochondrial F1-ATPase on stressed cells, that in combination with apolipoprotein A-I, can be recognized by the V{gamma}9/V{delta}2 T cell receptor [2 ]. This reactivity may be facilitated further by killer inhibitory receptors and activating receptors such as NKG2D [3 , 4 ].

The unconventional specificity for self and nonself "danger" signals bridges innate and acquired immunity and places V{gamma}9/V{delta}2 T cells at a key regulatory and effector position in the immune system. Once activated, they possess a broad, functional flexibility, as they may assume T helper cell type 1 (Th1)- or Th2-like effector functions, provide B cell help in secondary lymphoid tissues, and even act as professional antigen-presenting cells [5 6 7 8 ].

V{gamma}9/V{delta}2 T lymphocytes are rare in thymus and circulation at birth but increase during childhood in the blood, suggesting a positive selection in the periphery as a result of sustained antigenic stimulation [9 , 10 ]. In healthy adults, they typically constitute 0.5–5% of peripheral T cells, albeit in exceptional cases, seemingly stable levels of 20–40% are possible (our own unpublished observations). However, V{gamma}9/V{delta}2 T cell levels decline again with aging, with a reduced expansion and an altered cytokine production in elderly individuals [11 ].

We recently identified V{gamma}9/V{delta}2 T cell subsets with distinct migratory routes and effector functions, based on their expression of CD45RA and CD27. Naive {gamma}{delta} T cells (CD45RA+CD27+) and central memory {gamma}{delta} (TCM) cells (CD45RA–CD27+) express lymph node homing receptors and abound in secondary lymphoid tissue. Conversely, effector/memory {gamma}{delta} (TEM) cells (CD45RA–CD27–) and terminally differentiated {gamma}{delta} (TEMRA) cells (CD45RA+CD27–) are capable of homing to inflamed tissues and exerting proinflammatory and cytotoxic responses [6 ]. Using this classification, we here analyzed the frequencies and functional properties of V{gamma}9/V{delta}2 T cell subsets in male and female individuals at different ages.

Peripheral blood mononuclear cells (PBMC) from healthy blood donors were incubated with the following monoclonal antibodies in different combinations: anti-V{gamma}9 (Immu360), anti-V{delta}2 (Immu389), anti-CD3 (UCHT-1), and anti-CD45RA (2H4) from Beckman Coulter (Miami, FL) and anti-V{gamma}9 (7A5), anti-CD3 (SK7), and anti-CD27 (M-T271) from BD Biosciences (San Diego, CA). Data were acquired on a FACSCalibur instrument and analyzed using CellQuest software (BD Biosciences) or on an Epics XL flow cytometer supported with Expo32 ADC software (Beckman Coulter) [5 , 6 , 12 ]. Results were expressed as percentage of V{gamma}9/V{delta}2 T cells among CD3+ lymphocytes or as percentage of defined subsets among V{gamma}9/V{delta}2 T cells.

In line with earlier reports [9 , 11 ], the age-dependent changes in peripheral V{gamma}9/V{delta}2 T cell numbers in our study population resulted in a characteristic pattern, rising from birth to puberty and gradually decreasing beyond an age of 20–30 years (Fig. 1A ). Mean V{gamma}9/V{delta}2 T cell frequencies dropped from 3.5% of all peripheral CD3+ lymphocytes at the age of 2–15 years to 3.0% in young adults and to 1.5% in individuals above 30 years. This loss of V{gamma}9/V{delta}2 T cells with age appears to be a rather slow and steady process, estimated in a simple linear regression analysis to approximately –0.1% per year beyond the age of 20. That V{gamma}9/V{delta}2 T cell numbers drop indeed in individuals was confirmed by following nine healthy volunteers in a longitudinal study for up to 14 years (Fig. 1B ; and data not shown), where slopes of individual trend lines ranged from +0.06% (i.e., representing a slight increase with time) to –0.42% per year (i.e., representing a considerable drop with time), with a mean of –0.13% per year.


Figure 1
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  		Figure 1. Peripheral V{gamma}9/V{delta}2 T cell levels in healthy individuals. (A) Correlation between age and peripheral V{gamma}9/V{delta}2 T cell levels in all blood donors analyzed (n=320). Data points show values for individual men (solid symbols) and women (open symbols), determined as percentage of V{gamma}9+ cells (triangles) or V{delta}2+ cells (circles) among CD3+ lymphocytes. (B) Longitudinal analysis of peripheral V{gamma}9/V{delta}2 T cell levels in one male (solid symbols) and one female donor (open symbols), determined repeatedly over a period of 14 years as percentage of V{gamma}9+ cells among CD3+ lymphocytes. (C) Peripheral V{gamma}9/V{delta}2 T cell levels in three different age groups, 2–15 years (24 male, 20 female), 20–30 years (60 male, 67 female), and 30–60 years (51 male, 47 female), determined as percentage of V{delta}2+ cells among CD3+ lymphocytes. Error bars depict geometric means with standard errors for each group. Statistical differences were assessed using Mann-Whitney U-tests.

Unexpectedly, we also detected pronounced differences between the sexes, which held true for independent analyses of V{gamma}9+ T cells in healthy blood donors in Giessen, Germany, and for V{delta}2+ T cells in Palermo, Italy (Fig. 1A) . Although levels were virtually identical in female and male children and teenagers, we observed consistently higher numbers in women above 20–30 years than in men of the same age (Fig. 1C) .

This loss of peripheral V{gamma}9/V{delta}2 T cells in aging men is accompanied by remarkable changes in the distribution of functional subsets. Thus, TEM cells and terminally differentiated TEMRA cells seemed to disappear, thereby leaving TCM cells as the predominant V{gamma}9/V{delta}2 T cell population in the male circulation, while the proportions among TEM, TEMRA, and TCM cells apparently stay fairly constant in aging women (Fig. 2A ).


Figure 2
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  		Figure 2. Functional differences in V{gamma}9/V{delta}2 T cells between male (solid symbols) and female donors (open symbols). (A) Peripheral V{gamma}9/V{delta}2 T cell subsets in three different age groups: 2–15 years (20 male, 18 female), 20–30 years (40 male, 44 female), and 30–60 years (38 male, 41 female). Subsets were classified into the following categories: CD45RA+CD27+, naive T cells; CD45RA–CD27+, TCM cells; CD45RA–CD27–, TEM cells; and CD45RA+CD27–, TEMRA cells. (B) V{gamma}9/V{delta}2 T cell expansion after 7 days and interferon-{gamma} (IFN-{gamma}) secretion in response to isopentenyl pyrophosphate (IPP) in three different age groups: 2–15 years (10 male, 12 female), 20–30 years (10 male, 14 female), and 30–60 years (9 male, 12 female). Statistical differences were assessed using Mann-Whitney U-tests.

For a functional characterization of male and female PBMC, the V{gamma}9/V{delta}2 T cell expansion and IFN-{gamma} secretion in response to the HMB-PP analog IPP were determined as described previously [13 ]. Importantly, the drastic phenotypical conversion in men older than 30 years was mirrored by increased proliferative V{gamma}9/V{delta}2 T cell responses and reduced IFN-{gamma} secretion upon stimulation with IPP (Fig. 2B) . These data are in perfect accordance with our earlier findings that V{gamma}9/V{delta}2 TCM cells have a high capacity to proliferate in response to stimulation with IPP in vitro but are only poor producers of IFN-{gamma}; in contrast, V{gamma}9/V{delta}2 TEM cells and V{gamma}9/V{delta}2 TEMRA cells proliferate poorly but produce abundant amounts of IFN-{gamma} [6 , 13 ].

To our knowledge, this is the first report to describe a sex bias in {gamma}{delta} T cells. As the role of V{gamma}9/V{delta}2 T cells in pathogenesis and homeostasis remains unclear, it is premature to speculate whether this lymphocyte population renders women better or less-protected from infections than men. Differences between the male and female immune system do exist, as indicated by the distinct prevalence of certain autoimmune diseases. Recently, Sandberg and colleagues [14 ] reported differences in circulating V{alpha}24+ natural killer (NK) T cells and suggested that the number of regulatory and protective NKT cells might be expanded in women to control a sex-dependent predisposition to autoimmune disorders. An analogous, protective role is thinkable for V{gamma}9/V{delta}2 T cells, although female/male ratios were comparable in healthy donors and in relapsing-remitting multiple sclerosis patients (data not shown).

It seems that the differences in V{gamma}9/V{delta}2 T cell numbers only become manifest after puberty; hence, physiological or hormonal factors may be postulated. Data about the role of peripheral {gamma}{delta} T cells during the menstrual cycle are nonexistent though. Uterine {gamma}{delta} T cells (which belong predominantly to the V{gamma}1/V{delta}1 subset) were reported to remain at much the same density, whereas NK cells increase dramatically in numbers postovulation until a few days postmenstrually [15 , 16 ]. Yet, the idea that peripheral V{gamma}9/V{delta}2 T cells might, similarly as uterine NK cells, prepare the female immune system for the onset of pregnancy is in apparent conflict with the notion that elevated V{gamma}9/V{delta}2 T cell levels correlate with an increased risk of recurrent abortions [17 ].

Peripheral V{gamma}9/V{delta}2 T cell numbers not only reflect continuous microbial exposure during childhood [9 ] but also the influence of environmental factors in distinct geographic regions of the world [18 ]. Thus, our data may imply a higher antigenic exposure of women than of men throughout adulthood, be it a result of differences in the normal microbial flora of skin or urogenital tract or as consequence of increased susceptibility to infection.

At present, we can only speculate about the reason for the increasing deficiency in effector V{gamma}9/V{delta}2 T cells in men. However, it is noteworthy that dendritic cells from female blood were reported to express higher levels of interleukin (IL)-15 spontaneously [19 ], a cytokine for which we recently established a homeostatic role in maintaining the peripheral pool of V{gamma}9/V{delta}2 TEMRA cells [13 ]. Other cytokines preferentially expressed by female PBMC include IL-1{alpha}, IL-1ß, and IL-1RA [20 ]. Although a costimulatory role for IL-1ß on human V{gamma}9/V{delta}2 T cells has not been shown so far [21 ], it synergizes with IL-12 to induce IFN-{gamma} production by murine {gamma}{delta} T cells [22 ]. Clearly, a more thorough analysis of the role of V{gamma}9/V{delta}2 T cells in vivo will shed more light on the striking gender differences observed in the present study [23 ].

ACKNOWLEDGEMENTS

This work was supported in part by the University of Palermo, the Italian Ministry for University and Research (PRNI, MIUR), the Deutsche Forschungsgemeinschaft, and the Else Kröner-Fresenius-Stiftung. We gratefully acknowledge Simona Buccheri, Rosel Engel, Viviana Ferlazzo, Serena Meraviglia, Hans-Joachim Misterek, René Röhrich, and Marina Zafranskaya for their help.

Received November 8, 2005; revised December 12, 2005; accepted December 23, 2005.

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