Unique CD8� T cell-rich lymphoid aggregates in human uterine endometrium

Journal of Leukocyte Biology Volume 61, April 1997 427

Unique CD8� T cell-rich lymphoid aggregates inhuman uterine endometrium

Grant R. Yeaman,* Paul M. Guyre,t Michael W. Fanger,* Jane E. Collins,* Hillary D. White,*Wayne Rathbun� Kenneth A. Orndorff,t Jorge Gonzalez,t Judy E. Stern,� andCharles R. Wirat*Depajrtment� of Microbiology, t Physiology, 1 Pathology, and § Obstetrics & Gynecology, Dartmouth Medical School,

Lebanon, New Hampshire

Abstract: Using confocal scanning laser microscopy of

viable tissue sections, we have demonstrated organized

lymphoid aggregates (LA), that have a unique struc-

hire, in the stratum basalis of uterine endometrium.

These LA consist of a core of B cells surrounded by

more numerous T cells and an outer halo of monocytes/

macrophages. The T cells in the LA were almost ex-

clusively CD8’CD4 . These CD8� LA, in terms of

both their T cell and B cell components, were either

small or absent during the early proliferative stage

of the menstrual cycle, significantly larger in size at

mid-cycle and during the secretory phase, and absent

in post-menopausal women, suggesting that their de-

velopment is hormonally influenced. This new finding

ofa menstrual cycle-dependent, phenotypically unique,

organized immune cell structure may lead to new in-

sights into the mechanisms by which the endometrium

accepts a semiallogeneic graft while providing resis-

tance to infectious organisms. J. Leukoc. Rio!. 61:

427-435; 1997.

Key Words: repmductive immunology . mucosa � Tlymphocytes.

monocytes/macrophages . confocal scanning Inser micmscopy

INTRODUCTION

The mucosal immune system within the female reproduc-

tive tract is of particular importance because it is the first

site of immunological contact with many potentially patho-

genic bacteria and viruses. To be compatible with the repro-

ductive functions of these tissues, a local immune response

must protect against an infectious challenge, while at the

same time not mount a deleterious response to allogeneic

spermatozoa or an immunologically foreign fetal placental

unit [1]. The human non-pregnant uterus contains myeloid

and lymphoid cells that have the potential to function in

ways similar to their counterparts at other mucosal surfaces

and in the peripheral blood [2-7]. However, the distri-

bution of these cells within the female reproductive tract

appears to be uniquely regulated by estradiol and proges-

terone [8, 9]. Endometrial macrophages are distributed

diffusely throughout the uterine stroma and are also found

in small lymphoid aggregates that appear to increase in

number during the late secretory phase [3, 4, 7]. Macro-

phage numbers are surprisingly high, accounting for 5-

15% of the endometrial stromal cells [3, 5, 10]. A natural

killer (NK) cell population, phenotypically distinct from pe-

ripheral blood NK cells in that it is CD56 bright/CD16 dim,

is present in the uterine endometrium in large numbers

and increases markedly during the secretory phase and

during the first trimester of pregnancy [11-15; reviewed in

ref. 7]. The precise function of these cells within the fe-

male reproductive tract remains to be established. T cells

are found throughout the endometrium as isolated stromal

cells, intraepithelial lymphocytes, and in discrete lymphoid

follicles [2, 3, 6, 7, 10, 13, 16-18]. Our own work [Givan

et a!., unpublished resuitsi and that of others, show B cells

to be present in low but measurable numbers in the human

uterus [3, 7, 15, 19, 20]. The number of T cells relative

to other lymphoid cells does not increase significantly dur-

ing the menstrual cycle. However, the relative distribution

of T cells does change over the course of the menstrual cy-

cle. Discrete lymphoid follicles, or lymphoid aggregates, in

the uterine endometrium have long been recognized by his-

topathologists [reviewed in ref. 2] and were thought to oc-

cur in response to infection. More recently, Tabibzadeh

and others have presented evidence that these structures

develop during the menstrual cycle [17] and are composed

predominantly of T cells and some macrophages. It should

be noted that these structures are distinct from the NK cell

aggregates described by a number of authors [reviewed in

ref. 7J. To date, most immunophenotypic studies of human

uterine tissue have used single-color immunohistochemi-

cal staining of sequential frozen sections and have resulted

in little information about the extent of T cell aggregates

or the spatial relationships of the various cell types that

comprise them.

Abbreviations: LA, lymphoid aggregates; CSLM, confocal scanning

laser microscopy; NK, natural killer; PBS, phosphate-buffered saline;

Flit, fluorescein isothiocyanate; BSA, bovine serum albumin; PR Peyer’s

patches.

Correspondence: Dr. Grant R. Yeaman, Department of Microbiology,

Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH

03756.

Received November 6, 1996; revised January 6, 1997; accepted Jan-

uary 7, 1997.

428 Journal of Leukocyte Biology Volume 61, April 1997

In this study we report the cellular composition and dy-

namic changes in lymphoid aggregates (LA) located in the

stratum basalis of the human uterus throughout the men-

strual cycle by employing three-color immunofluorescent

staining of 30- to 70-�.tm-thick sections of freshly obtained

viable tissue analyzed by confocal scanning laser micros-

copy (CSLM). This approach permitted three-dimensional

modeling of these LA. Our results identify a unique popu-

lation of LA with a highly organized structure that includes

a B cell core surrounded by more numerous CD3+ T cells

and an outer mantle of macrophages. The size of these LA

fluctuated with the phase of the menstrual cycle, and with

few exceptions, the T cells in the aggregates were exclu-

sively CD8 �.

MATERIALS AND METHODS

Preparation of vibratome sections

Uterine endometrial tissue was obtained (with Institutional Review Board

approval) with informed consent from patients who had previously been

scheduled to undergo hysterectomy. For LA analysis, uterine endome-

trium were obtained from 9 postmenopausal women (average age 56,

range 42 to 70) and 23 cycling premenopausal women (average age 39.

range 25 to 52). One other patient included in the study was premeno-

pausal but not cycling (no. 707). This patient population is detailed in

Table 1 . Of the patients included in the study. none had a post-operative

diagnosis of malignant uterine disease. One patient (no. 445) had ovarian

cancer that had not spread to the Fallopian tube or the uterine endo-

metrium and two women had cervical carcinoma (nos. 707 and 724).

Single 150- to 300-mg samples of tissue were dissected out from equiv-

alent sites of the fundus in each uterus, distal to any gross pathology,

and placed immediately in sterile ice-cold phosphate-buffered saline

(PBS). The blocks of tissue were trimmed of excess myometrium, and

30- to 70-�.tm sections were cut using a vibratome (Vi000, Technical

Products International Inc. , St. Louis, MO). Sections were maintained

in ice-cold PBS throughout processing to prevent internalization of sur-

face markers. The stage of the menstrual cycle of the endometria was

determined in accordance with accepted histological practice using

hemotoxylin/eosin-stained paraffin sections. More precise evaluations of

proliferative phase endometria were carried out independently by two

pathologists who scored the degree of stromal edema and the relative

frequency of glandular and stromal mitoses; samples were classified as

early, middle, or late proliferative phase 1211. Secretory phase endo-

metria were not sub-staged.

Monoclonal antibodies

A panel of monoclonal antibodies, listed in Table 2, was used for direct

and indirect immunofluorescent staining: antibodies purified from hy-

bridoma cell culture supernatants (cell lines from American Type Cul-

ture collection, Rockville, MD) using HiTrap protein G superose col-

umns (Pharmacia Biotech Inc. , Piscataway, NJ) were labeled, where

indicated, with Cy3 or Cy5 Fluorolink protein labeling kits (Amersham

Life Sciences, Arlington Heights, IL) according to the manufacturer’s

TABLE 1. Summary of the Patient Population

Patient no. Age Uterine menstrual stage Uterine pathology Diagnosis

707 35 premenopausal (inactive) cervical cancer, HPV

477

358

380

445

502

522

526

578

627

60

57

56

57

53

42

70

45

63

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

polyp

leiomyomata/adenomyoma

leiomyomata

leiomyomata

leiomyomata


benign

benign

benign

ovarian cancer

endometriosis

benign

other-appendicitis

endometriosis

benign

368

373

382

424

470

475

506

5 1 5

529

534

704

724

728

736

775

41

37

25

47

41

52

44

41

29

31

3 1

35

41

38

37

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

leiomyomata

leiomyomata

hyalin sclerosis myometrium

leiomyomata


adenomyosis

leiomyomata

adenomyosis

leiomyomata

benign

benign

benign

benign

benign

benign

benign

benign

benign-inflammation

benign

endometriosis

cervical cancer

benign

benign

benign

387

446

484

563

564

678

697

698

37

40

48

39

44

3 1

41

43

secretory

secretory

secretory

secretory

secretory

secretory

secretory

secretory

leiomyomata

leiomyomata

leiomyomata

leiomyomata


inflammation

adenomyoma

uterus - premalignant

benign

benign

benign

benign

benign - inflammation

benign

benign

aCalt� Laboratories. San Francisco, CA.

b Antibodies were purified from cell culture supernatants of ATCC cell lines lAmerican Type Culture collection. Rockville, MD) and labeled with Cy3 or Cy5.

( Exalpha Corp. . Boston. MA.

‘1AMAC Inc.. Westbrook, ME.

�DAKO Corp.. Carpinteria. CA.

TABLE 2. Monoclonal Antibodies Used for Immunophenotyping

Yeaman et al. Unique human uterine lyniphoid aggregates 429

CD no.

CD3

CD3

CD4

CD4

CD8

CD8

CD14

CD14

CD I 9

CDI9

CD37

CD56

CD66b

Clone

�4.1

OKT3

S3.5

OKT4

3B5

OKT8

TUK 4

AML-223

SJ25-Ci

FMC63

WR17

562

531

Ber EP4

F8i 7

IVA12

P3

Supplier

Caltag#{176}

ATCCb

Caltag

ATCC

Caltag

ATCC

Caltag

ATCC

Caltag

ATCC

ATCC

Exalphac

AMACd

DAKOC

DAKO

ATCC

ATCC

Specificity

Pan T cell

Pan T cell

T helper subset

T helper subset

T suppressor/cytotoxic

T suppressor/cytotoxic

Monocyte/macrophage

Monocyte/macrophage

B cells

B cells

B cells

NK cell

Granulocytes

Epithelial cells

Pan HLA class II

Pan HLA class II

Mouse IgG1 isotype

Fluorochrome

FITC

Cy3, Cy5

FITC

Cy3, Cy5

FITC

Cy3, Cy5

FITC

Cy3, Cy5

FITC

Cy5

Cy5

FITC

FITC

FITC

FITC

Cy5

FITC, Cy3, Cy5

recommendations. Fluorescein isothiocyanate (FITC) -conjugated mouse

monoclonal antibodies were obtained from commercial suppliers as de-

noted in Table 2.

Three-color immunophenotyping

Three-color immunofluorescent staining of tissue sections was carried

out immediately after cutting. For direct staining. 2 �tg/IOO p1 each of

Flit-, Cy3-. and Cy5-labeled antibodies in PBS/1% bovine serum al-

bumin (BSA)/0.I% aside-containing human immunoglobulin (6 mg/mL

to block nonspecific and Fe-mediated binding) were added to sections

in 96-well plates and incubated overnight at 4#{176}Cin the dark with con-

tinuous gentle agitation. Unbound antibody was removed from the sec-

tions by aspiration followed by four 20-mm washes in PBS/1% BSA/0.I%

azide. Washed sections were then fixed overnight in the same buffer con-

taming 1% paraformaldehyde. Stained sections were wet-mounted in

anti-fade (Molecular Probes Inc. . Eugene, OR), sealed with nail varnish.

and stored at 4#{176}Cin the dark for up to 10 days before confocal imaging.

CSLM

Immunofluorescently labeled sections were optically sectioned using a

Bio-Rad MR1000 Confocal Scanning Laser Microscope system equipped

with a krypton/argon laser. Laser power, PMT gain, and enhancement

factors were then determined for the Flit, Cy3, and CyS channels by

use of the single fluorochrome-stained sections to ensure effective cross-

channel compensation. Three-color fluorescent sections were then eval-

uated for the presence of LAs. LAs were subjected to z sectioning using

a I- to 2-�tm z step at an appropriate confocality (typically an iris setting

between I and 2). The resulting image files were then used to create

three-dimensional reconstructions of the lymphoid aggregates.

RESULTS

Presence of basal LA at different stages of themenstrual cycle

Endometria were evaluated for the presence of basal LA

in sections stained with CD3. The presence of LA was

scored, on the basis of CD3 reactivity, as negative ( - ) if

there were no T cell clusters (i.e., accumulations ofT cells)

with a diameter of more than 2 T cells ( � 20 jim), small

( + ) if observed clusters had a diameter of 3-4 T cells

( � 20-40 �.tm), medium (+ +) if their diameter was be-

tween 5 and 10 T cells ( � 40-100 p.tm), and large (+ + +)

if their diameter was greater than 10 T cells ( 100 Rm).

The largest aggregates exceeded 20 cell diameters. Consid-

eration of the diameter of the T cell region in scoring the

size is important because the range of 0 to + + + suggests

a linear progression. However, the geometric nature of this

scale is emphasized by calculations of the number of cells

present in the different sizes of LA based on an average

cell diameter of 10.5 �im and assuming cell-cell contact.

Hence a score of zero represents fewer than 4 cells, where-

as large LA (+ + +) contain a minimum of 300 cells and

as many as 2400 cells.

The presence of CD3 � LA in postmenopausal, prolifer-

ative phase, and secretory phase endometna are summa-

rized in Table 3. Significant differences in the presence

and size of these LA were observed during the menstrual

cycle. During the secretory phase of the cycle, when an mi-tial rise in estradiol is followed by a concomitant rise in

both estradiol and progesterone, all tissues examined con-

tamed large LA. As indicated in Table 3, LA in uteri from

women at the secretory stage were significantly larger than

those seen in uteri from women at the proliferative stage

of the menstrual cycle (P = 0.009, Fisher’s exact test). In

contrast, aggregates of T cells were conspicuously absent

in sections from postmenopausal women compared with

premenopausal women of either secretory phase (P = 2 x

10-u, Fisher’s exact test) or in proliferative phase (P =

0.01 1 , Fisher’s exact test). Proliferative phase endometria

exhibited a high degree of variability in both presence and

size of LA, suggesting that LA develop during this stage

of the menstrual cycle. To further define this heterogeneity

TABLE 3. Influence of Menstrual Status

on Presence and Size of LA Detected Using Anti-CD3

LA Score

3 n=1 n=8

n=4

2.5

n=32

I .5

0.5

0

early mid late

Proliferative Secretory

Fig. 1. Lymphoid aggregates develop during the proliferative phase of

the menstrual cycle. Bars represent the mean LA size score ± SE for

each menstrual stage. The change in size correlates with stage ofthe men-

strual cycle from early proliferative to secretory phase (r = 0.85, P =

0.0003, Spearman rank correlation).


Patient

no.

Uterine

menstrual stage LA sizea

477

358

380

445

502

522

526

578

627

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

postmenopausal

-

-

-

-

-

-

-

-

-

368

373

382

424

470

475

506

5 1 5

529

534

704

724

728

736

775

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

proliferative

+

+

+ +

+

+ + +

-

+ + +

+

-

+ +

-

+ +

+ + +

+ + +

+ + +

P = 0.011

P = 0.009

P = 2 x 10�

387

446

484

563

564

678

697

698

secretory

secretory

secretory

secretory

secretory

secretory

secretory

secretory

+++

+ + +

+ + +

+ + +

+ + +

+ + +

+ 4- +

+ + +

707 premenopausal-

inactive

+ + +

a Key: - , no aggregates >20 �sm diameter; + . aggregates >20 �tm diameter

<40 lim; + + , aggregates >40 �im diameter <100 �tm; + + + . aggregates

>100 �.tm diameter.

during the proliferative phase, some endometria were fur-

ther staged as early, mid, or late proliferative phase (Fig. 1

and Table 4). The increase in LA size significantly corre-

lated with the progression of the menstrual cycle from early

proliferative phase through secretory phase (r = 0.85, P =

0.0003, Spearman rank correlation, Fig. 1). Three of nine

postmenopausal patients were on low-dose hormone re-

placement therapy at the time of surgery. Despite hormone

replacement, histological analysis indicated these uteri to

be inactive (postmenopausal), indicating that exogenous

hormone levels were too low to have a significant effect on

endometrial proliferation.

Phenotyping of the lymphoid aggregates

T cell phenotyping of LA was carried out using triple stain-

ing for CD3, CD4, and CD8. To preclude misinterpretation

of results that might be caused by different fluorochrome

intensities and color compensation, different sections were

stained with combinations of these antibodies such that

each marker was examined using each of the three fluoro-

chromes. Results using the different fluorochrome combina-

tions were comparable and consistently showed the CD3�

population to be uniformly CD8�CD4 (Fig. 2, A-D) in

all but three specimens. CD3�CD8� cells were also pres-

ent as isolated cells in the stroma and as intraepithelial lym-

phocytes (data not shown). Poor staining by the CD4 anti-

bodies was ruled out as a potential cause of the absence

of CD4� cells in LA because CD3�CD4� T cells were

clearly seen as individual cells dispersed throughout the

stroma in all sections. Within the LA, CD8�CD3� cells ac-

counted for nearly all CD3� cells. Where low-intensity

CD4 positivity was observed in these LA, it was always as-

sociated with the CDi4� macrophage population. In ad-

dition, endometria from three patients contained LA of

different phenotypes: specimen 707 contained LA with a

mixture of CD4� and CD8� T cells as well as LA that

were exclusively CD4� or CD8� (Fig. 3, C-D); number

728 contained CD8� LA and mixed CD4�, CD8� LA;

and number 697 contained LA that were either all CD8�

or all CD4�.

A core of CD19� B cells was a common feature ob-

served in LA in 7 of 12 specimens examined (Fig. 2, E-H).

B cell cores were found in all large LA (with the exception

of specimen 707) and the numbers of T and B cells were

proportional, suggesting that the B cell core develops con-

comitantly with the T cell component. In earlier cases, B

cell phenotyping was carried out using Flit-labeled anti-

CD19 alone. However, since CD19 expression is variable

depending on the developmental stage of the B cell, some

later tissues (specimen numbers greater than 500) were

stained with a combination of Cy5-conjugated anti-CD19

plus Cy5-conjugated anti-CD37 to maximize B cell detec-

tion, with a Cy3- or FITC-labeled anti-CD3 as one of the

Patient

no. Histological stage Size score T cell phenotype

T cells

HLA-II B cell score

Macrophage

halo

Macrophage

HLA-II

736 proliferative (undetermined) + + + all CD8� - + + ±

775 proliferative (undetermined) + + + all CD8� ND + + ND

728 proliferative (undetermined) + + + all CD8�,

mixed CD4�

and CD8�

ND + + ND

368 proliferative (early) + all CD8� ND ND ND ND

515 proliferative (early) + all CD8� - - + ±

534 proliferative (early) + + all CD8� - - + ±

373 proliferative (mid) + all CD8� - - + ±

424 proliferative (mid) + all CD8� - ND + ±

382 proliferative (mid) + + all CD8� - - + ±

470 proliferative (mid) + + + all CD8� - + + ±

506 proliferative (late) + + + all CD8� - + + ±

697 secretory (early) + + + all CD8� and

all CD4�

ND ND ND ND

387 secretory + + + all CD8� - ND + ±



678 secretory + + + all CD8� ND ND ND ND

698 secretory + + + all CD8� ND ND ND ND

446 secretory (late) + + + all CD8� - + + ±

484 secretory (late) + + + all CD8� - + + ±

707 Inactive premenopausal. Active + + + all CD8�, ND - ND ND

papilloma virus infection. all CD4�,

Combined estrogen and mixed CD4�

progesterone therapy. and CD8�

Cervical cancer.

TABLE 4. Cellular Composition and HLA-II Expression of LA

Yeaman et al. Unique human uterine lymphoid aggregates 431

ND = not done.

other two colors. Sections stained in this way still showed

variable sizes of B cell cores, with two specimens (775 and

728) showing extensive B cell cores similar in extent to

germinal centers (Fig. 3B). No CD3�, CD37� cells were

observed.

CD14� monocyte/macrophage cells were also common

to all LA examined. As seen in Figure 2, G and H, these

cells always formed a halo around the T cell mantle (Table

4). On the other hand, polymorphonuclear leukocytes, ex-

amined using anti-CD66b, were not found within the LA.

LA were located in the stratum basalis, at a depth ap-

proximating the base of the uterine epithelial glands. No

direct contact with the glandular epithelium could be iden-

tilled even for large LA in five of five specimens: two from

late proliferative, and three from secretory phase (Fig. 3A).

This is in contrast to aggregated NK cells that we and others

find predominantly located in the upper layers of the endo-

metrium in close contact with both the glandular and lu-

minal epithelium [7] and as scattered individual cells in

the stratum basalis.

MHC class II expression

Using IVA12, a pan HLA class II marker (HLA-DR, DP,

DQ), we evaluated the class II expression of LA cells. In

all cases examined (n = 13), the T cell population was low

or negative for class II expression. In contrast, in 13 of 13

specimens, approximately half of the macrophages form-

ing the halo around the T cells were class II bright (Fig.

3, E-H). The question of B cell HLA class II expression

was specifically addressed for only three of the specimens.

Surprisingly, only in one (736) were B cells intensely pos-

itive for HLA class II, in another (506) they were clearly

negative, whereas in the third (534) there was a mixture

of class II positive and negative B cells within the same LA.

More comprehensive analysis of class II and other mole-

cules involved in antigen presentation will clearly be impor-

tant for future studies.

DISCUSSION

Using triple-color immunofluorescent staining and CSLM

of viable tissue sections, we have defined the characteris-

tics of immune cells in unique lymphoid aggregates pres-

ent in the human uterine stratum basalis. Previously, im-

munophenotyping of the reproductive tract has largely been

limited to either single-color immunohistochemical tech-

niques, which are generally inadequate for the detection

of dual expression of antigens on individual cells, or flow-

cytometric analysis of mechanically or enzymatically dis-

rupted tissue. Our approach presents three major advan-

tages over more conventional techniques. First, no current

tissue fixation technique uniformly preserves all epitopes

recognized by monoclonal antibodies, leading to a restric-

tion in antigens that can be detected; use of fresh unfixed

tissue sections largely avoids this problem. Second, this ap-

Fig. 2. (A-D) Lymphoid aggregate T cells are CD3�CD4CD8�. Projected stack of eight 2-ptm optical sections through a proliferative phase lym-

phoid aggregate (A) CD4-Cy3; (B) CD3-FITC; (C) CD8-Cy5; (D) merged color image of (A-C). Notice the absence of CD4�CD3� cells. (E-H) Lym-

phoid aggregates have a B cell core and an outer halo of macrophages. Single optical slice in the z plane through the center of a lymphoid aggregate

present in a proliferative stage endometrial section. (E) CD3-Cy3; (F) CD19-FITC; (G) CD14-Cy5; (H) merged color image of (E-G).


Fig. 3. (A) LA are located between the

bases of the endometrial glands. Projected

stack ofeight 2-�.tm optical sections through

a dual-color image of a CD8� LA in a Se-

cretory phase endometrium showing LA

CD8 cells (Cy5. blue) and glandular epithe-

hum stained with Ber-EP4 (Flit, green).

(B) B cell cores have a germinal center

appearance in some large LA. Single low-

confocality ( = 10 rim) slice through the cen-

ter of a large LA in a proliferative phase tissue

showing a large B cell core (CD19�CD37

both labeled with Cy5. blue) surrounded

by T cells (CD3-FITC, green) and macro-

phages (CD14-Cy3, red). (C) A CD4� LA

in a premenopausal non-cycling endome-

trium. Three-color single slice through the

center of a LA from endometria no. 707:

CD3-Cy3 (red); CD4-FITC (green); CD8-

Cy5 (blue). Notice the predominance of

CD4�CD3� cells (yellow/orange). (D) A

mixed CD8�/CD4� LA in a premenopausal

non-cycling endometrium. Three-color sin-

gte slice through the center of a LA from

the same vibratome section as in (C): CD3-Cy3 (red); CD4-FITC (green); CD8-Cy5 (blue). Notice the presence of both CD3�, CD8� cells (purple)

and CD3�, CD8� (yellow). (E-H) Macrophages but not T cells are class II positive in LA. Single optical slice through the center of a proliferative

phase lymphoid aggregate. (E) CDI4-Cy3; (F) CD3-FITC; (G) pan HLA class II-Cy5; (H) merged color image of (E-H). Notice the lack of CD3/pan

HLA class II dual-positive cells (turquoise color in H) and the relative abundance of CD14/pan HLA class II dual-positive cells (purple color in H).

Yeaman et al. Unique human uterine lymphoid aggregates 433

434 Journal of Leukocyte Biology Volume 61. April 1997

proach permits co-localization of antigens on individual

cells but without the tissue disruption required for flow cy-

tometry. Third, z sectioning of thick vibratome sections a!-

lows three-dimensional reconstruction, giving more de-

tailed spatial information, including location of cell types

and cell-cell interactions within the tissue.

The CD8� LA described in this study are distinct from

large granular lymphocyte aggregates described by others

[reviewed in ref. 7]. They appear to be spread between

endometrial glands approximately equidistant from the

base of the glands in the stratum basalis, and in accor-

dance with previously published micrographs [16, 22],

often seem to be associated with blood vessels, as evi-

denced by phase-contrast appearance and smooth muscle

autofluorescence in the Flit channel. These CD8� LA

develop during the proliferative phase of the menstrual cy-

cle, reach their largest cell mass at mid-cycle (i.e., late pro-

liferative phase) and persist throughout the secretory phase.

This, taken in conjunction with their absence from uterine

endometrium of post-menopausal women, suggests that

their development is under the influence of sex hormones.

During the proliferative phase LA often have a central B

cell core that is surrounded by a mass of (usually) exclu-

sively CD8� T cells, surrounded in turn by an outer halo

of macrophages.

Our finding of LA development during the course of the

menstrual cycle extends the previous finding of Tabibzadeh

[17] who reported that BrDU incorporation by CD3� LA

cells in vibratome sections increased during the menstrual

cycle, particularly following ovulation. This proliferation ap-

peared to be interleukin-2 independent and may depend

on steroid hormones because lymphoid aggregate T cells

express estrogen receptors [23]. Progesterone has previ-

ously been shown to increase T cell numbers throughout

the uterus during the luteal phase of the cycle [24]. Our

findings show that LA seen in uteri during the secretory

phase of the menstrual cycle are significantly greater in size

than those seen in the early proliferative phase. This sug-

gests that estradiol and progesterone, both known to be el-

evated during this time, may act either directly or indirectly

to increase T cell numbers in the lymphoid aggregates. Al-

though others have noted a predominance of CD8� T cells

in both endometrial stroma and LA [3, 6, 10], our studies

show that in nearly all cases the T cell lymphoid aggregates

in the basalis region are exclusively of the CD3 �CD8�CD4

phenotype. CD3�CD8�CD4� cells were observed in the

stroma of the same tissues but not within the lymphoid ag-

gregates. All CD4 � reactivity within LA in these speci-

mens was confined to the CD14�, monocyte/macrophage

population.

Three of the tissues did not follow the above pattern in

that they displayed either exclusively CD4� and/or mixed

CD4�/CD8� LA in addition to the exclusively CD8� LA.

One patient (707) with this phenotype had both an active

human papilloma virus infection and an endometrium his-

tologically classified as inactive. The latter probably re-

sulted from concomitant high-dose estrogen and progester-

one therapy. This patient also had extensive cervical cancer.

No overt endometrial infections were noted for the other

two patients. One patient (728) was receiving progesterone

therapy and the other (697) had ovarian cysts. It is not

possible with only three cases to determine the underlying

differences leading to the formation of CD4� LA, but they

may result from an immunological response to infection or

neoplasia or from disruption of the endocrine balance.

The presence ofa defined B cell core in large LA demon-

strates a precise structural relationship between B cells and

CD8� T cells in the uterus. Although scattered B cells

have been noted in the same region of the stratum basalis

in two previous studies [3, 10], the findings presented here

demonstrate that the B cell core is a defined structural fea-

ture of the larger lymphoid aggregates. In contrast small

LA, identified most commonly in tissues from women at

the early proliferative stage of the menstrual cycle, lack B

cell cores. Whether this reflects a sequential construction

of LA in which chemokines initially attract CD8� cells fol-

lowed by B cell migration remains to be established. Macro-

phages form a halo around the periphery of the LA and

are seldom seen deep within the T cell or B cell regions.

In 13 of 13 cases, a high proportion CD14� cells, both sur-

rounding the LA and distributed throughout the stroma,

were found to be strongly HLA class II positive. In con-

trast, lymphoid aggregate T cells were uniformly negative

for class II expression. HLA class II expression in the B

cell core has not, as yet, been definitively examined. HLA

class II expression in human endometrium has previously

been examined by a number of investigators who uniformly

reported stromal HLA-DR positivity to have the same stain-

ing pattern as the macrophage population [3, 5, 16, 18,

25-27]. Two reports have specifically shown cells surround-

ing the basal LA to be strongly HLA-DR positive [3, 25].

Both of these studies also showed some cells within LA to

be HLA-DR positive, however, it is not possible to identify

the phenotype of the positive cells from these studies.

Uterine CD8� LA are also unique and distinct from

those found in secondary lymphoid tissues and at other

mucosal surfaces [28, 29]. For example, whereas primary

follicles in the periarteriolar lymphoid sheaths of the spleen

contain a B cell core surrounded by CD4� T cells, LA in

the uterus contain CD8�CD4 cells. The gastrointestinal

tract Peyer’s patches (PP) contain both CD4� and CD84

zones that contain B cells. Because PP are anatomically ad-

jacent to epithelial cells that line the intestinal lumen, they

are exposed to luminal antigens that pass through M cells

directly into the PP. In contrast, LA in the uterine stratum

basalis usually lack CD4� T cells and have no direct con-

tact with the uterine epithelial cells, as we find them equi-

distant between the bases of the epithelial glands. To date,

despite extensive investigation, M cells have not been found

in reproductive tract tissues. Also, in contrast to PP that

have macrophages distributed throughout, uterine LA are

surrounded by a halo of macrophages.

The functions of LA are presently unknown but their de-

velopment during the menstrual cycle and their absence

in postmenopausal women suggests that they are hormon-

ally influenced. It is possible that as LA these cells play an

Yeaman et a!. Unique human uterine lymphoid aggregates 435

immunoregulatory role, perhaps suppressing cell-mediated

immune responses during the postovulatory implantation

window to prevent immune responses against the semi-

allogeneic conceptus. In support of this hypothesis, our

group has recently shown that cytotoxic T cell function,

present throughout the female reproductive tract (Fallopi-

an tube, uterus, cervix, and vagina) is specifically sup-

pressed only in the endometrium during the secretory stage

of the menstrual cycle [301. Whether localized suppression

of cytotoxic T cell function is a consequence of LA forma-

tion remains to be established. Alternatively, LA may play

a central role in immune protection at the time of menstru-.

ation (5-7 days) when the outer third of the endometrium

is shed. During this time the protective barrier of epithelial

cells and the stromal immune cells of the decidual stroma

are lost, leaving the uterus uniquely vulnerable to infection

by viral and bacterial pathogens. We postulate that the

growth of LA during the cycle in the basalis stroma, which

is not shed during menses, is consistent with a consolida-

tion of T and B cells to prevent loss during menstruation.

Overall, our results indicate that LA in the uterus are

unique, as judged by their structure, location, and potential

endocrine control. Further studies are needed to identify

the events responsible for LA formation and the physiolog-

ical role these cells play in contributing to maternal im-

mune protection and fetal survival.

ACKNOWLEDGM ENTS

This work was supported by National Institutes of Health

Grant AI34478 (C. R. W.). Confocal scanning laser micros-

copy was performed in the Herbert C. Englert Cell Analy-

sis Laboratory, which was established with a grant from the

Fannie E. Rippel Foundation, and is supported in part by

the core grant of the Norris Cotton Cancer Center (CA-

23108).

The authors would like to thank the following individu-

als for their technical and clinical support: Dr. Alice Given,

Dr. Vincent Memoli, Dr. John Currie, Dr. Stephen Andrews,

Dr. Joan Barthold, Dr. Jackson Beecham, Dr. John Ket-

terer, Dr. Eileen Kirk, Dr. Benjamin Mahlab, Dr. Paul Man-

ganiello. Dr. Eric Sailer, Dr. Barry Smith, Dr. William

Young, Jaclyn Logren, Fran Reinfrank, Jeannette Sawyer,

Tracy Stokes, Joanne Lavin, Nancy Leonard, Kris Ramsey,

Tamara Krivit, Laura Wolf, Peter Seery, Maryalice Ach-

bach, Judy Rook, and Esther Colby.

REFERENCES

I . Harbour, D. V.. Blalock. J. E. (1989) Lymphocytes and lymphocyti’ hor.

mones in pregnancy. Prog. Neuroendocrinol. Immunol. 2. 55-63.2. Dallenbach-Hellweg. G. (1975) Hi.stopathology of the Endometnum, New

York: Springer-Verlag.3. Kamat. B., lsaacson. P. (1987)The immunocytochemical distribution of leo.

kocytic subpopulations in human endometrium. Am. J. Pat/wI. 127.66-73.

4. Bonatz, G., Hansmann, M., Buchholz, F., Mettler, L., Radzun, H., Semm,K. (1992) Macrophage. and lymphocyte-subtypes in the endometrium dur-

ing different phases of the ovarian cycle. mt. J. Gynecol. Obstet. 37, 29-36.

5. Laguens, G., Goni, J., Jr., Laguens, M., Goni, J., Laguens, R. (1990)Demonstration and characterization of HLA-DR positive cells in the stromaof human endometrium. J. Reprod. Immunol. 18. 179-186.

6. Morris, H., Edwards, J., Tiltman, A., Emms, M. (1985) Endometrial lym-phoid tissue: An immunohistological study. 2 Clin. Pa.thol. 38, 644-652.

7. Loke, Y. W., King, A. (1996) Human Implantation: Cell Biology and Im-

munology, New York: Cambridge University Press, 102-129.

8. Wira, C., Stern, J. (1992) Endocrine regulation ofthe mucosal immune sys-

tem in the female reproductive tract: Control of IgA, IgG, and secretory com-ponent during the reproductive cycle, at implantation and throughout preg-

nancy. In Hormones and Fetal Pathophysiology (J. Pasqualini and R.

Scholler, eds.), New York: Decker, 343-368.9. Wira, C.. Sandoe. C. (1977) Sex steroid hormone regulation of immuno-

globulin G (IgG) and A (IgA) in rat uterine secretions. Nature 268,534-536.

10. Marshall, R., Jones, D. (1988) An immunohistochemical study of lymphoid

tissue in human endometrium. hit. J. Gynecol. Pat/wI. 7. 225-235.1 1 . Hunt, J. S. (1994) Immunologically relevant cells in the uterus. Biol. Re-

prod. 50, 461-466.12. Chen, C-K.. Huang. S. C.. Chen, C-L., Yen, M-R., Hsu, H-C., Ho, H-N.

(1995) Increased expression of CD69 and HLA-DR but not of CD2S or

CD71 on endometrial T lymphocytes of nonpregnant women. Human Im-munol. 42, 227-232.

13. Lachapelle, M-H., Miron. P.. Hemmings, R., Baron, C., Roy, D. C. (1996)Flow-cytometric characterization o hematopoietic cells in non-pregnant ho-

man endometrium. Am. J. Repmd. Immunol. 35, 5-13.14. Lachapelle, M. W., Miron, P., Hemmings, R., Roy, D. C. (1996) Endome-

trial T, B, and NK cells in patients with recurrent spontaneous abortion.J. ImmunoL 156, 4027-4034.

15. King, A., Wellings, V., Gardner, L., Loke. Y. W. (1989) Immunocytochemi-

cal characterization of the unusual large granular lymphocytes in human

endometrium throughout the menstrual cycle. Human Immunol. 24, 195-205�

16. Tabibzadeh, S. S.. Poubouridis, D. (1990) Expression ofleukocyte adhesionmolecules in human endometrium. Am. .� Clin. Pathol. 93, 183-189.

1 7. Tabibzadeh, S. (1990) Proliferative activity of lymphoid cells in the humanendometrium throughout the menstrual cycle. J. Clin. Endocrinol. Metab.

70. 437-443.18. Bulmer, J., Lunny, D.. Hagin, S. (1988) Immunohistochemical characteriza-

tion of stromal leukocytes in nonpregnant human endometrium. Am. J. Re-

prod. Immunol. Microbiol. 17. 83-90.19. Witz, C. A., Montoya. I. A.. Dey. T. D.. Sehenken. R. S. (1994) Character-

ization of lymphocyte subpopulations and T cell activation in endometriosis.

Am. J. Reprod. Immunol. 32. 173-179.20. Fernandez-Shaw. S.. Clarke, M. T.. Hicks, B. Naish, C. E.. Barlow, D. H..

Starkey, P. M. (1995) Bone marrow-derived cell populations in uterine andectopic endometrium. Human Reprod. 10, 2285-2289.

21. Noyes, R. W.. Hertig. A. T. (1955) Dating the endometrial biopsy. Fertil.

Steril. 1. 3-25.22. Tabibzadeh, S. (1990) Immunoreactivity of human endometrium: correla-

tion with endometrial dating. Fertil. Steril. 54. 624-631.23. Tabibzadeh. S. S.. Satyaswaroop. P. G. (1989) Sex steroid receptors in lym-

phoid cells of human endometrium. Am. J. Pat/wI. 91, 656-663.24. Booker, S. S.. Jayanetti. C.. Karalak. S.. Hsiu. J. G., Archer. D. F. (1994)

The effect of progesterone on the accumulation of leukocytes in the humanendometrium. Am. J. Obstet. Gynecol. 171, 139-142.

25. Tabibzadeh, S. (1990) Evidence of T-cell activation and potential cytokine

action in human endometrium. J. Clin. Endocrinol. Metab. 71, 645-649.26. Tabibzadeh, S. (1991) Distinct subsets of stromal cells confined to unique

microenvironments in human endometrium throughout the menstrual cv-cle. Am. J. Repmd. Immunol. 26. 5-10.

27. Bjercke. S.. Brandtzaeg. P. (1993) Glandular distribution of immunoglob-

ulins, J chain, secretory component, and HLA-DR in the human endome-trium throughout the menstrual cycle. Human Reprod. 8. 1420-1425.

28. Picker. L. J., Siegelman. M. H. (1993) Lymphoid tissues and organs. InFundamental Immunology. 3rd ed. (W. E. Paul. ed). New York: Raven

Press, 145-197.29. Griebel. P. J.. Hem, W. R. (1996) Expanding the role of Peyer’s patches

in B-cell ontogeny. Immunol. Today 17. 30-39.30. White, H. D.. Crassi. K. M.. Givan, A. L.. Stern. J. E.. Green, W. R.. Wira.

C. R. (1997) CTL activity within the human female reproductive tract: influ-efl(’e of stage of the menstrual cycle and menopause. J. ImmunoL. in press.

Unique CD8� T cell-rich lymphoid aggregates in human uterine endometrium

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