|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Division of Hematology/Oncology, Children's
Hospital Medical Center, Cincinnati, OH; the Department of Pediatrics,
National Defense Medical College, Tokorozawa, Japan; and the Department
of Pathology, University of Nebraska, Omaha.
Mutations in the perforin gene have been described in some
patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and
patterns of perforin staining in cytotoxic lymphocytes (natural killer
[NK] cells, CD8+ T cells, CD56+ T cells) from
patients with HLH and their family members were studied. Eleven
unrelated HLH patients and 19 family members were analyzed
prospectively. Four of the 7 patients with primary HLH showed lack of
intracellular perforin in all cytotoxic cell types. All 4 patients
showed mutations in the perforin gene. Their parents, obligate carriers
of perforin mutations, had abnormal perforin-staining patterns.
Analysis of cytotoxic cells from the other 3 patients with primary HLH
and remaining family members had normal percentages of
perforin-positive cytotoxic cells. On the other hand, the 4 patients
with Epstein-Barr virus-associated HLH typically had depressed numbers
of NK cells but markedly increased proportions of CD8+ T
cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of
reduced NK function, may speed the identification of
life-threatening HLH in some families and direct further genetic
studies of the syndrome.
(Blood. 2002;99:61-66) Hemophagocytic lymphohistiocytosis (HLH) is a
life-threatening immune disorder characterized by fever, massive
hepatosplenomegaly, pancytopenia, hypertriglyceridemia,
hypofibrinogenemia, and, frequently, seizures.1
Historically HLH has been categorized clinically as primary or
secondary. The primary form, known as familial hemophagocytic lymphohistiocytosis (FHL), typically shows symptomatic presentation in
infancy and has an autosomal-recessive inheritance. The secondary form
is usually associated with Epstein-Barr virus (EBV) infection, and may
become apparent at an older age.
Regardless of which form is suspected, therapy with immunosuppressive
agents is addressed at the severity of symptoms (according to
HLH-94 treatment protocol).2 In patients with FHL, bone marrow transplantation is highly recommended before central nervous system involvement advances. However, it has been extremely difficult to distinguish the primary and secondary forms of HLH by clinical symptoms, documented infection, or clinical course unless families have
had more than one child with HLH. Even if the disease has been
triggered by EBV, it may still be FHL.
Recently, mutations in the perforin gene have been described in some
patients with HLH.3 This evidence suggests that defective or deficient perforin may be one of the causes of this disease. Perforin is a membranolytic protein that is expressed in the
cytoplasmic granules of cytotoxic T cells and natural killer (NK)
cells. Perforin is believed to be principally responsible for the
translocation of granzyme B from cytotoxic cells into target cells; the
granzyme B then migrates to the target cell nucleus to participate in
triggering apoptosis.4 The roles of perforin in immune
regulation still have not been well defined. However, it is easily
conceivable that without perforin, cytotoxic T cells and NK cells show
reduced or no cytolytic effect on target cells; this offers an
explanation of why patients with HLH have markedly decreased or absent
NK- cell function.
The objectives of our work were to validate a rapid technique for
the measurement of perforin in cytotoxic human lymphocytes, to
establish normal patterns of perforin staining in control subjects of
different ages, and to study the pattern of perforin staining in
HLH patients with and without mutation in the perforin genes, in
their family members, and in patients with other disorders with
life-threatening hemophagocytic complications.
Pediatric control samples
Patient and family member samples
Perforin staining Whole blood was first surface stained with the following antibodies: T-cell receptor  (TCR)-fluorescein
isothiocyanate, CD8-PerCP (BD Immunocytometry Systems, San
Jose, CA), and CD56-APC (Immunotech, Brea, CA) for 20 minutes at room
temperature. Red cells were then lysed for 10 minutes with 2 mL of
FACSlyse (BD Immunocytometry Systems) and washed. The resultant white
cell pellet was then permeabilized using Cytofix/Cytoperm (BD
Pharmingen, San Diego, CA) and stained with either phycoerythrin
(PE)-conjugated antiperforin or PE-conjugated mouse IgG2b (BD
Pharmingen) for 30 minutes at room temperature. After washing, cells
were resuspended in 1% paraformaldehyde and stored at 4°C prior to
analysis by flow cytometry. Normal ranges for age-matched controls have
been developed by studying 80 healthy individuals ranging from
1 month to 50 years of age, of which 41 were under 16 years of age.
Flow cytometric analysis Samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The following gates were used to distinguish the 3 populations of interest: CD8+ T cells were defined as being TCR+, CD8+, and CD56 ;
NK cells were defined as being TCR and
CD56+; CD56+ T cells were defined as being
TCR+ and CD56+. All populations were also
restricted to a lymphocyte gate based on forward versus side scatter.
The perforin-positive region was set using an isotype-matched negative
control, and the percent positive for each region was reported.
Detection of perforin mutation DNA was prepared from EBV-infected cell lines using standard methods. The coding region of the perforin gene was polymerase chain reaction (PCR) amplified from exon 2 and exon 3. Primers used for amplification were as follows: for exon 2, forward (F2) 5'-CCCTTCCATGTGCCCTGATAATC-3' and reverse (R2) 5'-AAGCAGCCTCCAAGT-TTGATTG-3', and for exon 3, forward (F3) 5'-CCAGTCCTAGTTCTGCCCACTTAC-3' and reverse (R3) 5'-GAACCCCTTCAGTCCAAGCATAC-3'. Direct DNA sequence analysis was performed using the ABI Prism 3700 Sequence Detection System (PE Biosystems, Foster City, CA) with the same primer pairs used for PCR amplification.
The technique described here has proved to be rapid and
reproducible. The proportion of perforin-expressing cytotoxic
lymphocytes was age related (Figure 1).
The percentage of NK cells staining with antiperforin antibody was
rather constant as age increased. In children (1-15 years) the
proportion of perforin-expressing NK cells was 86% ± 5%
compared to 92% ± 6% in adults (> 15 years). Perforin-containing CD8+ T cells increased with age. In
young children, especially in infants, the proportion of
perforin-expressing CD8+ T cells was very low. In children
the perforin expression of CD8+ T cells was
7% ± 5% compared to 18% ± 10% in adults. Perforin-containing CD56+ T cells also increased with age. In children the
proportion of CD56+ T cells expressing perforin was
23% ± 10% compared to 54% ± 23% in adult controls. A
characteristic perforin-staining pattern for a healthy adult is shown
in Figure 2A.
Eleven unrelated HLH patients (7 patients with primary HLH and 4 patients with presumed EBV-associated HLH) and 19 family members were prospectively analyzed with this technique. All but one EBV-associated HLH patient demonstrated absent NK function against K562 targets. Table 1 shows that 4 of the 7 patients
with presumed primary HLH showed lack of intracellular perforin in all
cytotoxic cell types (3 with complete and 1 with a partial defeciency).
All 4 patients with partial or complete perforin deficiency showed
missense or nonsense mutations. A typical example of complete perforin deficiency is shown in Figure 2B. Patient P10 showed a very low intensity of perforin staining (low mean channel fluorescence) in the
NK cells (Figure 2C). Analysis of cytotoxic cells from the other 3 patients with presumed primary HLH had normal percentages of
perforin-expressing cytotoxic cells for age.
Patients with EBV-associated HLH (Table
2) were generally older at diagnosis and
were more likely to have very low numbers of NK cells, and increased
proportions of CD8+ T cells expressing perforin (Figure
2D).
Table 3 lists other syndromes or diseases
associated with hemophagocytic complications. A patient with
Chediak-Higashi syndrome in accelerated phase showed values within the
normal range for her age. On the other hand, a patient with X-linked
lymphoproliferative disorder (XLP) showed a markedly increased
proportion of perforin-expressing CD8+ T cells. A patient
with juvenile rheumatoid arthritis (JRA) and macrophage
activation syndrome (MAS) showed decreased proportions of
perforin-expressing cytotoxic cell types.
Table 4 lists parents and asymptomatic
siblings of patients with primary HLH. The asymptomatic siblings of
patients with primary HLH (F (family member) 18, F19, and F20) show
extremely decreased NK function. All 3 unrelated siblings had low
percentages of perforin-positive T cells; however, in infancy healthy
controls also show low percentages of perforin-positive
CD8+ T cells. All 3 asymptomatic siblings of HLH patients
were proved not to have a perforin mutation. An investigation of 8 parents of patients with perforin deficiencies, who were documented to be carriers of a mutated allele, revealed that all but 1 parent had
reduced perforin expression in at least one of the 3 cytotoxic cell
types, with either a decreased proportion of perforin-expressing cells
and/or decreased mean channel fluorescence compared to healthy adults.
Most of the heterozygous carriers showed a slightly decreased percentage of perforin-positive NK cells and very low perforin expression in CD8+ and CD56+ T cells (Figure
3B-C). A similar pattern was seen in a
patient with JRA and MAS (Figure 3D). The father of patient P10, who is documented to be heterozygous for a mutated perforin gene, showed a
reduced proportion of perforin-expressing NK cells. Parents without
perforin mutations showed normal percentages of all perforin-positive cell types.
HLH frequently occurs in children under 2 years of age as a familial autosomal-recessive disorder.5 To test the utility of intracellular perforin staining by flow cytometry as a means of rapidly identifying children with HLH associated with genetic defects in perforin, we first needed to establish normal ranges in this age group. Perforin expression is mainly confined to NK cells,6
CD8+ T cells,7 CD56+ T
cells,8 Perforin expression in adult peripheral blood has been studied by
several investigators.10,11,13 Rutella et al11
showed that perforin positivity in NK cells
(CD3 Perforin expression in peripheral blood from healthy children has been reported only by Rukavina et al.10 They found that the perforin positivity in NK cells (CD56+ cells) from children was very similar to that of adult males, and that the perforin positivity in CD8+ cells increased with age. Our data concur with these findings. However, Rukavina et al10 reported that the average proportion of perforin-positive CD8+ T cells in children was 40%, which is much higher than our findings (7%). The discrepancy is difficult to explain, but may be due to the difference in numbers of specimens (n = 7 for Rukavina) or our selection method of normal samples. Our data for infants regarding the normal proportion of perforin-expressing CD8 cells (1%) are also lower than the cord blood data that was reported by Rukavina (5%),10 but similar to cord blood data published by Berthou (1%).13 We found that 4 of the 7 patients with primary HLH lacked intracellular perforin in all cytotoxic cell types. All of them showed biallelic mutations of the perforin gene. Two had an identical single homozygous nucleotide deletion that caused a frameshift and introduced a premature stop codon. This mutation has previously been reported in patients with HLH.3 In the 2 remaining patients, 4 missense mutations were identified, none of which have previously been reported. Patients with EBV-associated HLH were generally older at diagnosis. Three of the 4 patients studied here showed low NK functional activity, even though the majority of the NK cells expressed perforin. Therefore, their low NK activity may be related to their low numbers of NK cells. Nagao et al reported that some healthy people show low NK activity and that low NK activity can be due to a low percentage of NK cells, not to functional abnormality.14 We do not know at this time why these patients had low numbers of NK cells during the acute phase of disease. One patient recovered a normal proportion of NK cells as well as normal NK activity when he cleared the EBV infection and was no longer symptomatic with HLH. Patients with EBV-associated HLH also showed an increased proportion of perforin-expressing CD8+ T cells. Experimental in vitro studies have demonstrated that perforin expression in CD8+ T cells is induced by several cytokines, including interleukin-2 (IL-2)15 and/or IL-12, or when the cells are costimulated using anti-CD3 antibody.16 In patients with EBV infection, EBV-specific CD8+ T cells are strikingly induced, and these EBV-specific CD8+ T cells express perforin.17 Our preliminary data suggest that EBV-transformed B cell lines (autologous or allogeneic) are also effective in increasing the proportions of perforin-expressing CD8+ T cells in vitro. However, study of 4 healthy adolescents with primary EBV infection (infectious mononucleosis) showed that the subjects maintained normal percentages of NK cells and normal proportions of total perforin-positive CD8 T cells (unpublished data, March 2001). Thus, the mechanism of induction of perforin expression in CD8+ T cells in patients presenting with HLH requires further study. However, our preliminary results suggest that quantitation of NK cells and intracellular perforin staining of CD8+ T cells can potentially distinguish EBV-related HLH from primary HLH. Studies of perforin from parents of patients with primary HLH have revealed that in all 4 families where the proband had perforin deficiency, the parents had abnormal perforin-staining patterns. The most common parental pattern was a normal percentage of perforin-positive NK cells (although mean channel fluorescent intensity was usually decreased), but a very decreased proportion of perforin-expressing CD8+ T and CD56+ T cells, similar to the profile observed in healthy infants. The ability of heterozygous carriers of PRF1 mutations to generate cytotoxic T cell responses remains to be defined. We also had the opportunity to test NK function and perforin expression in cytotoxic cells obtained from infant siblings of patients who had died of HLH in the past. The samples (F18, F19, and F20) were submitted for these studies by their referring physicians in an effort to "screen" for possible HLH prior to onset of symptoms. In all 3 unrelated cases no prior information regarding the possibility of familial perforin defects was available. Our analyses showed that all the infants had normal proportions of perforin-positive cytotoxic cells for age but decreased NK function. Subsequent mutational analysis proved that none of these children carried mutations of the PRF1 gene. Since asymptomatic adult carriers of PRF1-negative HLH often have decreased NK function,18 these children may be carriers for the other gene(s) etiologic for HLH. Alternatively, they could be affected. Additional 8-month follow-up indicates that none have yet developed HLH. In conclusion, the present study suggests that a flow cytometric assay examining 3 different cytotoxic cell populations constitutes a rapid and sensitive approach for detection of HLH patients and carriers of HLH secondary to perforin deficiency. As more cases of HLH are studied to define those with and without perforin mutations, it should be possible to analyze whether any features of the clinical phenotype are associated with perforin deficiency. This technique appears to provide diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome. We recommend that all patients with clinical criteria consistent with HLH be studied for NK number and function, as well as having perforin staining of T and NK cytotoxic cells carried out. This screening battery will help confirm the diagnosis of HLH for all patients (decreased or absent NK function) and is highly likely to identify the one third of patients with HLH due to perforin deficiency. In families where perforin deficiency has been documented and the perforin-staining pattern in an affected individual has been determined, this technique can be reliably applied to screen asymptomatic newborn siblings for HLH, in the event that parental mutational analysis of PRF1 has not already been done.
The authors are very grateful to all the participating families and physicians for their generous cooperation in this study and to David Lee, Sue Vergamini, Carol Moore, Terri Ellerhorst, and Darryl Hake for excellent technical support.
Supported by the Ryan Marrocco Memorial Fund, the Zachary Carter Fund for HLH Research, and a grant from the Histiocytosis Association of America.
Submitted May 3, 2001; accepted August 21, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alexandra H. Filipovich, Division of Hematology/Oncology, Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229-3039; e-mail: filil0{at}chmcc.org.
1. Loy TS, Diaz-Arias AA, Perry MC. Familial erythrophagocytic lymphohistiocytosis. Semin Oncol. 1991;18:34-38[Medline] [Order article via Infotrieve]. 2. Henter JI, Arico M, Egeler RM, et al. HLH-94: a treatment protocol for hemophagocytic lymphohistiocytosis. HLH study Group of the Histiocyte Society. Med Pediatr Oncol. 1997;28:342-347[CrossRef][Medline] [Order article via Infotrieve].
3.
Stepp SE, Dufourcq-Lagelouse R, Le Deist F, et al.
Perforin gene defects in familial hemophagocytic lymphohistiocytosis.
Science.
1999;286:1957-1959 4. Pinkoski MJ, Heibein JA, Barry M, Bleackley RC. Nuclear translocation of granzyme B in target cell apoptosis. Cell Death Differ. 2000;7:17-24[CrossRef][Medline] [Order article via Infotrieve]. 5. Gencik A, Signer E, Muller H. Genetic analysis of familial erythrophagocytic lymphohistiocytosis. Eur J Pediatr. 1984;142:248-252[CrossRef][Medline] [Order article via Infotrieve].
6.
Kawasaki A, Shinkai Y, Kuwana Y, et al.
Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells.
Int Immunol.
1990;2:677-684 7. Garcia-Sanz JA, Plaetinck G, Velotti F, et al. Perforin is present only in normal activated Lyt2+ T lymphocytes and not in L3T4+ cells, but the serine protease granzyme A is made by both subsets. Embo J. 1987;6:933-938[Medline] [Order article via Infotrieve]. 8. Musha N, Yoshida Y, Sugahara S, et al. Expansion of CD56+ NK T and gamma delta T cells from cord blood of human neonates. Clin Exp Immunol. 1998;113:220-228[CrossRef][Medline] [Order article via Infotrieve].
9.
Nakata M, Smyth MJ, Norihisa Y, et al.
Constitutive expression of pore-forming protein in peripheral blood gamma/delta T cells: implication for their cytotoxic role in vivo.
J Exp Med.
1990;172:1877-1880
10.
Rukavina D, Laskarin G, Rubesa G, et al.
Age-related decline of perforin expression in human cytotoxic T lymphocytes and natural killer cells.
Blood.
1998;92:2410-2420 11. Rutella S, Rumi C, Lucia MB, Etuk B, Cauda R, Leone G. Flow cytometric detection of perforin in normal human lymphocyte subpopulations defined by expression of activation/differentiation antigens. Immunol Lett. 1998;60:51-55[CrossRef][Medline] [Order article via Infotrieve]. 12. Kawamura T, Kawachi Y, Moroda T, et al. Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen. Immunology. 1996;89:68-75[CrossRef][Medline] [Order article via Infotrieve].
13.
Berthou C, Legros-Maida S, Soulie A, et al.
Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes.
Blood.
1995;85:1540-1546 14. Nagao F, Yabe T, Xu M, Okumura K. Phenotypical and functional analyses of natural killer cells from low NK activity individuals among healthy and patient populations. Nat Immun. 1995;14:225-233[Medline] [Order article via Infotrieve]. 15. Kataoka K, Naomoto Y, Kojima K, et al. Flow cytometric analysis on perforin induction in peripheral blood mononuclear cells with interleukin-2 or OK-432. J Immunother. 1992;11:249-256.
16.
Smyth MJ, Ortaldo JR, Shinkai Y, et al.
Interleukin 2 induction of pore-forming protein gene expression in human peripheral blood CD8+ T cells.
J Exp Med.
1990;171:1269-1281 17. Callan MF, Fazou C, Yang H, et al. CD8+ T-cell selection, function, and death in the primary immune response in vivo. J Clin Invest. 2000;106:1251-1261[Medline] [Order article via Infotrieve]. 18. Sullivan KE, DeLaat CA, Douglas SD, Filipovich AH. Defective killer cell function in patients with Hemophagocytic Lymphohistiocytosis and first degree relatives. Pediatr Res. 1998;44:465-468[Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
T cells