Immunophenotype of Skine Lymphocitic Infiltrate (CD 8)

7/29/2019 Immunophenotype of Skine Lymphocitic Infiltrate (CD 8)

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C L I N I C A L A N D L A B O R A T O R Y I N V E S T I G A T I O N S

BJDBritish Journal of Dermatology

Immunophenotype of skin lymphocytic infiltrate in patients

co-infected with Mycobacterium leprae and human

immunodeficiency virus: a scenario dependent on CD8+

andor CD20+ cellsC. Massone, C. Talhari,* S. Talhari,* A.M.G. Brunasso, T.M. Campbell, P. Curcic, L. Cerroni andR. Ribeiro-Rodrigues

Department of Dermatology, Medical University of Graz, Graz, Austria*Department of Dermatology, Tropical Medicine Foundation of Amazonas, Manaus, AM, BrazilDepartment of Dermatology, Galliera Hospital, Genoa, ItalyDermatology Research Centre, University of Queensland, School of Medicine, Brisbane, Qld, AustraliaDepartment of Pathology and Nucleo de Doencas Infecciosas, Universidade Federal do Esprito Santo, Av. Marechal Campos 1468, Vitoria, ES, 29043900,Brazil

CorrespondenceRodrigo Ribeiro-Rodrigues.

E-mail: [email protected]

Accepted for publication

26 April 2011

Funding sources

None.

Conflicts of interest

None declared.

DOI 10.1111/j.1365-2133.2011.10412.x

Summary

Background Leprosy occurs rarely in human immunodeficiency virus (HIV)-positivepatients. In contrast to tuberculosis, there has been no report to date of anincrease in HIV prevalence among patients with leprosy or of differences inleprosys clinical spectrum. While several studies describe the systemic immuneresponse profile in patients co-infected with HIV and leprosy, the local immuneskin response has been evaluated in only a small number of case reports andlimited series of patients.Objective To investigate the interaction between Mycobacterium leprae and HIV infec-tion in the skin.Methods We investigated the presence and frequency of cells positive for CD4,CD8, CD20, TIA-1, FOXP3 and CD123 in lymphocytic infiltrates from 16 skin

biopsies taken from 15 patients with HIVleprosy co-infection.Results CD4+ cells were absent in infiltrates from 6 (38%) skin biopsies and presentin 10 (62%) cases at low levels (< 116%) of the lymphocytic infiltrate. CD8+was the predominant phenotype in the infiltrate (994%), followed by TIA-1,expressed by > 75% of CD8+ cells. FOXP3+ cells were also present, representing34% of the lymphocytic infiltrate. CD20+ cells were detected in 75% of the cases;however, in two cases (12%) these cells represented 2550% of the infiltrate,while in the other 10 cases (62%) they were present only focally (< 25% of theinfiltrate). CD123+ cells were not observed in any of the studied specimens.Conclusions Data presented here suggest that cell-mediated immune responses toM. leprae are preserved at the site of disease and that in the absence of CD4+ cells,CD8+FOXP3+ and CD20+ cells may be involved in granuloma formation.

Leprosy occurs rarely in human immunodeficiency virus(HIV)-positive patients, and only a small number of casereports and limited series of patients in this subgroup werepublished in the literature during the 1990s. In contrast totuberculosis, to date neither an increase in HIV prevalenceamong patients with leprosy nor differences in leprosys clini-cal spectrum has been reported.1

The introduction of highly active antiretroviral therapy(HAART), because of its involvement with immune reconsti-

tution inflammatory syndrome (IRIS), warranted a new inves-tigation into the occurrence of leprosy in these cases.However, quite unexpectedly, clinical and histological datafrom these studies demonstrated that patients co-infected withHIV and leprosy did not differ from patients with leprosywho were HIV-negative; only a higher frequency of reactionstates [type 1 reaction (T1R)] was observed in the former.2

Unlike cases of tuberculosis, in which an impaired granuloma-tous response is frequently observed, it was reported that in

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patients with HIVleprosy, the capacity to elicit a normalgranulomatous response against Mycobacterium leprae is preserved,even when peripheral blood CD4 cell counts are low (theso-called granuloma paradox).3

While reports regarding the profile of systemic immuneresponse in patients with HIVleprosy are found in theliterature, data on local skin immunity against M. leprae isscarce.111 Furthermore, parameters such as cytotoxic markers

or recently defined phenotypes, such as T-regulatory cells(Tregs) and plasmacytoid dendritic cells (PDCs), have neverbeen investigated in skin biopsies of patients with HIVleprosy.

Despite its intrinsic limitations, this study is a first effort tofurther the knowledge concerning the interaction between M.leprae and HIV infection in the skin.

Patients and methods

Data from 15 patients (male to female ratio 9 : 6; mean age3573 years; median age 32 years; age range 2059 years)

were obtained from the database of the Pathology Departmentat the Amazonas Tropical Medicine Foundation in Manaus,Brazil. A total of 16 biopsy specimens were available for thestudy. In all but one case, specimens were obtained and pro-cessed at the time of the diagnosis. In a single case, two speci-mens were obtained, one before and one after multidrugtherapy initiation; therefore we analysed data from 16 skinbiopsies from 15 patients. HIV infection and acquired immunedeficiency syndrome (AIDS) cases were defined according toguidelines established by the Brazilian Ministry of Health.12

Leprosy was treated according to World Health Organizationguidelines.13 The Institutional Review Board of the TropicalMedicine Foundation of Amazonas in Manaus, Brazil approvedthis study.

Histopathology

Biopsy specimens were fixed in 10% buffered formalin andsubsequently embedded in paraffin. Sections were stained withhaematoxylineosin for routine histopathological evaluation.All specimens were stained with modified FiteFaraco stain foracid-fast bacilli; the bacterial index of the granuloma wasassessed with a logarithmic scale according to Ridley.14

Immunohistology

Immunophenotypic analysis was performed on routinelyfixed, paraffin-embedded tissue sections according to a previ-ously described three-step immunoperoxidase technique usingmonoclonal antibodies.15 Microwave enhancement was usedfor all the antibodies. Second and third antibodies wereobtained from BD Biosciences, Erembodegem, Belgium(CD123); AbD Serotec, Martinsried, Germany (FOXP3);Dako, Glostrup, Denmark (CD4, CD8, CD20); Novocastra,Newcastle, U.K. (CD4) and Coulter, Fullerton, CA, U.S.A.(TIA-1).

Analysis

Immunohistochemical staining was quantified visually by twoof the investigators (C.M. and R.R.-R.), who, in a blindedfashion, separately counted the numbers of cells positive forCD4, CD8, FOXP3 and CD123 in the dermis of the samples.Each specimen was examined first at a scanning magnification( 40) in order to identify the relevant areas (where the infil-

trate was present). Subsequently, each of the selected areaswas examined in detail at high magnification ( 400). Thecell counts were separately averaged for each sample giving anapproximate percentage in proportion to the infiltrate. CD20and TIA-1 positivity was quantified as negative ()) or positive(+) with the following scale: +, 025% positive cells; ++,2050% positive cells; +++, > 75% positive cells.

Controls

Two biopsies of discoid lupus erythematosus (positive controlfor CD123) and two biopsies of lichenoid dermatitis (positive

control for FOXP3) were selected. Endothelial cell stainingwas also used as an internal control for CD123. Histiocyteswere used as an internal control for CD4.

Statistical analysis

Analysis was performed using the STATA software package(version 10.1; Stata-Corp LP, College Station, TX, U.S.A.).Standard descriptive statistics, such as mean and standard devi-ation, were computed. Differences in FOXP3 and CD123 werecompared using the ANOVA test corrected using the Bonferroniformula. All P-values are two sided and P < 005 was consid-ered statistically significant.

Results

Study population characteristics

Cases were categorized as follows: (i) indeterminate leprosy(I), two specimens; (ii) tuberculoid leprosy (TT), one speci-men; (iii) borderline tuberculoid (BT), five specimens; (iv)BT with T1R, three specimens; (v) BT-T1R with IRIS, twospecimens; (vi) mid-borderline leprosy (BB) with T1R withIRIS, one specimen; (vii) borderline lepromatous (BL), onespecimen; and (viii) BL with IRIS, one specimen. AIDS was

reported in 12 (80%) patients with HIVleprosy, and four(33%) out of the 12 patients developed IRIS after HAARTintroduction. Six (40%) patients had T1R at the time of thediagnosis (Table 1).

Immunohistochemistry results

Immunohistochemistry data are summarized in Table 1. CD4+cells were absent in six (38%) and present in 10 (62%)biopsies. In four out of the 10 cases, CD4 cell counts were< 1% of the total lymphocytic infiltrate, and in the remaining

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six cases the mean number of CD4+ cells was 1 16% of thetotal lymphocytic infiltrate. CD4 staining was repeated twicein order to confirm these results. In all the specimens studied,most of the infiltrate (994%) was composed of CD8+ cells,and > 75% of CD8+ cells co-expressed TIA-1. FOXP3+ cellswere also found in all specimens (mean 34% of the lympho-cytic infiltrate, range 15%). CD20+ cells were detected in 12specimens (75%): in two cases (12%), these cells represented2550% of the infiltrate, while in the other 10 cases (62%)they were only focally present (< 25% of the infiltrate). The

control cases (lichen planus) showed an overall mean FOXP3

reactivity of 15%. Despite focal positivity in endothelial cellsused as a positive control, CD123+ cells were not detected inany of the studied specimens (Figs 16).

While in TT and BT cases with or without T1R, FOXP3+cells were detected both inside and around the granulomas, inBL cases these cells were randomly distributed within thediffuse macrophage-rich infiltrates.

No statistical correlation between the number of CD4+cells, CD8+ cells and FOXP3+ cells or the presence of a T1R,HAART therapy and a CD4+ cell count in the peripheral blood

and the clinical form of the disease was observed.

Table 1 Patients clinical data and immunohistochemical results

PatientSex age(years)

Leprosy HIVclassification

CD4 blood(cells lL)1)

Viral load(copies mL)1)

CD4(%)

CD8(%)

FOXP3c(%) TIA-1 CD123 CD20

1 F 48 BT-T1R 290 9500 0 100 4 ++ +++ 0 ++2a M 25 BL IRIS 77 158 100 1 99 4 +++ 0 +2b BT-T1R IRIS Undetected Undetected 0 100 2 +++ 0 +

3 M 32 BT 20 26000 0 100 5 +++ 0 )

4 F 31 BT 457 34 851 1 100 5 +++ 0 +5 M 22 BL 6 183 < 1 99 5 +++ 0 +6 F 30 BT 342 22000 0 100 3 +++ 0 +7 F 20 BT 372 30 424 < 1 99 2 +++ 0 +8 M 48 BT-T1R IRIS 210 8614 < 1 99 3 +++ 0 +9 F 33 BT 223 5432 0 100 3 +++ 0 )

10 M 48 BT-T1R 39 17530 < 1 99 4 ++ +++ 0 + ++11 F 21 I 278 20 624 0 100 2 +++ 0 +12 M 44 BB-T1R IRIS 120 20 177 2 98 4 +++ 0 +13 M 57 BT-T1R 536 Undetected 1 99 3 +++ 0 +14 M 59 TT 175 15 723 1 99 4 +++ 0 )15 M 28 I 770 Undetected 1 99 1 +++ 0 )

HIV, human immunodeficiency virus; BT, borderline tuberculoid; T1R, type 1 reaction; BL, borderline lepromatous; IRIS, immune reconsti-tution inflammatory syndrome; I, indeterminate leprosy; BB, mid-borderline leprosy; TT, tuberculoid leprosy.

(a)

(c)

(b)

(d)

Fig 1. Borderline tuberculoid leprosy.

Immunohistochemical staining of (a) CD8;

(b) TIA-1; (c) CD4: note absence of positive

cells; and (d) FOXP3: regulatory T cells are

present around and within the granuloma.

Original magnification: (a, b) 200;

(c, d) 400.

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Discussion

Our data demonstrate that CD8+ cells were more frequentthan CD4+ cells in the infiltrate of TT and BT HIVleprosyco-infected patients, regardless of the presence of T1R.Although a limited number of multibacillary patients wereenrolled (two BL and one BB), immunohistochemical analysisdemonstrated that CD8+ cells also predominated in thesepatients. In contrast to previous results from HIV-negativepatients with leprosy,16,17 CD8+ cells expressing a cytotoxicphenotype (TIA-1) accounted for the vast majority of cellswithin the infiltrate in patients with HIVleprosy. On the

other hand, CD4+ lymphocytes were either absent (38% ofthe cases) or present at very low frequency (62% of the speci-mens), regardless of the clinical form of leprosy, HAART ther-apy (IRISleprosy or not) or peripheral blood CD4 cell count.The number of positive cells in the dermis of the samples wasdetermined manually and expressed as a rough percentage inproportion to the total lymphocytic infiltrate. The discrepancybetween the literature and our data may be explained if oneconsiders that previous studies were conducted on smaller ser-ies of patients than ours,111,1620 and that the same markerswere not investigated (as they became available only recently).

Unfortunately, only a limited number of immunohisto-chemical studies investigating CD4 and CD8 in HIVleprosyare available and their results remain controversial. In thepresent work, only a small number of cases was analysed, dueto the scarcity of patients with HIVleprosy co-infection inBrazil; although a limitation, it did not hamper the interpreta-tion of the data. It has been shown in other studies that lepro-matous lesions contained few CD8+ T cells while lesions fromHIV-positive patients with BT leprosy showed extensive CD4+T-cell infiltration.1820 Conversely, other authors were not ableto find CD4+ cells in BT or TT cases, even after severalattempts using CD4-specific monoclonal antibodies from dif-

ferent suppliers.21

In our work, a similar pattern was observedand despite the use of anti-CD4 monoclonal antibodies fromtwo different manufacturers, CD4+ cells were either notdetected or found at a low frequency.

Natural Tregs (CD25+FOXP3+ cells) are purported tomaintain tolerance, suppressing the function of autoreactive Tcells in different cutaneous diseases.22 FOXP3+ cells weredetected in all our cases at an average of 34% cells per lym-phocytic infiltrate, confirming previous results from our groupon HIV-negative leprosy cases.17 Although the use of FOXP3expression as the sole marker for Tregs may be considered a

(a) (b)

(c) (d)

(e) (f)

Fig 2. Borderline tuberculoid leprosy with

type 1 reaction. Histopathology showing (a)

multifocal granulomatous infiltrate in the

upper and mid-dermis with periadnexal and

perineural distribution (haematoxylineosinstaining) and (b) epithelioid granulomas

surrounded and infiltrated by lymphocytes

(haematoxylineosin staining).

Immunohistochemical staining of (c) CD4:

note absence of positive cells; (d) CD8:

positive cells are seen within and around the

granulomas; (e) TIA-1: the positive cells have

the same distribution as the CD8 positive

cells; and (f) CD20: focal aggregate of B

lymphocytes. Original magnification:

(a) 40; (be) 100.

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limitation in the study, detection of nuclear expression ofFOXP3 has been broadly used as a relatively accurate assess-ment of quantitative Treg function.23 Moreover, it seems thatin the skin, frequency of FOXP3+ cells is not influenced bythe clinical type of leprosy, the presence of T1R, CD4+ T-cellcounts in peripheral blood or HIV viral load. Colocalization ofFOXP3+ and CD8+ cells was identified visually through theassessment of marker expression proximity (overlay) in paraf-fin-embedded tissue samples. When images from our CD8,CD4 and FOXP3 stainings were overlayed, it became evidentthat FOXP3+ cells had a CD8+ rather than a CD4+ phenotype,as cells expressing CD8 and FOXP3 markers were detected inthe same areacell. Recently, CD8+FOXP3+ Tregs were identi-fied within tuberculous lymphadenitis granulomas, suggestingthat they may be involved in the active immunosuppressionobserved at the infection site.24 The occurrence of virus-spe-

cific CD8+FOXP3+ Tregs was also described in HIV-positiveindividuals.25

Unfortunately, studies on circulating CD4+ and CD8+ Tregsin patients co-infected with HIV and leprosy are still lacking.Although Treg cells have been defined as a key populationduring HIV-1 infection, longitudinal studies investigating Tregfrequency in HIV-1 infection have shown divergent results.26

Consequently, the role of Tregs in HIV-1 pathogenesis remainsunclear. After treatment with HAART, CD4+ T-cell numbersrecover but their function remains persistently suppressed.26

Hence, progressive and nonprogressive HIV-1 disease repre-

sent a broad range of immunological scenarios with potential-

ly different outcomes for Tregs.26

Data from several studies indicate that Treg cells are essen-tial modulators of immunity against different pathogens.27 Inleishmaniasis, Tregs have been shown to be pivotal for estab-lishing and maintaining persistent infection.27 In tuberculosis,Treg frequency is approximately threefold higher in patientswith pulmonary tuberculosis compared with healthy subjects,and Mycobacterium tuberculosis-specific interferon-c levels inM. tuberculosis-stimulated whole blood culture supernatants frompatients with tuberculosis are depressed.28 Both macrophages,CD8+ cytolytic and FOXP3+ T cells have been reported to

(a)

(b)

Fig 3. Borderline tuberculoid leprosy with type 1 reaction.

(a) Immunohistochemical staining of CD4: absence of positive cells.

(b) Nuclear FOXP3 staining. (a, b) Original magnification 400.

(a)

(b)

(c)

Fig 4. Mid-borderline leprosy with type 1 reaction and immune

reconstitution inflammatory syndrome. Immunohistochemical staining

of (a) CD4: few positive cells; the presence of background staining

makes the evaluation difficult; and (b) CD8. (c) Nuclear FOXP3

staining. Original magnification: (a, c) 400; (b) 200.

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surround granulomas in children with tuberculous lymphade-nitis.24 However, CD8+ T cells present in the parafollicularareas of granulomas expressed low levels of perforin and gra-nulysin, which may be the result of the elevated number of

CD4+FOXP3+ T cells in the granulomatous lesion. Skewing ofthe immune response toward a regulatory phenotype mayresult in an uncoordinated effector T-cell response that reducesgranule-mediated killing of M. tuberculosis-infected cells andsubsequent disease control.24 Recently, CD8+ T cells with aregulatory phenotype (CD8+FOXP3+) were described withinthe tumour microenvironment.29 Effector CD8+ T cellsmigrate into the tumours microenvironment and continue toexpand according to antigen load and the cytokine milieu.29

Later, as the tumour regresses and the antigen load dimin-ishes, changes in the cytokine milieu induce an upregulation

of FOXP3 expression by a subpopulation of effector CD8+ Tcells. Additionally, in sarcoidosis, Tregs suppress the initialsteps of granuloma formation, but they have no positive influ-ence on lesion regression.30

B lymphocytes in leprosy skin lesions may be crucial toreduce tissue damage resulting from the vigorous hostresponse mounted against M. leprae infection or for optimalbacterial containment through the development and mainte-nance of granulomas.31 We detected CD20+ cells in 12 speci-mens (75%). In two BT leprosy with T1R cases, CD20+ cellsrepresented 2550% of the infiltrate, while in the other 10cases (62%) they were present only focally (< 25% of theinfiltrate). Although data from both experimental and humantuberculosis suggest that B cells may play an important role ingranuloma formation and inflammatory progression, in lep-

rosy this hypothesis requires further investigation, as a stronggranulomatous response is observed in TT patients despitehaving few B cells within their lesions.3133 Iyer et al., using alesional organotypic skin culture model, showed the existenceof a microenvironment conducive to the differentiation andmaturation of B cells in granulomatous lesions in patients withleprosy and demonstrated the presence of different function-ally active B-cell stages, which could secrete anti-M. leprae-specific antibodies.31 Tyagi et al. suggested that augmented lev-els of M. leprae antigens and specific antibodies detected at thelepromatous pole may lead to an increase in the development

(a)

(b)

(c)

Fig 5. Tuberculoid leprosy. Immunohistochemical staining of (a)

CD4: few focally positive cells; and (b) CD8. (c) Nuclear FOXP3

staining. Original magnification: (a, c) 400; (b) 200.

(a)

(b)

Fig 6. Borderline lepromatous and immune reconstitution

inflammatory syndrome. (a) Immunohistochemical staining of CD4:

only one single positive cell. (b) Nuclear FOXP3 staining. (a, b)

Original magnification: 400.

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of immune complexes, which in turn may suppress specificT-cell responses.34

Recently, we reported the absence of PDCs in skin biopsiesfrom 20 HIV-negative patients with leprosy encompassing theentire clinical spectrum of the disease.17 In the present work,we extend previous results showing that PDCs are notinvolved in the skin immunopathogenesis of leprosy, regard-less of HIV status.

Our novel findings can be summarized into a few keypoints: (i) unlike HIV-negative patients with leprosy, patientsco-infected with HIV and leprosy present an almost exclusiveCD8+ cytotoxic infiltrate at both tuberculoid and lepromatouspoles of the disease; (ii) skin biopsy data from patients withHIVleprosy demonstrate that lymphocytic infiltrates are simi-lar in these patients regardless of the presence or not of IRIS;(iii) B lymphocytes can be found in skin lesions of patientswith HIVleprosy; (iv) FOXP3 and CD123 expression inpatients with HIVleprosy is similar to that in HIV-negativepatients with leprosy; and (v) Tregs in patients with HIVleprosy are probably CD8+ rather than CD4+.

The interaction between HIV and M. leprae seems to be muchmore complicated than previously hypothesized or than thatobserved between HIV and tuberculosis. Although the presentstudy may have limitations, data presented here suggest thatcell-mediated immunity to M. leprae is preserved at the site ofdisease, contrasting with tuberculosis, in which the host granu-lomatous response is impaired by HIV co-infection. The exactmechanism of this paradox, however, is yet to be elucidated.

Whats already known about this topic?

While there are several reports regarding the profile ofsystemic immune response in patients with humanimmunodeficiency virus (HIV) and leprosy, only lim-ited data are available on local skin immunity.

What does this study add?

CD8+FOXP3+ cells, in the absence of CD4+ cells,may be involved in granuloma formation.

Immunohistochemistry data on skin biopsies frompatients with HIVleprosy are presented, includingfor the first time TIA-1, CD20, FOXP3 and CD123.

Highly active antiretroviral therapy does not affectcutaneous immune responses in leprosy occurring inHIV-positive patients.

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Immunophenotype of Skine Lymphocitic Infiltrate (CD 8)

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