| Abstract|| |
X-linked hyperimmunoglobulin M (HIGM) syndrome may increase the susceptibility of patients to disseminated cryptococcal infections primarily due to CD40L deficiency that causes defective cross talk between T- and B-cells, thus preventing class switching. In HIGM syndrome, serum IgM levels are elevated with severe reduction in serum immunoglobulin G (IgG) and IgA levels. In addition, the expression of CD40L (CD154) on in vitro-activated T-cells is severely reduced or absent. Here, we describe a rare, and perhaps, the first reported case in India of a 3-year-old male child with X-linked HIGM immunodeficiency syndrome who developed disseminated Cryptococcosis. Evaluation of the serum IgG profile of the patient revealed increased serum IgM levels with reduced IgG and IgA levels. Both the frequency and the function of T-cells, primarily CD40L on activated T-cells, showed weak expression suggestive of HIGM syndrome.
Keywords: Disseminated cryptococcosis, hyperimmunoglobulin M syndrome, immunodeficiency
|How to cite this article:|
Mohanty SK, Thakral D, Gupta D, Kumar P, Mitra DK. Diminished CD40L expression on T-cells in a case of disseminated cryptococcosis. Indian J Pathol Microbiol 2018;61:137-40
|How to cite this URL:|
Mohanty SK, Thakral D, Gupta D, Kumar P, Mitra DK. Diminished CD40L expression on T-cells in a case of disseminated cryptococcosis. Indian J Pathol Microbiol [serial online] 2018 [cited 2021 Jul 25];61:137-40. Available from: https://www.ijpmonline.org/text.asp?2018/61/1/137/228194
| Introduction|| |
Primary immunodeficiencies (PIDs) are a heterogeneous group of inherited disorders that can affect cells of the innate or adaptive immune system. Hyperimmunoglobulin M (HIGM) syndrome is one of the rarest PIDs characterized by defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM), resulting in a defect in switching from IgM to IgG, IgA, or IgE. More than 90% of the patients present clinically, within 4 years of age. The most common type of inherited HIGM is the X-linked form with a deficiency in expression of CD40 ligand (CD40L, CD154) on T-cells on activation. CD40L on activated T-cells binds to CD40 on B-cells and initiates class switching as well as generation of memory B-cells in response to T-cell-dependent antigens. Mutations of CD40, the receptor for CD40L, also cause a rare autosomal recessive form of HIGM with a clinical phenotype similar to CD40L deficiency. Clinically, most patients present with an increased susceptibility to infections, especially by encapsulated bacterial, opportunistic fungal, as well as viral infections. Here, we report a rare case of a 3-year-old male child with X-linked HIGM syndrome who developed disseminated cryptococcosis.
| Case Report|| |
A 3-year-old male child was admitted to the pediatric ward of this tertiary care hospital with presenting complaints of persistent low-grade fever over the past 1 month, not responding to conventional antipyretics and antibiotics. The child was the only sibling, out of a nonconsanguineous marriage. He was asymptomatic with normal developmental milestones until about 2 years of age. Perinatal history of the child was uneventful, and although his mother gave a history of him suffering from recurrent fever with upper respiratory tract infections over the past 1 year, he never required hospital admission. On admission at our hospital, a general physical examination revealed multiple subcentrimetric, hyperpigmented, and maculopapular skin lesions over the trunk. He was febrile and had generalized lymphadenopathy and mild icterus. The liver was enlarged and tender. The child had no meningeal signs on presentation. During the course of admission, the child was investigated thoroughly as a case of pyrexia of unknown origin. Routine laboratory tests revealed the following: hemoglobin – 7.1 g%, total leukocyte count – 3,500/cumm, differential leukocyte count – neutrophils (67%), lymphocytes (26%), monocytes (03%), eosinophils (04%), and platelet count 3.98 × 106/cumm. Serum bilirubin was 2.5 mg/dl, and hepatic enzymes were raised, aspartate aminotransferase (AST) – 132 IU/L (reference range – <40 IU/L), AST – 78 IU/L (reference range – <40 IU/L), and alkaline phosphatase – 1850 U/L (15–270 U/L). Coagulation profile (prothrombin time, activated partial thromboplastin time, and international normalized ratio) was within normal limits. Peripheral smear as well as rapid immunochromatographic test for malaria, serology workup for Leishmania, HIV, and hepatitis viruses were negative. Urine routine microscopy revealed budding yeast forms and the urine culture grew Cryptococcus neoformans. Blood culture (BACTEC) and sputum culture also grew C. neoformans. Fine-needle aspiration cytology cervical lymph node aspirated scanty and mucoid material, which revealed budding yeast forms surrounded by a halo. Special histological stains such as periodic acid–Schiff and mucicarmine identified the organism as C. neoformans. Histopathology from liver biopsy, skin lesion biopsy, lymph node biopsy, and bone marrow aspiration was consistent with disseminated cryptococcosis [Figure 1]a and [Figure 1]b. Cerebrospinal fluid (CSF) was positive for Cryptococcus by latex agglutination test and CSF culture also grew C. neoformans. Computed Tomography (CT) chest/abdomen revealed multiple enlarged homogeneously enhancing mediastinal, mesenteric, and retroperitoneal lymph nodes with vascular encasement. Multiple lung nodules in the perilymphatic and peribronchial region were noted. CT head showed mild cerebral atrophy, although no evidence of meningitis or cryptococcomas was seen.
|Figure 1: (a) Periodic acid–Schiff-stained section from the skin lesion (×20 magnification) shows normal epidermal lining. The subepidermal tissue shows scattered inflammatory cells dispersed in gelatinous and mucoid background. (b) At higher magnification (×40), numerous yeast forms of Cryptococcus neoformans are seen with prominent magenta capsules|
Click here to view
Owing to the unusual and disseminated form of this infection in the absence of other secondary causes of immunosuppression, a PID disorder was suspected, and the sample was sent for immunological profiling in our immunological laboratory. Serum IgG – 120 mg/dl (reference range – 45–900 mg/dl) and IgA – 15 mg/dl (reference range – 20–100 mg/dl) were reduced with elevated IgM – 290 mg/dl (reference range – 20–200 mg/dl) levels. A CD40L defect was suspected, and we performed in vitro T-cell stimulation assay to evaluate its expression by flow cytometry (FACS Aria; BD, San Jose, USA). We observed severely reduced expression of CD40 L (CD154) on T-cells obtained from the peripheral blood of the patient after in vitro stimulation with phorbol myristate acetate. Cells were stained with fluorescently labeled antibodies against cell surface markers followed by acquisition by BD FACS Aria. Data analysis was done by FlowJo software [Figure 2]. Since the patient was a male child, his mother's sample was also evaluated for expression of CD154 to determine possible carrier status. CD40L expression was observed only on approximately 50% of the T-cell population, whereas the rest failed to express, suggesting the “X”-linked inheritance pattern of this condition. Based on the clinical presentation and supported by investigation findings, a diagnosis of HIGM syndrome was offered. The child was treated with intravenous immunoglobulin (IVIG) 400 mg/kg 3 monthly, IV Amphotericin B deoxycholate (0.7 mg/kg/day), oral tablet fluconazole (6 mg/kg/day) for 2 weeks, and prophylactic antibiotics to which he responded well. His serum IgG levels normalized and he was discharged after 3 weeks with subsequent follow-up in the pediatric outpatient department.
|Figure 2: Cell surface expression of CD40L (CD154) on CD3+ CD4+ T-cells from patient with hyperimmunoglobulin syndrome. (a) Lymphocytes were gated by forward versus side scatter, and CD3+ T-cells were further gated on activated CD4+ T-cells expressing CD69 as depicted in the gating strategy. The CD40L expression in stimulated cells (CD154) was overlayed on unstimulated cells in the (b) healthy control and (c) patient as shown in the histograms|
Click here to view
| Discussion|| |
The International Union of Immunological Societies PID expert committee in 2015 have classified HIGM as a Category III disorder comprising of “predominantly antibody deficiencies.” This condition was first described by Rosen et al., and later the WHO gave it the nomenclature of “syndrome immunodeficiency with HIGM.” The disease phenotype was initially thought to be due to an intrinsic failure of B-cells to mature by undergoing CSR and SHM. However, later, it was recognized that defective T-cell cross talk with the B-cells prevents their sequential class switch. This form of disease is the most common form of HIGM and accounts for 65%–70% of all cases. It is inherited as an X-linked recessive condition, wherein there is a mutation of the CD154 (CD40L, tumor necrosis factor [TNF] SF5) gene, which encodes for the CD40L molecule expressed transiently on the surface of activated CD4+ T-cells (HIGM Type 1). CD40 expressed on B-cells are the receptors for CD154, and this defective cross talk between CD40 and CD154 leads to the impaired isotype switch in the B-cells. The less common forms of HIGM are inherited as autosomal recessive forms and affect both males and females equally. These are CD40 deficiency (HIGM Type 3), activation-induced cytidine deaminase deficiency, where there is a mutation in the activation-induced cytidine deaminase (AICDA) gene (HIGM Type 2), and uracil N-glycosylase deficiency (HIGM Type 5). Another X-linked recessive form of this disease is associated with a mutation in the gene encoding nuclear factor kappa B (NF-κB) essential modulator (HIGM Type 6) that is necessary for the activation of the signaling molecule NF-κB. These patients typically present with a skin condition called ectodermal dysplasia. Type 4 HIGM is genetically undefined.
Although most patients are diagnosed before age 4 years, over one-half of patients develop symptoms of immunodeficiency by the age of 1 year and nearly all develop symptoms by 4 years of age. The patients are prone to opportunistic infections, especially encapsulated bacteria, fungus, and viruses. The most common presentation is sinopulmonary infection, but the gastrointestinal, central nervous system, hepatobiliary, and skin involvement also occurs. Patients may also present with autoimmune manifestations such as autoimmune hemolytic anemia, arthritis, thrombocytopenia, and hypothyroidism. There is an increased risk of malignancies, especially hepatocellular carcinoma.
Although ubiquitous in exposure, C. neoformans is an encapsulated yeast that rarely causes clinical infections in the immunocompetent host. It usually affects those having a defect in cell-mediated immunity.
Disseminated cryptococcosis is defined by (1) a positive culture from at least two different sites or (2) a positive blood culture. In our patient, positive cultures were obtained from blood and bone marrow, CSF, and lymph node aspirate.
Due to the intrinsic challenges faced by our country in the diagnosis of PIDs, the incidence of individual PIDs is largely underestimated and hence unknown. A study of PIDs in two major pediatric centers in northern India reveals an incidence of X-linked HIGM varying between 1.3% and 2.4%. Shah et al. have reported two cases of HIGM who presented with liver abscesses; however, the diagnosis was based only on basic immunoglobulin profile, and no flow cytometry or genetic analysis was carried out. Nag et al. reported a case of X-linked HIGM with bronchiectasis and Pneumocystis jirovecii pneumonia in a 5-year-old male child.
Owing to the disseminated and invasive nature of this otherwise innocuous fungus in a 3-year-old male child who was nonreactive for HIV compelled the clinician to investigate further for a PID disease. In view of the reduced levels of IgG and IgA with mildly elevated IgM levels, the patient was suspected as a case of HIGM syndrome, and he and his mother were subjected to flow cytometric analysis for the expression of CD40L (CD154). The analysis revealed a weak expression of CD40L on the surface of the activated CD4 T-lymphocytes and a carrier status in the mother.
Although classified primarily as a purely humoral defect, patients with HIGM Type 1 and Type 3 are known to present with opportunistic infections such as pneumocystis, toxoplasma, and Cryptococcus. It is probably because in vitro studies have shown that the CD40L–CD40 cross talk is essential for the secretion of interleukin-12 and interferon-γ in response to this organism. Due to the absence of CD40L, the macrophages are unable to activate the T-cells through the APCs, thus significantly blunting the Th1 response, which is primarily responsible for launching the immune response against these fungal elements.
CD40L expression is reduced on activated CD4+ T-cells (expressing CD69), with <15% of CD4+ cells expressing CD40L following stimulation; in contrast, the comparable value for healthy controls is >80%. CD4 T-cells from mothers of XHIGM patients exhibited a duality of CD40L expression (with approximately 50% of CD4 cells positive for CD40L), suggesting their carrier status.
In this case, the percentage of CD4+ T-cells expressing CD69 and CD40L poststimulation was markedly reduced (~17%) in contrast to the percentage expressing CD69 activation marker (approximately 80%). However, the affirmative diagnosis of XHIGM syndrome requires the identification of a mutation affecting the CD40L/TNF receptor gene. The mutational study, however, could not be performed in our hospital due to its nonavailability.
| Conclusion|| |
Although cases of disseminated cryptococcosis are known, it is highly uncommon in children. Cases have been reported in India, but perhaps, this is the first reported case of HIGM syndrome in a child presenting with disseminated cryptococcosis. This case suggests that patients with any disseminated fungal infection should be suspected for PID, especially HIGM, and accordingly evaluated. It also highlights the invaluable role of modern laboratory techniques such as flow cytometry in the early diagnosis of such cases.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Notarangelo LD, Hayward AR. X-linked immunodeficiency with hyper-IgM (XHIM). Clin Exp Immunol 2000;120:399-405.
Bousfiha A, Jeddane L, Al-Herz W, Ailal F, Casanova JL, Chatila T, et al.
The 2015 IUIS phenotypic classification for primary immunodeficiencies. J Clin Immunol 2015;35:727-38.
Rosen FS, Kevy SV, Merler E, Janeway CA, Gitlin D. Recurrent bacterial infections and dysgamma-globulinemia: Deficiency of 7S gamma-globulins in the presence of elevated 19S gamma-globulins. Report of two cases. Pediatrics 1961;28:182-95.
Mayer L, Kwan SP, Thompson C, Ko HS, Chiorazzi N, Waldmann T, et al.
Evidence for a defect in “switch” T cells in patients with immunodeficiency and hyperimmunoglobulinemia M. N
Engl J Med 1986;314:409-13.
Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, et al.
Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2). Cell 2000;102:565-75.
Yehia BR, Eberlein M, Sisson SD, Hager DN. Disseminated cryptococcosis with meningitis, peritonitis, and cryptococcemia in a HIV-negative patient with cirrhosis: A case report. Cases J 2009;2:170.
Gupta S, Madkaikar M, Singh S, Sehgal S. Primary immunodeficiencies in India: A perspective. Ann N
Y Acad Sci 2012;1250:73-9.
Shah I, Rahangdale A, Bhatnagar S. Liver abscesses and hyper IgM syndrome. J Family Med Prim Care 2013;2:206-8.
] [Full text]
Nandan D, Nag VK, Trivedi N, Singh S. X-linked hyper-IgM syndrome with bronchiectasis. J Lab Physicians 2014;6:114-6.
] [Full text]
Grewal IS, Xu J, Flavell RA. Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand. Nature 1995;378:617-20.
Dipendra Kumar Mitra
Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]