There are no known criteria for definitive diagnosis of LGE but it resists conventional plaque removal therapies and oral hygiene measures and this distinguishes it from acute marginal gingivitis. LGE is now described as a candidal infection. Though the microbiology of LGE has not been fully described, C. albicans and C. dubliniensis have been isolated from LGE lesions in both children and adults. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999;

Foscarnet 24 – 40mg/kg 8 hourly (very severe cases only) for 14-21days.

Acyclovir 200 – 400mg tabs 6 hourly for 10-14days

episodes. (Ramos-Gomez et al., 1999)

Fig. 9. Primary herpetic gingivostomatitis.

plaque that is present on the teeth. (Flaitz & Hicks 1999).

**4.3 Linear gingival erythema (LGE)**

1996).

**Treatment**

1999).

Velegraki et al 1999).

hexahydrate).

Fig. 10. Linear gingival erythema.

#### **4.4 Major salivary gland enlargement**

Salivary gland disease in HIV infected children presents as either xerostomia or major salivary gland enlargement. Major salivary gland enlargement refers to lymphocyte mediated salivary gland disease in cases of HIV infection. It may affect the parotid and/or submandibular glands. It was first seen in paediatric HIV infection. It is also called Diffuse Infiltrative Lymphocytosis syndrome or Sjogren syndrome-like disease. (Flaitz & Hicks 2003). (Figure 11).

Major salivary gland enlargement is more common in children than in adults with HIV infection and the prevalence in HIV infected children varies widely in different studies with up to 58% of them being affected. The aetiology of major salivary gland enlargement is unknown but infection with EBV or HIV or the interaction between these two viruses is suspected to be responsible. (Flaitz & Hicks 2003; Fonseca et al., 2000; Katz et al., 1993; Valdez et al., 1994).

Fig. 11. Parotid gland enlargement.

The parotid gland is most commonly affected with parotid swelling in 10 – 30% of HIV infected children. There is unilateral or bilateral diffuse soft tissue swelling resulting in facial disfigurement. It is sometimes painful, and may be associated with lymphoid interstitial pneumonitis and diffuse lymphadenopathy. There may be concurrent enlargement of the palatine tonsils, resulting in partial airway obstruction, difficulty with swallowing and sleep apnoea. Parotid swelling has been associated with slower progression

Oral Manifestations of Paediatric HIV Infection 179

This presents as 1-3 shallow, oval or round small ulcers less than 5mm in diameter covered with a pseudo membrane surrounded by an erythematous halo. (Figure 12). They are self limiting and typically heal within 2weeks without scarring. (Flaitz & Hicks 2003; Ramos-

These are oval or round, similar to minor recurrent aphthous ulcers but much larger 1– 2cm in diameter, are fewer in number, and may persist for weeks at a time. They are painful and may interfere with mastication and swallowing. They tend to occur on the soft palate, buccal mucosa, tonsillar area, and tongue. They have well delineated to irregular borders and depressed bases and are often covered by a thick, tenacious, fibrinous exudate. Scaring is a

These appear as clusters or crops of tiny recurrent aphthous ulcers, 1 to 2mm in diameter, which may coalesce. They tend to occur in the soft palate, buccal mucosa, tonsillar area, and tongue. Diagnosis is usually clinical, based on the size, site, appearance and duration of the

A definitive criterion for all three types of RAU is the response to treatment with steroid agents. Tissue cultures for viral, fungal and mycobacterial organisms and incisional biopsy are necessary for persistent lesions to rule out other causes of ulcers. (Flaitz & Hicks 2003;

A definitive criterion for all the three types is the response to treatment with steroid agents. Tissue cultures for viral, fungal and mycobacterial organisms and incisional biopsy are necessary for persistent lesions to rule out other causes of ulcers. Topical steroid therapy is the first choice. In severe cases, a short course of prednisolone is indicated. Antifungal agents to prevent oropharyngeal candidiasis are added when steroids are used. These steroids include fluocinomide, clobetasol propionate and dexamethasone elixir. (Ramos-

**4.5.1 Minor recurrent aphthous ulcers**

Gomez et al 1999).

Fig. 12. Minor Aphthous Ulcer.

common finding in this condition.

Ramos-Gomez et al 1999).

**4.5.4 Treatment**

Gomez et al 1999).

ulcers.

**4.5.3 Herpetiform recurrent aphthous ulcers**

**4.5.2 Major recurrent aphthous ulcers**

to AIDS: having a median time to death of 5. 4years as against 3. 4years among patients with oral candidiasis. (Chetty et al., 1999; Flaitz & Hicks 2003; Katz et al., 1993; Ramos-Gomez et al 1999).

There are no established criteria for the definitive diagnosis of salivary gland enlargement (SGE) and the exact pathophysiology remains uncertain. Theories concerning the origin of SGE include lymphoepithelial lesions, cysts involving salivary parenchyma, interglandular lymph nodes, and an inflammatory infiltrate similar to Sjogren's syndrome. Greenspan described the possible relationship between SGE and T-lymphocyte CD8+ cell infiltration in the gland. In addition, genetic loci have been linked to SGE in children, specifically HLADR5 and HLA-DR11, suggesting a genetic predisposition to this condition. (Greenspan & Greenspan 1996; Pinto& De Rossi 2004)

#### **Treatment**

Salivary gland swelling is usually left untreated because it is usually asymptomatic. However, there are a number of dental management concerns and serious complications in children related to infections of the salivary glands becoming life threatening at a faster rate than in adults. The paediatric dentist should be aware of the possible complications in the paediatric HIV/AIDS population. Often the salivary gland disorders or their potential sequelae can be a predictor of HIV disease progression. Consultation with the child's infectious disease provider, including ordering complete blood counts, should give an indication of the child's immune status. Assessment of salivary function should be performed at the initial dental exam. Subjective complaints of dryness in children are often equivocal, so the clinician should be familiar with examination of the Stensen's and Wharton's ducts to verify flow. Palpation of the major glands and milking of the major glands should be a standard part of the comprehensive head and neck examination of the HIV Positive paediatric patient.

Salivary gland disease can present as benign enlargement with or without xerostomia, xerostomia alone, or parotitis or more ominous conditions such as malignancy. In children, serious complications related to salivary gland infections can become life-threatening at a faster rate than in adults. The clinician should be aware of the etiologic agents that can cause parotitis (bacterial/viral). Knowledge of the child's immune status will often serve as a guide towards diagnosis (i. e., cytomegalovirus or paramyxovirus in immune-compromised children). Thorough examination of salivary ducts can provide valuable information on aetiology-(purulence in saliva, thick mucous saliva) and guide the clinician to adequate treatment. The antibiotic of choice is clindamycin, in a dosage of 8-25mg/kg/day. A therapeutic alternative antibiotic is penicillin, considering the gastrointestinal effects of clindamycin. (Pinto& De Rossi 2004).

#### **4.5 Recurrent aphtous ulcers (RAU)**

The prevalence of RAU in HIV infected children ranges from 0-7% similar to HIV infected adults. In general RAU tend to recur more frequently in the HIV infected child; and with immunosuppression, major aphthous ulcers are also more likely to develop. (Flaitz & Hicks 2003).

RAU presents as painful ulcers of rapid onset with marked predilection for non keratinised mucosa especially the labial and buccal mucosa, ventral tongue and soft palate. The pharyngeal and oesophageal mucosa may be involved in the most extensive cases. Three clinical varieties have been described based on size, number and duration of lesions present.

HIV Infection in the Era of Highly Active 178 Antiretroviral Treatment and Some of Its Associated Complications

to AIDS: having a median time to death of 5. 4years as against 3. 4years among patients with oral candidiasis. (Chetty et al., 1999; Flaitz & Hicks 2003; Katz et al., 1993; Ramos-Gomez et

There are no established criteria for the definitive diagnosis of salivary gland enlargement (SGE) and the exact pathophysiology remains uncertain. Theories concerning the origin of SGE include lymphoepithelial lesions, cysts involving salivary parenchyma, interglandular lymph nodes, and an inflammatory infiltrate similar to Sjogren's syndrome. Greenspan described the possible relationship between SGE and T-lymphocyte CD8+ cell infiltration in the gland. In addition, genetic loci have been linked to SGE in children, specifically HLADR5 and HLA-DR11, suggesting a genetic predisposition to this condition. (Greenspan

Salivary gland swelling is usually left untreated because it is usually asymptomatic. However, there are a number of dental management concerns and serious complications in children related to infections of the salivary glands becoming life threatening at a faster rate than in adults. The paediatric dentist should be aware of the possible complications in the paediatric HIV/AIDS population. Often the salivary gland disorders or their potential sequelae can be a predictor of HIV disease progression. Consultation with the child's infectious disease provider, including ordering complete blood counts, should give an indication of the child's immune status. Assessment of salivary function should be performed at the initial dental exam. Subjective complaints of dryness in children are often equivocal, so the clinician should be familiar with examination of the Stensen's and Wharton's ducts to verify flow. Palpation of the major glands and milking of the major glands should be a standard part of the comprehensive head and neck examination of the

Salivary gland disease can present as benign enlargement with or without xerostomia, xerostomia alone, or parotitis or more ominous conditions such as malignancy. In children, serious complications related to salivary gland infections can become life-threatening at a faster rate than in adults. The clinician should be aware of the etiologic agents that can cause parotitis (bacterial/viral). Knowledge of the child's immune status will often serve as a guide towards diagnosis (i. e., cytomegalovirus or paramyxovirus in immune-compromised children). Thorough examination of salivary ducts can provide valuable information on aetiology-(purulence in saliva, thick mucous saliva) and guide the clinician to adequate treatment. The antibiotic of choice is clindamycin, in a dosage of 8-25mg/kg/day. A therapeutic alternative antibiotic is penicillin, considering the gastrointestinal effects of

The prevalence of RAU in HIV infected children ranges from 0-7% similar to HIV infected adults. In general RAU tend to recur more frequently in the HIV infected child; and with immunosuppression, major aphthous ulcers are also more likely to develop. (Flaitz & Hicks

RAU presents as painful ulcers of rapid onset with marked predilection for non keratinised mucosa especially the labial and buccal mucosa, ventral tongue and soft palate. The pharyngeal and oesophageal mucosa may be involved in the most extensive cases. Three clinical varieties have been described based on size, number and duration of lesions present.

al 1999).

**Treatment**

& Greenspan 1996; Pinto& De Rossi 2004)

HIV Positive paediatric patient.

clindamycin. (Pinto& De Rossi 2004).

**4.5 Recurrent aphtous ulcers (RAU)**

2003).

#### **4.5.1 Minor recurrent aphthous ulcers**

This presents as 1-3 shallow, oval or round small ulcers less than 5mm in diameter covered with a pseudo membrane surrounded by an erythematous halo. (Figure 12). They are self limiting and typically heal within 2weeks without scarring. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999).

Fig. 12. Minor Aphthous Ulcer.

#### **4.5.2 Major recurrent aphthous ulcers**

These are oval or round, similar to minor recurrent aphthous ulcers but much larger 1– 2cm in diameter, are fewer in number, and may persist for weeks at a time. They are painful and may interfere with mastication and swallowing. They tend to occur on the soft palate, buccal mucosa, tonsillar area, and tongue. They have well delineated to irregular borders and depressed bases and are often covered by a thick, tenacious, fibrinous exudate. Scaring is a common finding in this condition.

#### **4.5.3 Herpetiform recurrent aphthous ulcers**

These appear as clusters or crops of tiny recurrent aphthous ulcers, 1 to 2mm in diameter, which may coalesce. They tend to occur in the soft palate, buccal mucosa, tonsillar area, and tongue. Diagnosis is usually clinical, based on the size, site, appearance and duration of the ulcers.

A definitive criterion for all three types of RAU is the response to treatment with steroid agents. Tissue cultures for viral, fungal and mycobacterial organisms and incisional biopsy are necessary for persistent lesions to rule out other causes of ulcers. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999).

#### **4.5.4 Treatment**

A definitive criterion for all the three types is the response to treatment with steroid agents. Tissue cultures for viral, fungal and mycobacterial organisms and incisional biopsy are necessary for persistent lesions to rule out other causes of ulcers. Topical steroid therapy is the first choice. In severe cases, a short course of prednisolone is indicated. Antifungal agents to prevent oropharyngeal candidiasis are added when steroids are used. These steroids include fluocinomide, clobetasol propionate and dexamethasone elixir. (Ramos-Gomez et al 1999).

Oral Manifestations of Paediatric HIV Infection 181

cases. The final stage of NUP is marked by severe gingival recession resulting from rapid bone loss and soft tissue necrosis. (Figures 13 &14) Pocketing may be minimal and tissue destruction may extend across the mucogingival junction. The more extensive disease may contribute to premature exfoliation of primary teeth and aborted development or early loss of primary teeth. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999). Diagnosis of NUP is based

This presents as an acute and painful ulceronecrotic lesion on the oral mucosa. The lesion starts from the oral mucosa and may extend to alveolar bone and contiguous soft tissues. The underlying bone may be exposed. Histologic examination reveals the features of nonspecific ulceration. No specific microbial organism has been identified as the cause of NS.

Irrigation with 10%Betadine providone -iodine or 1:4 hydrogen peroxide or

The patient should be re-evaluated after one week of treatment and the medication should

Human Papilloma Virus (HPV) induced oral lesions associated with HIV infection include Verucca vulgaris, condyloma acuminatum, focal epithelial hyperplasia, and koilocytic

Gentle debridement of affected areas to minimize bleeding and pain

Chlorhexidine gluconate (Peridex), 0. 12% to aid debridement.

Metronidazole (Flagyl) 125 mg tablets 8hourly for 7days

Erythromycin enteric coated 250mg tablet 8hourly for 7 days

Amoxicillin (Amoxil) 250 mg 8hrly for 7days

on clinical presentations.

Fig. 14. Necrotising Ulcerative Periodontitis (NUP)

(Flaitz & Hicks 2003; Ramos-Gomez et al 1999).

**5.1.4 Treatment for NUG, NUP and NS**

Oral hygiene instruction

If the patient is allergic to penicillin:

be repeated if response is not satisfactory. Supportive therapy: Multivitamins

**Systemic Treatment**

**5.2 Viral infections**

**5.2.1 Human Papilloma Virus**

**Local treatment**

**5.1.3 Necrotising stomatitis (NS)**

#### **5. Oral lesions less commonly associated with paediatric HIV infection**

#### **5.1 HIV associated periodontal diseases**

These include:

In children, HIV associated periodontal diseases appear to be associated with both immunodeficiency and malnutrition4. HIV infected children from developing countries appear to be more susceptible to necrotising periodontal diseases. A declining immune system with CD4 + cell counts below 200 cells/mm is associated with necrotising ulcerative periodontitis and necrotising stomatitis. (Flaitz & Hicks 2003; Greenspan 1994; Ramos-Gomez et al 1999).

Fig. 13. Necrotising Ulcerative Periodontitis (NUP).

#### **5.1.1 Necrotising ulcerative gingivitis (NUG)**

This is uncommon in HIV infected children. It is reported to present with the destruction of one or more interdental papillae accompanied by necrosis, ulceration and/or sloughing. Destruction is limited to the marginal gingival tissues. In the acute stage, the gingival tissues appear fiery red and swollen, and is accompanied by yellowish grey necrotic tissue that bleeds easily. Patients are reported to experience bleeding on brushing, pain and a characteristic halitosis. Symptoms subside gradually over 3 – 4 weeks but recurrences are common.

There may be lymphadenopathy, fever and malaise. Permanent gingival scarring with interdental papillary crater contributes to an increased risk of recurrence. (Flaitz & Hicks 2003). Diagnosis is based on clinical features described above.

#### **5.1.2 Necrotising ulcerative periodontitis (NUP)**

NUP has been reported in 0-4% of children. It presents as severe soft tissue necrosis along with destruction of the periodontal attachment and bone over a short period of time. Patients often experience spontaneous gingival bleeding or bleeding when brushing and severe, deep, aching pain in the jawbone. The jawbone may be exposed in the most severe

HIV Infection in the Era of Highly Active 180 Antiretroviral Treatment and Some of Its Associated Complications

In children, HIV associated periodontal diseases appear to be associated with both immunodeficiency and malnutrition4. HIV infected children from developing countries appear to be more susceptible to necrotising periodontal diseases. A declining immune system with CD4 + cell counts below 200 cells/mm is associated with necrotising ulcerative periodontitis and necrotising stomatitis. (Flaitz & Hicks 2003; Greenspan 1994; Ramos-

This is uncommon in HIV infected children. It is reported to present with the destruction of one or more interdental papillae accompanied by necrosis, ulceration and/or sloughing. Destruction is limited to the marginal gingival tissues. In the acute stage, the gingival tissues appear fiery red and swollen, and is accompanied by yellowish grey necrotic tissue that bleeds easily. Patients are reported to experience bleeding on brushing, pain and a characteristic

There may be lymphadenopathy, fever and malaise. Permanent gingival scarring with interdental papillary crater contributes to an increased risk of recurrence. (Flaitz & Hicks 2003).

NUP has been reported in 0-4% of children. It presents as severe soft tissue necrosis along with destruction of the periodontal attachment and bone over a short period of time. Patients often experience spontaneous gingival bleeding or bleeding when brushing and severe, deep, aching pain in the jawbone. The jawbone may be exposed in the most severe

halitosis. Symptoms subside gradually over 3 – 4 weeks but recurrences are common.

**5. Oral lesions less commonly associated with paediatric HIV infection**

**5.1 HIV associated periodontal diseases**

 Necrotising ulcerative gingivitis (NUG) Necrotising ulcerative periodontitis (NUP)

Fig. 13. Necrotising Ulcerative Periodontitis (NUP).

Diagnosis is based on clinical features described above.

**5.1.2 Necrotising ulcerative periodontitis (NUP)**

**5.1.1 Necrotising ulcerative gingivitis (NUG)**

Necrotising stomatitis (NS)

These include:

Gomez et al 1999).

cases. The final stage of NUP is marked by severe gingival recession resulting from rapid bone loss and soft tissue necrosis. (Figures 13 &14) Pocketing may be minimal and tissue destruction may extend across the mucogingival junction. The more extensive disease may contribute to premature exfoliation of primary teeth and aborted development or early loss of primary teeth. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999). Diagnosis of NUP is based on clinical presentations.

Fig. 14. Necrotising Ulcerative Periodontitis (NUP)

#### **5.1.3 Necrotising stomatitis (NS)**

This presents as an acute and painful ulceronecrotic lesion on the oral mucosa. The lesion starts from the oral mucosa and may extend to alveolar bone and contiguous soft tissues. The underlying bone may be exposed. Histologic examination reveals the features of nonspecific ulceration. No specific microbial organism has been identified as the cause of NS. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999).

#### **5.1.4 Treatment for NUG, NUP and NS**

#### **Local treatment**

#### **Systemic Treatment**

If the patient is allergic to penicillin:

Erythromycin enteric coated 250mg tablet 8hourly for 7 days

The patient should be re-evaluated after one week of treatment and the medication should be repeated if response is not satisfactory.

Supportive therapy: Multivitamins

#### **5.2 Viral infections**

#### **5.2.1 Human Papilloma Virus**

Human Papilloma Virus (HPV) induced oral lesions associated with HIV infection include Verucca vulgaris, condyloma acuminatum, focal epithelial hyperplasia, and koilocytic

Oral Manifestations of Paediatric HIV Infection 183

This presents as a persistent oral ulcer, which may mimic aphthous ulcers, recurrent herpes simplex virus infection, necrotising stomatitis and ulceration not otherwise specified. Occasionally it may present as a brightly erythematous gingivitis. CMV ulcers in the oral cavity usually occur with disseminated CMV disease; therefore the patient should be examined for the systemic disease or other organ involvement including CMV retinitis, colitis, pneumonitis and progressive neurologic disease. Definitive diagnosis can be made

VZV infection can present as Herpes Zoster or Varicella (chicken pox). HIV infected children are at risk for persistent, recurrent, chronic infections with VZV. Children with advanced immunosuppression seem to be at increased risk for recurrent disease and more severe manifestations of VZV. Chronic VZV infection has been described in HIV-infected children and adults with low CD4+ counts. (Gershon et al., 1997; Morris et al., 1996; Ramos-Gomez et

The Center for Disease Control and Prevention has classified an HIV-infected child with herpes zoster involving at least 2 distinctive episodes or more than one dermatome as being moderately symptomatic. Herpes Zoster infection results from a reactivation of latent varicella – zoster virus. Fevers, complaints of sensitive teeth, earache or headache as well as pain or paresthesia are prodromal symptoms. A well-delineated unilateral maculo-papular rash that becomes pustular ulcerated follows. Involvement of the second and third branches of the trigeminal nerve produces oral lesions on both keratinised and non keratinsed oral mucosa that extend to the midline. Frequently concurrent vesicles and crusted skin lesions overlie the affected dental quadrant. Most cases heal without complications in children except for facial skin scarring. Definitive diagnosis is by virus antigen typing with laboratory immunologic

This is a virally induced lesion of the skin, vagina, and rarely, the oral cavity. Lesions are small, discrete, and dome shaped. Their colour ranges from pearly white to skin colour. The lesions may number in the hundreds. Definitive diagnosis is made when molluscum bodies, which are virally transformed epithelial cells are seen, when the core of the lesion is

Xerostomia is a common symptom of HIV-infected individuals and has many potential

It is more common in HIV infected children than HIV-infected adults. The causes of xerostomia include HIV infection itself, therapeutic antiviral and antimicrobial drugs, prophylactic medications, antiretrovirals (such as didanosine), gamma globulin, or lymphocytic infiltration of the major salivary glands. Clinical features include dry mouth (Figure 16) and severely reduced salivary flow rates. Reduced salivary flow results in a

through culture and biopsy. (Flaitz et al., 1996; Ramos-Gomez et al., 1999).

tests. (Caldwell et al., 1994; Flaitz & Hicks 2003; Ramos-Gomez et al 1999).

**5.2.5 Cytomegalovirus (CMV)**

**5.2.6 Varicella Zoster Virus (VZV)**

al., 1999).

**5.2.7 Herpes Zoster**

**5.2.8 Molluscum contagiosum**

expressed and stained. (Ramos-Gomez et al 1999).

**5.3 Xerostomia**

causes.

dysplasia which are referred to as oral warts. Oral HPV infections are rare in children. Oral warts present as raised, irregular, flesh coloured lesions.

#### **5.2.2 Focal epithelial hyperplasia (Heck's disease)**

This is a benign condition associated with HPV types 13 and 32 and is usually seen in children. The lesions appear as multiple, non-tender papules, plaques or nodules with grainy pink mucosal surfaces. The labial, buccal and lateral tongue, mucosa are most often involved, with occasional lesions on the palatal and gingival mucosa. (Figure 15).

#### **Treatment**

In most cases, conservative excision is treatment of choice. Other methods of treatment include laser ablation, topical podophyllin resin, systemic interferon, and cimetidin. Despite aggressive therapy and multiple treatment modalities, recurrences are common when oral warts are widespread. (Flaitz & Hicks 2003; Howell et al 1996; Ramos-Gomez et al., 1999).

Fig. 15. Focal Epithelial hyperplasia (Heck's disease).

#### **5.2.3 Verruca vulgaris**

This typically affects the skin, especially the hands and perioral skin. Oral mucosal involvement due to inoculation from these extraoral sites. Intraoral involvement is uncommon and is usually limited to isolated areas of the lip vermillion, labial mucosa and anterior tongue. They present as white, rough, conical or papillary papules or nodules. Treatment is as for Focal Epithelial Hyperplasia above.

#### **5.2.4 Condyloma acuminatum**

It typically presents as a large sessile, pink nodule with short, blunt surface projections. Multiple lesions may be found that tend to coalesce, forming a discrete, well-delineated enlargement. Diagnosis is usually clinical. Definitive diagnosis can be made by excisional biopsy.

Treatment is as for Focal Epithelial Hyperplasia and oral warts.

#### **5.2.5 Cytomegalovirus (CMV)**

HIV Infection in the Era of Highly Active 182 Antiretroviral Treatment and Some of Its Associated Complications

dysplasia which are referred to as oral warts. Oral HPV infections are rare in children. Oral

This is a benign condition associated with HPV types 13 and 32 and is usually seen in children. The lesions appear as multiple, non-tender papules, plaques or nodules with grainy pink mucosal surfaces. The labial, buccal and lateral tongue, mucosa are most often

In most cases, conservative excision is treatment of choice. Other methods of treatment include laser ablation, topical podophyllin resin, systemic interferon, and cimetidin. Despite aggressive therapy and multiple treatment modalities, recurrences are common when oral warts are widespread. (Flaitz & Hicks 2003; Howell et al 1996; Ramos-Gomez et al., 1999).

This typically affects the skin, especially the hands and perioral skin. Oral mucosal involvement due to inoculation from these extraoral sites. Intraoral involvement is uncommon and is usually limited to isolated areas of the lip vermillion, labial mucosa and anterior tongue. They present as white, rough, conical or papillary papules or nodules.

It typically presents as a large sessile, pink nodule with short, blunt surface projections. Multiple lesions may be found that tend to coalesce, forming a discrete, well-delineated enlargement. Diagnosis is usually clinical. Definitive diagnosis can be made by excisional

involved, with occasional lesions on the palatal and gingival mucosa. (Figure 15).

warts present as raised, irregular, flesh coloured lesions.

**5.2.2 Focal epithelial hyperplasia (Heck's disease)**

Fig. 15. Focal Epithelial hyperplasia (Heck's disease).

Treatment is as for Focal Epithelial Hyperplasia above.

Treatment is as for Focal Epithelial Hyperplasia and oral warts.

**5.2.3 Verruca vulgaris**

biopsy.

**5.2.4 Condyloma acuminatum**

**Treatment**

This presents as a persistent oral ulcer, which may mimic aphthous ulcers, recurrent herpes simplex virus infection, necrotising stomatitis and ulceration not otherwise specified. Occasionally it may present as a brightly erythematous gingivitis. CMV ulcers in the oral cavity usually occur with disseminated CMV disease; therefore the patient should be examined for the systemic disease or other organ involvement including CMV retinitis, colitis, pneumonitis and progressive neurologic disease. Definitive diagnosis can be made through culture and biopsy. (Flaitz et al., 1996; Ramos-Gomez et al., 1999).

#### **5.2.6 Varicella Zoster Virus (VZV)**

VZV infection can present as Herpes Zoster or Varicella (chicken pox). HIV infected children are at risk for persistent, recurrent, chronic infections with VZV. Children with advanced immunosuppression seem to be at increased risk for recurrent disease and more severe manifestations of VZV. Chronic VZV infection has been described in HIV-infected children and adults with low CD4+ counts. (Gershon et al., 1997; Morris et al., 1996; Ramos-Gomez et al., 1999).

#### **5.2.7 Herpes Zoster**

The Center for Disease Control and Prevention has classified an HIV-infected child with herpes zoster involving at least 2 distinctive episodes or more than one dermatome as being moderately symptomatic. Herpes Zoster infection results from a reactivation of latent varicella – zoster virus. Fevers, complaints of sensitive teeth, earache or headache as well as pain or paresthesia are prodromal symptoms. A well-delineated unilateral maculo-papular rash that becomes pustular ulcerated follows. Involvement of the second and third branches of the trigeminal nerve produces oral lesions on both keratinised and non keratinsed oral mucosa that extend to the midline. Frequently concurrent vesicles and crusted skin lesions overlie the affected dental quadrant. Most cases heal without complications in children except for facial skin scarring. Definitive diagnosis is by virus antigen typing with laboratory immunologic tests. (Caldwell et al., 1994; Flaitz & Hicks 2003; Ramos-Gomez et al 1999).

#### **5.2.8 Molluscum contagiosum**

This is a virally induced lesion of the skin, vagina, and rarely, the oral cavity. Lesions are small, discrete, and dome shaped. Their colour ranges from pearly white to skin colour. The lesions may number in the hundreds. Definitive diagnosis is made when molluscum bodies, which are virally transformed epithelial cells are seen, when the core of the lesion is expressed and stained.

(Ramos-Gomez et al 1999).

#### **5.3 Xerostomia**

Xerostomia is a common symptom of HIV-infected individuals and has many potential causes.

It is more common in HIV infected children than HIV-infected adults. The causes of xerostomia include HIV infection itself, therapeutic antiviral and antimicrobial drugs, prophylactic medications, antiretrovirals (such as didanosine), gamma globulin, or lymphocytic infiltration of the major salivary glands. Clinical features include dry mouth (Figure 16) and severely reduced salivary flow rates. Reduced salivary flow results in a

Oral Manifestations of Paediatric HIV Infection 185

criteria for oral hairy leukoplakia are the presence of Epstein –Barr virus (EBV) in the lesions, to be determined by laboratory histopathology and in-situ DNA hybridization.

A combined infection with HIV and mycobacterium tuberculosis is common in LINCs and developing countries. However, secondary and primary tuberculosis lesions of the oral cavity are rare. The lesions present as painless, non-healing ulcerations on the buccal mucosa, hard palate, gingivae and/or tongue. Occasionally, tuberculosis may present with tongue involvement manifesting as macroglossia or a mass in the cheek. In most cases the mucosal lesions are difficult to recognize, but enlarged regional lymph nodes may call

A chest radiograph and a Purified Protein Derivative test with appropriate controls should be obtained. Diagnosis is usually confirmed by identification of acid-fast bacilli in tissue sections or culture**.** The oral lesions respond to treatment with use of anti-tuberculosis drugs. (Ceballos-Selobrena et al., 1996; Grupta et al., 1997; Hathiram et al., 1997; Haller & Ginsberg 1997; Kolokotronis et al., 1996; Ramesh 1997; Phelan et al., 1997; Ramos-Gomez et

Gingival disease not specifically associated with HIV infection is called conventional gingivitis. Reported prevalence in HIV infected children is 7% and 40%. Clinically, it presents as gingival inflammation in the absence of attachment loss, necrosis or gingival

(Barasch et al., 2000; Gelbier et al., 2000). An association between the prevalence of gingivitis in HIV infected children and age has been reported. (As low as 6% in less than 1 year olds, 55% in 1year olds, 85% in 2year olds, 87% in 3year olds and 66% in 4year olds). (Ramos et

**7.2 Over retained primary teeth and delayed eruption of primary and permanent teeth**  Delayed eruption of teeth has been reported among children with HIV-infection. Poor general health in some children may be an associated factor. Delayed tooth development has been reported in 31% of paediatric AIDS patients. (Valdez et al 1994). Delayed dental development in paediatric HIV patients has been linked to lower CD4 counts. In the report by Ramos-Gomez et al 2000; it was observed that at age 3, there was an average of two fewer teeth per child among children whose average CD4 count was 200/mm3 than children whose average CD4 count was 800/mm. A study on 173 HIV infected children, delayed eruption was reported in 43% males and 41% females. Accelerated eruption in 11% males and 13% females, while 46% of both males and females have normal eruption pattern. Over

There is anecdotal evidence of vulnerability of HIV infected children to dental caries. However, some researchers have drawn attention to comparable caries prevalence in

retained primary teeth was observed in 25% of the children. (Flaitz et al., 2000).

**7. Oral conditions with increased severity in paediatric HIV infection**

**7.1 Gingivitis and periodontitis (increased gingival and plaque indices)**

(Flaitz & Hicks 2003; Morris et al., 1996; Ramos-Gomez et al 1999).

**6.4 Tuberculosis related ulcers**

attention to the process.

erythematous banding.

al 1999).

al., 2000).

**7.3 Dental caries**

mucosa that is desiccated and is at higher risk for opportunistic infections such as candidiasis and increased caries. Xerostomia may appear with or without parotid swelling. No definitive diagnostic criteria exist for xerostomia. (Flaitz & Hicks 2003; Frezzini et al., 2006; Ramos-Gomez et al 1999).

Fig. 16. Dry lips with crusting, resulting from xerostomia.

#### **6. Oral lesions strongly associated with HIV infection but rare in children**

#### **6.1 Kaposi's sarcoma**

This is much less common in HIV infected children than adults. The incidence of HIV associated cancers in symptomatic children is less than 2%. However, among HIV infected paediatric population, it is more common in Africa, Latin America and Eastern Europe, where endemic Kaposi's sarcoma is more prevalent in the non-HIV infected population. Human herpes virus – 8 has been identified in all forms of Kaposi's sarcoma. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999; Ranganathan & Hemalatha 2006). Kaposi's sarcoma presents as one or more erythematous, slightly bluish or violaceous macules or swellings with or without ulceration. It is predominantly seen on the palate or gingiva. A definitive criterion is characteristic histologic appearance on biopsy. (William 1993).

#### **6.2 Non-Hodgkin's lymphoma**

This is far less common in HIV infected children than adults. This presents as a firm, elastic often somewhat reddish or purplish swelling with or without ulceration. The gingiva, palatal mucosa and fauces are more commonly affected. A definitive criterion is the characteristic histological appearance on biopsy, supported by appropriate immuno-cytochemical or molecular biological investigations. (Ramos-Gomez et al 1999; William 1993).

#### **6.3 Oral Hairy Leukoplakia (OHL)**

This is an opportunistic infection caused by the Epstein-Barr virus (EBV) and is a marker for increasing immunodeficiency. It is a common oral manifestation of HIV infection in adults with a point presence of approximately 20% but it is documented in only 2% of children infected with HIV. It presents as white non-removable lesions with corrugated surface appearing bilaterally on the lateral border of the tongue. These lesions may appear on the ventral and dorsal surfaces of the tongue and more rarely, on the buccal mucosa. Definitive criteria for oral hairy leukoplakia are the presence of Epstein –Barr virus (EBV) in the lesions, to be determined by laboratory histopathology and in-situ DNA hybridization. (Flaitz & Hicks 2003; Morris et al., 1996; Ramos-Gomez et al 1999).

#### **6.4 Tuberculosis related ulcers**

HIV Infection in the Era of Highly Active 184 Antiretroviral Treatment and Some of Its Associated Complications

mucosa that is desiccated and is at higher risk for opportunistic infections such as candidiasis and increased caries. Xerostomia may appear with or without parotid swelling. No definitive diagnostic criteria exist for xerostomia. (Flaitz & Hicks 2003; Frezzini et al.,

**6. Oral lesions strongly associated with HIV infection but rare in children**

This is much less common in HIV infected children than adults. The incidence of HIV associated cancers in symptomatic children is less than 2%. However, among HIV infected paediatric population, it is more common in Africa, Latin America and Eastern Europe, where endemic Kaposi's sarcoma is more prevalent in the non-HIV infected population. Human herpes virus – 8 has been identified in all forms of Kaposi's sarcoma. (Flaitz & Hicks 2003; Ramos-Gomez et al 1999; Ranganathan & Hemalatha 2006). Kaposi's sarcoma presents as one or more erythematous, slightly bluish or violaceous macules or swellings with or without ulceration. It is predominantly seen on the palate or gingiva. A definitive criterion is

This is far less common in HIV infected children than adults. This presents as a firm, elastic often somewhat reddish or purplish swelling with or without ulceration. The gingiva, palatal mucosa and fauces are more commonly affected. A definitive criterion is the characteristic histological appearance on biopsy, supported by appropriate immuno-cytochemical or

This is an opportunistic infection caused by the Epstein-Barr virus (EBV) and is a marker for increasing immunodeficiency. It is a common oral manifestation of HIV infection in adults with a point presence of approximately 20% but it is documented in only 2% of children infected with HIV. It presents as white non-removable lesions with corrugated surface appearing bilaterally on the lateral border of the tongue. These lesions may appear on the ventral and dorsal surfaces of the tongue and more rarely, on the buccal mucosa. Definitive

2006; Ramos-Gomez et al 1999).

**6.1 Kaposi's sarcoma**

**6.2 Non-Hodgkin's lymphoma**

**6.3 Oral Hairy Leukoplakia (OHL)**

Fig. 16. Dry lips with crusting, resulting from xerostomia.

characteristic histologic appearance on biopsy. (William 1993).

molecular biological investigations. (Ramos-Gomez et al 1999; William 1993).

A combined infection with HIV and mycobacterium tuberculosis is common in LINCs and developing countries. However, secondary and primary tuberculosis lesions of the oral cavity are rare. The lesions present as painless, non-healing ulcerations on the buccal mucosa, hard palate, gingivae and/or tongue. Occasionally, tuberculosis may present with tongue involvement manifesting as macroglossia or a mass in the cheek. In most cases the mucosal lesions are difficult to recognize, but enlarged regional lymph nodes may call attention to the process.

A chest radiograph and a Purified Protein Derivative test with appropriate controls should be obtained. Diagnosis is usually confirmed by identification of acid-fast bacilli in tissue sections or culture**.** The oral lesions respond to treatment with use of anti-tuberculosis drugs. (Ceballos-Selobrena et al., 1996; Grupta et al., 1997; Hathiram et al., 1997; Haller & Ginsberg 1997; Kolokotronis et al., 1996; Ramesh 1997; Phelan et al., 1997; Ramos-Gomez et al 1999).

#### **7. Oral conditions with increased severity in paediatric HIV infection**

#### **7.1 Gingivitis and periodontitis (increased gingival and plaque indices)**

Gingival disease not specifically associated with HIV infection is called conventional gingivitis. Reported prevalence in HIV infected children is 7% and 40%. Clinically, it presents as gingival inflammation in the absence of attachment loss, necrosis or gingival erythematous banding.

(Barasch et al., 2000; Gelbier et al., 2000). An association between the prevalence of gingivitis in HIV infected children and age has been reported. (As low as 6% in less than 1 year olds, 55% in 1year olds, 85% in 2year olds, 87% in 3year olds and 66% in 4year olds). (Ramos et al., 2000).

#### **7.2 Over retained primary teeth and delayed eruption of primary and permanent teeth**

Delayed eruption of teeth has been reported among children with HIV-infection. Poor general health in some children may be an associated factor. Delayed tooth development has been reported in 31% of paediatric AIDS patients. (Valdez et al 1994). Delayed dental development in paediatric HIV patients has been linked to lower CD4 counts. In the report by Ramos-Gomez et al 2000; it was observed that at age 3, there was an average of two fewer teeth per child among children whose average CD4 count was 200/mm3 than children whose average CD4 count was 800/mm. A study on 173 HIV infected children, delayed eruption was reported in 43% males and 41% females. Accelerated eruption in 11% males and 13% females, while 46% of both males and females have normal eruption pattern. Over retained primary teeth was observed in 25% of the children. (Flaitz et al., 2000).

#### **7.3 Dental caries**

There is anecdotal evidence of vulnerability of HIV infected children to dental caries. However, some researchers have drawn attention to comparable caries prevalence in

Oral Manifestations of Paediatric HIV Infection 187

Dental caries status in primary dentition of HIV infected children has been reported to be considerably greater than that for the United States paediatric population. (Hicks et al., 2000.) During the 30 month period longitudinal study, there was an almost two fold increase in primary tooth surface caries for the 2-9 year olds. Caries free status in the primary dentition declined from 60% at baseline to 37% at the 30months period. Among 5 to 11 year olds, dmfs and dmft remained relatively stable while the proportion of caries free individuals declined from 72% at baseline to 50% at 18 months. Caries free status decreased with age, lower CD4 percentage and from moderate to severe immunosuppression. (Hicks et al., 2000). A high prevalence of Candida colonization in HIV positive /AIDS children with untreated dental caries has been reported. (Domaneschi et al., 2010). This further reinforces the importance of

Caries and periodontal disease are among the most common infections known to humans. Both are initiated by oral bacteria and are modulated by the host response. Disease occurs as a result of an imbalance between the provoking bacteria and host response. It is logical to expect, therefore, that alterations of either the associated aetiological factors or the host response to these factors and or agents should result in a change in the clinical presentation

Oral manifestations are common and prevalent in paediatric HIV infection and have been found to be the earliest indicators of HIV infection. Early intervention in HIV disease is crucial for each individual according to his/her risk and seroconversion status. Comprehensive oral examination should be done at regular intervals especially in LINCs and developing countries where techniques to diagnose, and the drugs to treat HIV infection are not uniformly distributed. The use of oral lesions as predictors of disease progression could be of immense importance. Primary oral health care for HIV infected children should include a careful oral examination at regular intervals to ensure early detection and intervention for pseudomembranous candidiasis and other infections and to prevent more deterioration of the immune system and further opportunistic infections. Preventive oral health care especially where treatment is progression unavailable, can improve a child's overall health. Though these measures cannot stop HIV disease progression, in the absence of medications, improved diagnosis of the oral manifestations can enhance case management, ensure better oral health outcomes, reduce morbidity and

The authors wish to thank the Staff of the Special Paediatric Outpatient Clinic of Lagos University Teaching Hospital and Peadiatric HIV Outpatient Clinic of Massey Children Hospital, Lagos Nigeria for their cooperation and assistance and in particular the parents and children for their consent to participate with the investigations and approval to make

Abrams EJ. (2000). Opportunistic Infections and Other Clinical Manifestations of HIV

Disease in Children. Pediatr Clin North Am 47: 95 -96

oral health care in interdisciplinary health unit that manage these patients.

of disease. (Fine et al., 2003).

improve quality of life of HIV-infected children.

**9. Acknowledgement**

**10. References**

use of their clinical photographs.

**8. Conclusions**

infected and non-infected children. Other studies have shown slightly higher prevalence of dental caries in children with HIV. The suggestion that caries development could be directly linked to immunosuppression is yet to be substantiated, although the possibility does exist. Other factors may be responsible for the high caries level in these children, and these include infant feeding practices, the long term use of sugar containing medicines, high consumption of carbohydrate and sugar intake required to prevent or treat any failure to thrive.

A number of studies have reported on the prevalence of dental caries among HIV infected children. (Elderidge & Gallagher 2000; Flaitz et al., 2000; Gelbier et al., 2000; Hicks et al 2000; Obileye et al., 2009, Sowole et al 2009; Tofsky et al 2000; Valdez et al 1994). In a study on dental caries prevalence and dental health behavior in HIV infected children, 63% of the children had past dental caries experience. (Elderidge & Gallagher 2000) This is similar to a previous report by Valdez et al; it was observed that 60% of paediatric AIDS patients had clinical evidence of current or past caries.

In the primary dentition of HIV infected children, the anterior caries (early childhood caries, nursing bottle or baby bottle tooth decay) pattern is common. The pattern of caries observed in this group of children may be as a result of poor oral hygiene practices, inappropriate use of a nursing bottle containing high sucrose liquids at bed time, medications containing high sucrose content, xerostomia induced by medication or HIV infection, the need for high caloric and carbohydrate/sucrose diets, and alterations in saliva viscosity, cytokines, protease inhibitors and immunoglobulin. (Flaitz & Hicks 2003). (Figures 17 & 18)

Fig. 17. Dental caries on mandibular primary molars.

Fig. 18. Dental caries on maxillary primary canines and molars.

HIV Infection in the Era of Highly Active 186 Antiretroviral Treatment and Some of Its Associated Complications

infected and non-infected children. Other studies have shown slightly higher prevalence of dental caries in children with HIV. The suggestion that caries development could be directly linked to immunosuppression is yet to be substantiated, although the possibility does exist. Other factors may be responsible for the high caries level in these children, and these include infant feeding practices, the long term use of sugar containing medicines, high consumption of carbohydrate and sugar intake required to prevent or treat any failure to

A number of studies have reported on the prevalence of dental caries among HIV infected children. (Elderidge & Gallagher 2000; Flaitz et al., 2000; Gelbier et al., 2000; Hicks et al 2000; Obileye et al., 2009, Sowole et al 2009; Tofsky et al 2000; Valdez et al 1994). In a study on dental caries prevalence and dental health behavior in HIV infected children, 63% of the children had past dental caries experience. (Elderidge & Gallagher 2000) This is similar to a previous report by Valdez et al; it was observed that 60% of paediatric AIDS patients had

In the primary dentition of HIV infected children, the anterior caries (early childhood caries, nursing bottle or baby bottle tooth decay) pattern is common. The pattern of caries observed in this group of children may be as a result of poor oral hygiene practices, inappropriate use of a nursing bottle containing high sucrose liquids at bed time, medications containing high sucrose content, xerostomia induced by medication or HIV infection, the need for high caloric and carbohydrate/sucrose diets, and alterations in saliva viscosity, cytokines,

protease inhibitors and immunoglobulin. (Flaitz & Hicks 2003). (Figures 17 & 18)

thrive.

clinical evidence of current or past caries.

Fig. 17. Dental caries on mandibular primary molars.

Fig. 18. Dental caries on maxillary primary canines and molars.

Dental caries status in primary dentition of HIV infected children has been reported to be considerably greater than that for the United States paediatric population. (Hicks et al., 2000.) During the 30 month period longitudinal study, there was an almost two fold increase in primary tooth surface caries for the 2-9 year olds. Caries free status in the primary dentition declined from 60% at baseline to 37% at the 30months period. Among 5 to 11 year olds, dmfs and dmft remained relatively stable while the proportion of caries free individuals declined from 72% at baseline to 50% at 18 months. Caries free status decreased with age, lower CD4 percentage and from moderate to severe immunosuppression. (Hicks et al., 2000). A high prevalence of Candida colonization in HIV positive /AIDS children with untreated dental caries has been reported. (Domaneschi et al., 2010). This further reinforces the importance of oral health care in interdisciplinary health unit that manage these patients.

Caries and periodontal disease are among the most common infections known to humans. Both are initiated by oral bacteria and are modulated by the host response. Disease occurs as a result of an imbalance between the provoking bacteria and host response. It is logical to expect, therefore, that alterations of either the associated aetiological factors or the host response to these factors and or agents should result in a change in the clinical presentation of disease. (Fine et al., 2003).

#### **8. Conclusions**

Oral manifestations are common and prevalent in paediatric HIV infection and have been found to be the earliest indicators of HIV infection. Early intervention in HIV disease is crucial for each individual according to his/her risk and seroconversion status. Comprehensive oral examination should be done at regular intervals especially in LINCs and developing countries where techniques to diagnose, and the drugs to treat HIV infection are not uniformly distributed. The use of oral lesions as predictors of disease progression could be of immense importance. Primary oral health care for HIV infected children should include a careful oral examination at regular intervals to ensure early detection and intervention for pseudomembranous candidiasis and other infections and to prevent more deterioration of the immune system and further opportunistic infections. Preventive oral health care especially where treatment is progression unavailable, can improve a child's overall health. Though these measures cannot stop HIV disease progression, in the absence of medications, improved diagnosis of the oral manifestations can enhance case management, ensure better oral health outcomes, reduce morbidity and improve quality of life of HIV-infected children.

#### **9. Acknowledgement**

The authors wish to thank the Staff of the Special Paediatric Outpatient Clinic of Lagos University Teaching Hospital and Peadiatric HIV Outpatient Clinic of Massey Children Hospital, Lagos Nigeria for their cooperation and assistance and in particular the parents and children for their consent to participate with the investigations and approval to make use of their clinical photographs.

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of Nigeria, 2006

**11**

Nicole F. Bernard1,2,3,10 et al.\*

*Montréal, Québec,*

*Canada*

**Natural Killer Cells from HIV Infected Slow**

**Progressors Who Carry the Protective**

*for the Canadian Cohort of HIV Infected Slow Progressors 1Research Institute of the McGill University Health Centre*

> *3Division of Clinical Immunology and Allergy, McGill University Health Centre, Toronto, Ontario*

**HLA-B\*27 Allele and Inhibitory KIR3DL1**

**Receptors Have Elevated Poly-Functional Potential Compared to Bw6 Homozygotes**

*2Division of Experimental Medicine, McGill University, Clinique du Quartier*

NK cells are a key component of the innate immune system, which can act early in defences against virally infected and tumor cells (Robertson and Ritz 1990; Bottino, Moretta, and Moretta 2006; Bancroft 1993). They have the capacity to secrete proinflammatory cytokines and lyse their targets without prior antigen sensitization (Cooper, Fehniger, and Caligiuri 2001). They are also involved in regulation of the adaptive immune response through their

Carlos Melendez-Pena1,2, Philomena Kamya1,2, Christos M. Tsoukas1,2,3, Mohamed-Rachid Boulassel4,

*3Division of Clinical Immunology and Allergy, McGill University Health Centre, Montréal, Québec, Canada 4Immunodeficiency Service and Division of Hematology, Royal Victoria Hospital, Canada*

*8National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United*

*9AIDS Research Program, St. Paul's Hospital, University of British Columbia, Vancouver, BC, Canada 10Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Québec, Canada*

interaction with dendritic cells (DCs)(Sanabria et al. 2008; Smyth et al. 2005).

Jean-Pierre Routy1,4, Réjean Thomas5, Pierre Côté6, Colin Kovacs7, Stephen A. Migueles8,

Mark Connors8, Martin Potter1, Marianne Harris9 and Cecile L. Tremblay10 *1Research Institute of the McGill University Health Centre, Montréal, Québec, Canada 2Division of Experimental Medicine, McGill University, Montréal, Québec, Canada*

*McGill University Health Center, Montreal, Quebec, Canada*

*5Clinique L'Actuel, Montréal, Québec, Canada 6Clinique Quartier Latin, Montréal, Québec, Canada 7Maple Leaf Medical Clinic, Toronto, Canada*

**1. Introduction**

*States of America*

 \* *10Centre de Recherche du Centre Hospitalier de l'Université de Montréal*

### **Natural Killer Cells from HIV Infected Slow Progressors Who Carry the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated Poly-Functional Potential Compared to Bw6 Homozygotes**

#### Nicole F. Bernard1,2,3,10 et al.\*

*for the Canadian Cohort of HIV Infected Slow Progressors 1Research Institute of the McGill University Health Centre 2Division of Experimental Medicine, McGill University, Clinique du Quartier 3Division of Clinical Immunology and Allergy, McGill University Health Centre, Toronto, Ontario 10Centre de Recherche du Centre Hospitalier de l'Université de Montréal Montréal, Québec, Canada*

#### **1. Introduction**

HIV Infection in the Era of Highly Active 192 Antiretroviral Treatment and Some of Its Associated Complications

Soares, LF., de Araújo Castro, GFB, de Souza IPR & Pinheiro M. (2004). Pediatric HIV-

Sowole CA, Orenuga OO, Chikte SA (2009). Access to oral health care and treatment needs

Sullivan (2003). Prevention of Mother – to- Child Transmission of HIV—What Next? J *AIDS*

Swendeman D, Ingram BL, Rotheram-Borus MJ. (2009) Common elements in self-

Tofsky N, Nelson EM, Lopez RN, Catalanotto FA, Fine DH, Katz RV. Dental Caries in HIV-

Ukpebor M, Braimoh OB. (2007). HIV/AIDS; Oral Complications and Challenges, The Nigerian Experience. *Benin Journal of Postgraduate Medicine* 2007; 9(1):44-54 UNAIDS Joint United Nations Programme on HIV/AIDS (UNAIDS). *Report on the Global*

UNAIDS Joint United Nations Programme on HIV/AIDS (UNAIDS). *Report on the Global*

UNAIDS. *AIDS* (2006). *Epidemic Update - Special report on HIV/AIDS - December 2006.* Geneva:

Velegraki A, Nicolatou O, Theodoridou A, et al. Paediatric AIDS – Related Linear Gingival

William DM (1993). In: EC – Clearing House on Oral Problems Related to HIV Infection of

World Health Organization. (2003) Global Health-Sector Strategy for HIV/AIDS 2003-2007.

World Health Organization (2003) *The World Health Report,* Shaping the Future. Geneva:

Working Group on Mother To Child Transmission of HIV. Rates of mother- to- child

Wright AA. (2003) A Study of Oral Manifestations of HIV/AIDS and Response to

Yengopal V, Bhayat A, Coogan M. (2011). Pediatric Oral HIV Research in the Developing

Zijenah LS, Katzenstein DA (2002). Immunopathogenesis of AIDS In: *AIDS in Africa*., Essex

Erythema: A Form Erythematous of Candidiasis? *J Oral Pathol Med* 1999; 28: 178 -

the Immunodeficiency Virus: Classification and Diagnostic Criteria for Oral

transmission of HIV-1 in Africa, America and Europe: Results from 13 perinatal studies. *Journal of Acquired Immune Deficiency Syndrome and Human Retrovirology*

Management at the General Hospital Lagos, Nigeria (Dissertation) Lagos, Nigeria:

M, Mboup S, Phyllis J, Richard K, Marlick G, Tiou SD. Pp 35,Khuser Academic

18(1):

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2003; 34: S67-72.

UNAIDS; 2006.

Geneva: *WHO,* 2003.

WHO, 2003

1995; 8: 506-510

182.

*Care*. 2009; 21(10):1321-34.

*Clinic.* 2009 9(2): 141-146, maio/ago

*AIDS Epidemic* (2002). Geneva: *UNAIDS*, 2002

*AIDS Epidemic* (2004). Geneva: *UNAIDS*, 2004. UNAIDS/WHO (2005) *Report on the Global AIDS Epidemic* 2005. Geneva

UNAIDS (2009) *Report on the Global AIDS Epidemic Update* 2009. Geneva

Lesions in HIV Infection. *J Oral Pathol Med* 1993; 22: 289 - 291.

National Postgraduate Medical College of Nigeria, 2003;48

World. *Adv Dent Res.* 2011; 23(1): 61-6.

/Plenum Publishers, New York.

related oral manifestations – a five-year retrospective study. *Braz. Oral Res*, 2004;

of HIV positive paediatric patients *Brazilian Oral Research and Integrated Pediatric*

management of HIV and other chronic illnesses: an integrative framework. *AIDS*

Infected Children versus Household Peers: Two-Year Findings. *Pediatr Dent* 2000;

NK cells are a key component of the innate immune system, which can act early in defences against virally infected and tumor cells (Robertson and Ritz 1990; Bottino, Moretta, and Moretta 2006; Bancroft 1993). They have the capacity to secrete proinflammatory cytokines and lyse their targets without prior antigen sensitization (Cooper, Fehniger, and Caligiuri 2001). They are also involved in regulation of the adaptive immune response through their interaction with dendritic cells (DCs)(Sanabria et al. 2008; Smyth et al. 2005).

Mark Connors8, Martin Potter1, Marianne Harris9 and Cecile L. Tremblay10

<sup>\*</sup> Carlos Melendez-Pena1,2, Philomena Kamya1,2, Christos M. Tsoukas1,2,3, Mohamed-Rachid Boulassel4, Jean-Pierre Routy1,4, Réjean Thomas5, Pierre Côté6, Colin Kovacs7, Stephen A. Migueles8,

*<sup>1</sup>Research Institute of the McGill University Health Centre, Montréal, Québec, Canada 2Division of Experimental Medicine, McGill University, Montréal, Québec, Canada*

*<sup>3</sup>Division of Clinical Immunology and Allergy, McGill University Health Centre, Montréal, Québec, Canada 4Immunodeficiency Service and Division of Hematology, Royal Victoria Hospital, Canada*

*McGill University Health Center, Montreal, Quebec, Canada*

*<sup>5</sup>Clinique L'Actuel, Montréal, Québec, Canada*

*<sup>6</sup>Clinique Quartier Latin, Montréal, Québec, Canada*

*<sup>7</sup>Maple Leaf Medical Clinic, Toronto, Canada*

*<sup>8</sup>National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America*

*<sup>9</sup>AIDS Research Program, St. Paul's Hospital, University of British Columbia, Vancouver, BC, Canada 10Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Québec, Canada*

Natural Killer Cells from HIV Infected Slow Progressors Who Carry

**2. Materials and methods**

to the ethical guidelines of the authors' institutions.

**2.1 Study population**

**2.2 MHC and KIR typing**

the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated... 195

example in humans of an MHC class I antigen with an NK education potency that is superior to that of most other HLA-Bw4 molecules. HLA-B\*27 is another allele considered to be protective in the context of HIV infection (Kaslow et al. 1996; Carrington and Bontrop 2002; Goulder, Edwards et al. 1997; Trachtenberg et al. 2003). Like HLA-B\*57, its protective effect is mediated at least in part through immune pressure exerted by CD8+ T cells (Schneidewind et al. 2009; Goulder, Phillips et al. 1997; den Uyl, van der Horst-Bruinsma, and van Agtmael 2004; Goulder et al. 2001). In this report we questioned whether HLA-B\*27, like HLA-B\*57, could also act as a ligand for 3DL1 NK receptors that was superior to other HLA-Bw4 alleles in terms of its ability to educate NK cells for functional potential. *HLA-B\*27* is found at a higher frequency among the approximately 5% of HIV infected individuals that we have classified as Slow Progressors (SP) compared with HIV infected individuals exhibiting a typical rate of disease progression or uninfected subjects (Carrington, Martin, and van Bergen 2008; Kaslow, Dorak, and Tang 2005). SP are defined by either exhibiting spontaneous control of viremia or maintaining CD4 counts >400 cells/mm3 for at least 7 years post-infection (Madec et al. 2005; Deeks and Walker 2007). In order to determine whether HLA-B\*27, like HLA-B\*57, could educate NK cells from *3DL1* hmz individuals for superior functional potential we compared the functional potential of NK cells from *3DL1 hmz+B\*27* (*3DL1+B\*27*) carriers with that from *3DL1 hmz* who were *Bw6 hmz* (*3DL1+Bw6*) or who expressed at least 1 *Bw4* alleles other than *B\*57* or *B\*27* (*3DL1+Bw4*). NK cells from *3DL1+B\*27* carriers had a significantly higher functional potential than those from *3DL1+Bw6.* When the functional potential of NK cells from carriers of the 3DL1 NK receptor /HLA-B ligand pairs \**h/\*y+B\*57*, *3DL1+B\*27* and *3DL1+Bw4* were compared we observed decreasing levels in the functional potential where NK cells responded to missing self with a *h/\*y+B\*57* > *3DL1+B\*27* > *3DL1+Bw4* hierarchy.

A total of 51 HIV-infected SP were studied. Forty four were from the Canadian Cohort of HIV Infected Slow Progressors and 7 were from a cohort followed at the National Institutes of Allergy and Infectious Diseases (NIAID) (Migueles et al. 2008). The term SP was used here to define treatment naïve HIV infected subjects who maintained absolute CD4 counts above 400 cells/mm3 for more than 7 years or who were followed for at least 1 year with VL <3000 copies/ml of plasma. Information on CD4, CD8 T cell counts, VL and duration of infection at time of testing, *3DL1* genotype and *HLA-B* allotype of the study population is provided in Table 1. All individuals in the study populations are *3DL1 hmz* to eliminate the possible confounding effect on NK function of expressing the activating 3DS1 receptor, which is an allele at this locus (Martin et al. 2007; Boulet et al. 2008; Yawata et al. 2006). The study population is classified into 4 groups: group 1 (n=12) carry the *3DL1+B\*27* genotype, group 2 (n=13) are *3DL1+Bw4*, group 3 (n=14) are *3DL1+Bw6* and group 4 (n=12) are *\*h/\*y+B\*57*. Informed consent was obtained from all study subjects and research conformed

All subjects were typed for MHC class I alleles by sequence-based typing using kits from Atria Genetics (South San Francisco, CA) and Assign software to interpret sequence information for allele typing (Conexio Genetics, Perth, Australia) as previously described

Activation of NK cells is regulated through the integration of signals from a number of activating and inhibitory receptors(Lanier 2005). Many of the inhibitory receptors use major histocompatibility complex (MHC) class I or class I-like proteins as their ligands(Lanier 2005). The interaction between inhibitory NK receptors and their ligands during NK cell development is important in educating these cells for subsequent function and for avoiding reactivity to normal cells expressing self MHC class I (Kim et al. 1969; Anfossi et al. 2006). In humans one large family of NK receptors are encoded by the Killer Immunoglobulin-like Receptors (KIR) region that maps to chromosome 19q13.4 (Lanier 2005). The most polymorphic locus among the KIR region genes is *KIR3DL1*, which encodes both inhibitory KIR3DL1 (3DL1) and activating KIR3DS1 (3DS1) alleles (Norman et al. 2007). *3DL1* alleles can be further classified according to their expression levels on the cell surface into high (*\*h*), low/intermediate (*\*l*) and null (*\*004*) (not cell surface expressed) alleles (Yawata et al. 2006; Norman et al. 2007; Gardiner et al. 2001; Pando et al. 2003). Genotypes homozygous for *3DL1* can be divided into 2 groups: *\*h/\*y*, where *\*y* can be either another *\*h* allele or *\*004* with no *\*l* alleles, and *\*l/\*x*, where *\*x* can be an *\*l*, *\*x* or *\*004* allele (Martin et al. 2007). 3DL1 receptors recognize a subset of MHC class I HLA-B molecules known as Bw4. HLA-Bw4 differ from the remaining HLA-Bw6 antigens encoded at this locus, which do not interact with 3DL1, in the amino acids present between positions 77 and 83 of the HLA heavy chain (Wan et al. 1986). Bw4 allotypes with isoleucine at position 80 (Bw4\*80I) have been reported to be better ligands for many of the 3DL1 alleles (Cella et al. 1994). However, there is evidence that Bw4 antigens with threonine at position 80 (Bw4\*80T), particularly HLA-B\*2705, interact strongly with certain 3DL1 receptors (Luque et al. 1996).

Epidemiological studies have reported that several *KIR/HLA* combinations are associated with slower progression to AIDS and suppression of viral load (VL) (Martin et al. 2007). Compared to *Bw6* homozygotes (hmz) the *3DL1/HLA-B* combination having the most potent influence on slowing time to AIDS and VL control is *3DL1\*h/\*y* with *HLA-B\*57* (*\*h/\*y*+*B\*57*)(Martin et al. 2007). Previous work from our group showed that NK cells from individuals carrying this genotype combination demonstrated higher functional potential than those from carriers of either the NK receptor genotype or *HLA-B\*57* alone or from *Bw6 hmz* (Boulet et al. 2010). In these studies functional potential was defined as the percent contribution of NK cells secreting interferon-γ (IFNγ) and tumor necrosis factor-α (TNF-α) and expressing CD107a, a marker of degranulation to the total response to stimulation with the HLA devoid K562 cell line. Furthermore, NK cells from carriers of *\*h/\*y*+*B\*57* had higher functional potential than carriers of *3DL1\*h/\*y* with other *Bw4* alleles(Boulet et al. 2010). HLA-B\*57 is an HLA antigen considered to be protective in the context of HIV infection(Kaslow, Dorak, and Tang 2005; Altfeld et al. 2003; Leslie et al. 2004; Carrington, Martin, and van Bergen 2008). While the protective effect conferred by HLA-B\*57 is mediated at least in part through CD8+ T cell recognition of HIV epitopes restricted by this antigen, epidemiological studies and our results support the possibility that HLA-B\*57's protective effect may also be mediated through its ability to educate NK cells for superior functional potential (Martin et al. 2002; Martin et al. 2007; Leslie et al. 2004; Miura et al. 2009; Leslie et al. 2005).

Murine models have shown using single MHC class I transgenic mice that MHC class I molecules can differ from each other in the potency of their NK education signals, which directly translates into activation potency upon encountering cells lacking that MHC ligand (Brodin, Karre, and Hoglund 2009). Since NK cells from *\*h/\*y+B\*57* carriers had higher functional potential than carriers of *3DL1\*h/\*y* with other *Bw4* alleles, HLA-B\*57 may be an example in humans of an MHC class I antigen with an NK education potency that is superior to that of most other HLA-Bw4 molecules. HLA-B\*27 is another allele considered to be protective in the context of HIV infection (Kaslow et al. 1996; Carrington and Bontrop 2002; Goulder, Edwards et al. 1997; Trachtenberg et al. 2003). Like HLA-B\*57, its protective effect is mediated at least in part through immune pressure exerted by CD8+ T cells (Schneidewind et al. 2009; Goulder, Phillips et al. 1997; den Uyl, van der Horst-Bruinsma, and van Agtmael 2004; Goulder et al. 2001). In this report we questioned whether HLA-B\*27, like HLA-B\*57, could also act as a ligand for 3DL1 NK receptors that was superior to other HLA-Bw4 alleles in terms of its ability to educate NK cells for functional potential. *HLA-B\*27* is found at a higher frequency among the approximately 5% of HIV infected individuals that we have classified as Slow Progressors (SP) compared with HIV infected individuals exhibiting a typical rate of disease progression or uninfected subjects (Carrington, Martin, and van Bergen 2008; Kaslow, Dorak, and Tang 2005). SP are defined by either exhibiting spontaneous control of viremia or maintaining CD4 counts >400 cells/mm3 for at least 7 years post-infection (Madec et al. 2005; Deeks and Walker 2007). In order to determine whether HLA-B\*27, like HLA-B\*57, could educate NK cells from *3DL1* hmz individuals for superior functional potential we compared the functional potential of NK cells from *3DL1 hmz+B\*27* (*3DL1+B\*27*) carriers with that from *3DL1 hmz* who were *Bw6 hmz* (*3DL1+Bw6*) or who expressed at least 1 *Bw4* alleles other than *B\*57* or *B\*27* (*3DL1+Bw4*). NK cells from *3DL1+B\*27* carriers had a significantly higher functional potential than those from *3DL1+Bw6.* When the functional potential of NK cells from carriers of the 3DL1 NK receptor /HLA-B ligand pairs \**h/\*y+B\*57*, *3DL1+B\*27* and *3DL1+Bw4* were compared we observed decreasing levels in the functional potential where NK cells responded to missing self with a *h/\*y+B\*57* > *3DL1+B\*27* > *3DL1+Bw4* hierarchy.

#### **2. Materials and methods**

#### **2.1 Study population**

HIV Infection in the Era of Highly Active 194 Antiretroviral Treatment and Some of Its Associated Complications

Activation of NK cells is regulated through the integration of signals from a number of activating and inhibitory receptors(Lanier 2005). Many of the inhibitory receptors use major histocompatibility complex (MHC) class I or class I-like proteins as their ligands(Lanier 2005). The interaction between inhibitory NK receptors and their ligands during NK cell development is important in educating these cells for subsequent function and for avoiding reactivity to normal cells expressing self MHC class I (Kim et al. 1969; Anfossi et al. 2006). In humans one large family of NK receptors are encoded by the Killer Immunoglobulin-like Receptors (KIR) region that maps to chromosome 19q13.4 (Lanier 2005). The most polymorphic locus among the KIR region genes is *KIR3DL1*, which encodes both inhibitory KIR3DL1 (3DL1) and activating KIR3DS1 (3DS1) alleles (Norman et al. 2007). *3DL1* alleles can be further classified according to their expression levels on the cell surface into high (*\*h*), low/intermediate (*\*l*) and null (*\*004*) (not cell surface expressed) alleles (Yawata et al. 2006; Norman et al. 2007; Gardiner et al. 2001; Pando et al. 2003). Genotypes homozygous for *3DL1* can be divided into 2 groups: *\*h/\*y*, where *\*y* can be either another *\*h* allele or *\*004* with no *\*l* alleles, and *\*l/\*x*, where *\*x* can be an *\*l*, *\*x* or *\*004* allele (Martin et al. 2007). 3DL1 receptors recognize a subset of MHC class I HLA-B molecules known as Bw4. HLA-Bw4 differ from the remaining HLA-Bw6 antigens encoded at this locus, which do not interact with 3DL1, in the amino acids present between positions 77 and 83 of the HLA heavy chain (Wan et al. 1986). Bw4 allotypes with isoleucine at position 80 (Bw4\*80I) have been reported to be better ligands for many of the 3DL1 alleles (Cella et al. 1994). However, there is evidence that Bw4 antigens with threonine at position 80 (Bw4\*80T), particularly HLA-

B\*2705, interact strongly with certain 3DL1 receptors (Luque et al. 1996).

Leslie et al. 2005).

Epidemiological studies have reported that several *KIR/HLA* combinations are associated with slower progression to AIDS and suppression of viral load (VL) (Martin et al. 2007). Compared to *Bw6* homozygotes (hmz) the *3DL1/HLA-B* combination having the most potent influence on slowing time to AIDS and VL control is *3DL1\*h/\*y* with *HLA-B\*57* (*\*h/\*y*+*B\*57*)(Martin et al. 2007). Previous work from our group showed that NK cells from individuals carrying this genotype combination demonstrated higher functional potential than those from carriers of either the NK receptor genotype or *HLA-B\*57* alone or from *Bw6 hmz* (Boulet et al. 2010). In these studies functional potential was defined as the percent contribution of NK cells secreting interferon-γ (IFNγ) and tumor necrosis factor-α (TNF-α) and expressing CD107a, a marker of degranulation to the total response to stimulation with the HLA devoid K562 cell line. Furthermore, NK cells from carriers of *\*h/\*y*+*B\*57* had higher functional potential than carriers of *3DL1\*h/\*y* with other *Bw4* alleles(Boulet et al. 2010). HLA-B\*57 is an HLA antigen considered to be protective in the context of HIV infection(Kaslow, Dorak, and Tang 2005; Altfeld et al. 2003; Leslie et al. 2004; Carrington, Martin, and van Bergen 2008). While the protective effect conferred by HLA-B\*57 is mediated at least in part through CD8+ T cell recognition of HIV epitopes restricted by this antigen, epidemiological studies and our results support the possibility that HLA-B\*57's protective effect may also be mediated through its ability to educate NK cells for superior functional potential (Martin et al. 2002; Martin et al. 2007; Leslie et al. 2004; Miura et al. 2009;

Murine models have shown using single MHC class I transgenic mice that MHC class I molecules can differ from each other in the potency of their NK education signals, which directly translates into activation potency upon encountering cells lacking that MHC ligand (Brodin, Karre, and Hoglund 2009). Since NK cells from *\*h/\*y+B\*57* carriers had higher functional potential than carriers of *3DL1\*h/\*y* with other *Bw4* alleles, HLA-B\*57 may be an A total of 51 HIV-infected SP were studied. Forty four were from the Canadian Cohort of HIV Infected Slow Progressors and 7 were from a cohort followed at the National Institutes of Allergy and Infectious Diseases (NIAID) (Migueles et al. 2008). The term SP was used here to define treatment naïve HIV infected subjects who maintained absolute CD4 counts above 400 cells/mm3 for more than 7 years or who were followed for at least 1 year with VL <3000 copies/ml of plasma. Information on CD4, CD8 T cell counts, VL and duration of infection at time of testing, *3DL1* genotype and *HLA-B* allotype of the study population is provided in Table 1. All individuals in the study populations are *3DL1 hmz* to eliminate the possible confounding effect on NK function of expressing the activating 3DS1 receptor, which is an allele at this locus (Martin et al. 2007; Boulet et al. 2008; Yawata et al. 2006). The study population is classified into 4 groups: group 1 (n=12) carry the *3DL1+B\*27* genotype, group 2 (n=13) are *3DL1+Bw4*, group 3 (n=14) are *3DL1+Bw6* and group 4 (n=12) are *\*h/\*y+B\*57*. Informed consent was obtained from all study subjects and research conformed to the ethical guidelines of the authors' institutions.

#### **2.2 MHC and KIR typing**

All subjects were typed for MHC class I alleles by sequence-based typing using kits from Atria Genetics (South San Francisco, CA) and Assign software to interpret sequence information for allele typing (Conexio Genetics, Perth, Australia) as previously described

Natural Killer Cells from HIV Infected Slow Progressors Who Carry

**Subjects**

**2.3 Cells**

**2.4 NK cell functional potential**

**ID gender1 Age2 Time**

the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated... 197

**infected2 Group3** *HLA-B1 HLA-B2 KIR*

for KIR3DL1 alleles, 5 cells/mm3, 6 VL= log10 viral load copies/ml plasma.

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque Pharmacia Upsala, Sweden) from whole blood obtained by venipuncture into tubes containing EDTA anti-coagulant or by leukaphersis as previously described (Boulassel et al. 2003). Cells were cryopreserved in 10% DMSO (Sigma-Aldrich, St.

Cryopreserved PBMC were thawed and resuspended at 106 cells/ml in RPMI 1640 (Wisent) that contained 10% FBS (Wisent), 2mM L-glutamine and 50 IU penicillin and 50µg/ml streptomycin (Wisent). Brefeldin A (at 5µg/ml, Sigma-Aldrich), Monensin (at 6µg/ml, Golgi-Stop; BD Biosciences, Mississauga, Ontario, Canada) and anti-CD107a–FITC mAb (BD Biosciences) were added to the cells. One million PBMCs were stimulated with media alone or HLA devoid K562 cells (American Type Culture Collection Manassas, VA) at a PBMC to K562 cell ratio of 5:1 or with 1.25µg/ml phorbol 12-myristate 13-acetate (PMA); 0.25µg/ml ionomycin, (Sigma-Aldrich) as a positive control for 6 hours at 37oC in a humidified 5% CO2

Louis, MO) with 90% fetal bovine serum (FBS, Wisent, St. Bruno, Quebec, Canada).

3008 M 53 4.1 3 B07:02 B14:02 \*h/\*y 447 455 3.3 3009 M 29 4.2 3 B07:02 B14:02 3DL1 hmz 516 788 2.89 3010 F 61 14.3 3 B07:02 B18:01 \*l/\*x 810 1050 1.7 3011 F 39 6.6 3 B42:01 B45:01 \*h/\*y 620 408 2.69 3012 M 39 9.9 3 B14:02 B40:06 \*h/\*y 720 1820 2.59 3013 M 46 7.0 3 B07:02 B07:05 \*h/\*y 970 1210 1.94 3014 M 62 17.5 3 B14:01 B81:01 \*h/\*y 650 2110 4.46 4001 F 39 3.0 4 B57 B7 \*h/\*y 1443 895 1.7 4002 F 55 23.8 4 B57 B57 \*h/\*y 277 385 1.7 4003 M 58 20.5 4 B57 B15 \*h/\*y 883 590 1.7 4004 M 41 11.2 4 B40:01 B57:01 \*h/\*y 1200 860 1.7 4005 M 61 11.0 4 B40:02 B57:01 \*h/\*y 770 990 3.9 4006 M 45 14.9 4 B07:02 B57:01 \*h/\*y 650 1460 4.21 4007 F 39 2.6 4 B35:01 B57:01 \*h/\*y 530 NA 1.7 4008 M 24 2.0 4 B07:02 B57:01 \*h/\*y 680 880 3.1 4009 M 49 10.0 4 B57 B13 \*h/\*y 955 881 1.7 4010 M 58 18.0 4 B57 B44 \*h/\*y 1329 1243 1.7 4011 M 46 21.0 4 B57 B81 \*h/\*y 1362 1055 1.7 4012 M 36 7.0 4 B57 B52 \*h/\*y 780 739 1.7 Table 1. Study population characteristics. 1 M=male/ F=female, 2 in years, 3 1= 3DL1+B\*27; 2=3DL1+Bw4; 3=3DL1+Bw4; 4=\*h/\*y+B\*57. The 2 subjects with the 1a designation carry both a B\*27 and B\*57 allele, 4 the individual classified as 3DL1 hmz has not been allotyped

**genotype4 CD45 CD85 VL6**

(Boulet et al. 2010). *HLA-Bw6 hmz* subjects lacked any *HLA-Bw4* alleles at the *HLA-A* or *B*  locus. *3DL1/S1* genotyping was performed using two sets of primers specific for the *3DL1* and *3DS1* alleles at the 3DL1 locus as previously described (Boulet et al. 2008)*.* Subjects were subsequently *3DL1* allotyped by identifying single nucleotide polymorphisms (SNP) corresponding to high frequency *3DL1* alleles as previously described (Boulet et al. 2010). In our study we categorized *3DL1\*005, \*006, \*007, \*053, \*054* as *\*l* alleles, *3DL1\*001, \*002, \*008, \*009, \*015, \*020* as *\*h* alleles and *\*004* as a null allele.

Table 1. Study population characteristics. 1 M=male/ F=female, 2 in years, 3 1= 3DL1+B\*27; 2=3DL1+Bw4; 3=3DL1+Bw4; 4=\*h/\*y+B\*57. The 2 subjects with the 1a designation carry both a B\*27 and B\*57 allele, 4 the individual classified as 3DL1 hmz has not been allotyped for KIR3DL1 alleles, 5 cells/mm3, 6 VL= log10 viral load copies/ml plasma.

#### **2.3 Cells**

HIV Infection in the Era of Highly Active 196 Antiretroviral Treatment and Some of Its Associated Complications

(Boulet et al. 2010). *HLA-Bw6 hmz* subjects lacked any *HLA-Bw4* alleles at the *HLA-A* or *B*  locus. *3DL1/S1* genotyping was performed using two sets of primers specific for the *3DL1* and *3DS1* alleles at the 3DL1 locus as previously described (Boulet et al. 2008)*.* Subjects were subsequently *3DL1* allotyped by identifying single nucleotide polymorphisms (SNP) corresponding to high frequency *3DL1* alleles as previously described (Boulet et al. 2010). In our study we categorized *3DL1\*005, \*006, \*007, \*053, \*054* as *\*l* alleles, *3DL1\*001, \*002, \*008,*

**infected2 Group3** *HLA-B1 HLA-B2 KIR*

1001 M 59 2.9 1 B15:01 B27:05 \*h/\*y 760 900 1.7 1002 M 59 14.6 1 B15:01 B27:05 \*h/\*y 788 2498 4.12 1003 M 61 23.5 1 B27:02 B67:01 \*h/\*y 670 1050 4.7 1004 M 49 20.2 1a B27:05 B57:01 \*h/\*y 830 1700 3.91 1005 M 37 2.5 1 B27:05 B40:02 \*h/\*y 1040 1090 1.7 1006 M 40 3.7 1 B14:02 B27:05 \*h/\*y 820 570 1.7 1007 M 71 5.2 1 B27:03 B51:01 \*l/\*x 590 114 1.7 1008 F 31 1.2 1a B27:05 B57:01 \*l/\*x 489 672 1.7 1009 F 59 13.2 1 B27:03 B49:01 \*l/\*x 692 627 1.7 1010 M 38 2.3 1 B15:01 B27:05 \*l/\*x 396 936 1.7 1011 M 63 25.0 1 B27:05 B51:01 \*h/\*y 340 748 1.7 1012 M 41 6.4 1 B07:02 B27:05 \*l/\*x 710 970 3.25 2001 M 35 6.5 2 B15:02 B51:02 \*l/\*x 500 NA 3.24 2002 F 39 15.9 2 B49:01 B58:02 \*h/\*y 400 510 3.54 2003 M 15 8.0 2 B38:01 B51:01 \*h/\*y 596 1614 2.08 2004 M 45 16.2 2 B07:02 B51:01 \*h/\*y 440 350 3.54 2005 F 46 14.7 2 B07:02 B38:01 \*l/\*x 720 720 1.7 2006 M 47 17.0 2 B39:01 B35:01 \*h/\*y 546 572 5.21 2007 M 46 1 2 B53:01 B58:01 \*l/\*x 870 550 1.7 2008 M 49 7.3 2 B44:03 B53:01 \*l/\*x 360 1610 2.8 2009 F 40 2.8 2 B07:02 B13:01 \*h/\*y 1487 712 3.76 2010 F 50 6.9 2 B15:03 B44:02 \*h/\*y 590 1650 3.73 2011 F 44 22.2 2 B44:02 B52:02 \*h/\*y 420 1180 3.15 2012 F 32 5.1 2 B14:02 B44:03 \*l/\*x 715 384 1.7 2013 F 36 6.3 2 B49:01 B53:01 \*h/\*y 535 793 2.43 3001 M 34 3.0 3 B07:02 B14:02 \*l/\*x 680 890 2.85 3002 M 40 14.4 3 B14:02 B15:01 \*h/\*y 343 804 1.7 3003 M 40 4.7 3 B14:02 B14:02 \*h/\*y 350 730 3.41 3004 M 40 3.4 3 B07:02 B07:02 \*h/\*y 985 605 3.3 3005 M 46 5.6 3 B15:10 B41:01 \*l/\*x 530 390 1.7 3006 F 42 4.0 3 B07:05 B35:01 \*l/\*x 919 1557 1.7 3007 F 57 3.1 3 B07:02 B42:01 \*l/\*x 636 663 1.7

**genotype4 CD45 CD85 VL6**

*\*009, \*015, \*020* as *\*h* alleles and *\*004* as a null allele.

**ID gender1 Age2 Time**

**Subjects**

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque Pharmacia Upsala, Sweden) from whole blood obtained by venipuncture into tubes containing EDTA anti-coagulant or by leukaphersis as previously described (Boulassel et al. 2003). Cells were cryopreserved in 10% DMSO (Sigma-Aldrich, St. Louis, MO) with 90% fetal bovine serum (FBS, Wisent, St. Bruno, Quebec, Canada).

#### **2.4 NK cell functional potential**

Cryopreserved PBMC were thawed and resuspended at 106 cells/ml in RPMI 1640 (Wisent) that contained 10% FBS (Wisent), 2mM L-glutamine and 50 IU penicillin and 50µg/ml streptomycin (Wisent). Brefeldin A (at 5µg/ml, Sigma-Aldrich), Monensin (at 6µg/ml, Golgi-Stop; BD Biosciences, Mississauga, Ontario, Canada) and anti-CD107a–FITC mAb (BD Biosciences) were added to the cells. One million PBMCs were stimulated with media alone or HLA devoid K562 cells (American Type Culture Collection Manassas, VA) at a PBMC to K562 cell ratio of 5:1 or with 1.25µg/ml phorbol 12-myristate 13-acetate (PMA); 0.25µg/ml ionomycin, (Sigma-Aldrich) as a positive control for 6 hours at 37oC in a humidified 5% CO2

Natural Killer Cells from HIV Infected Slow Progressors Who Carry

were from *3DL1*+*B\*27* individuals than from *3DL1+Bw6*.

Mann-Whitney *U* test) (**Figure 2A**).

concentrated on the tri-functional NK subset in subsequent analyses.

standard deviation.

**from** *3DL1+Bw6*

*3DL1+B\*27* **(n=12)**

the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated... 199

**Duration of infection1** 10.1 ± 8.9 10.0 ± 6.5 7.2 ± 4.8 12.1 ± 7.6

Table 2. Summary of study population characteristics. I In years, 2 Cells/mm3, 3 Means ±

**3.2 NK cells from** *3DL1+B\*27* **carriers have a higher functional potential than those**

To investigate whether NK cells from HIV-infected SPs carrying the *3DL1+B\*27* genotype for an NK receptor/HLA-B ligand pair differ in their NK cell functional potential compared to *Bw6 hmz* with no ligand for 3DL1 receptors, we measured the frequency of NK cells expressing CD107a and secreting IFN- and TNF- from K562-stimulated NK cells using eight colour multi-parametric flow cytometry. In this analysis CD107a expressing NK cells were the sum of the mono-, bi- and tri-functional functional subsets expressing CD107a. This was also the case for IFN-γ and TNF-α secreting NK cells. As shown in Figure 1A the median (range) frequency of NK cells from *3DL1*+*B\*27* versus *3DL1+Bw6* carriers expressing CD107a was 6.92% (3.01%, 17.15%) versus 4.27% (0.74%, 13.32%), secreting IFN-γ was 8.91% (4.25%, 21.52%) versus 5.17% (0.55%, 25.92%) and secreting TNF-α was 0.92% (0.11%, 3.67%) versus 0.23% (0.01%, 3.06) (p<0.05 for all comparisons; Mann-Whitney test). Therefore, the frequency of NK cells with any of these functions was significantly greater when NK cells

Since we simultaneously measured these three functions following K562 stimulation we were able to assess the frequency of seven possible NK cell functional profiles and their percent contribution to the total K562 response. Figure 1B shows the percent contribution of each NK cell functional profile in PBMC from 12 *3DL1+B\*27* and 14 *3DL1+Bw6* subjects. Of the seven possible NK cell functional profiles, only the percent contribution of tri-functional NK cells to the total K562 response was significantly higher in the *3DL1+B\*27* group versus *3DL1+Bw6* SPs (2.99% [0.00%, 6.43%]) and (0.52% [0.00%, 7.36%]) for 3DL1*+B\*27* and *3DL1+Bw6*, respectively, p=0.019; Mann-Whitney *U* test). Based on these results we

We next compared the K562 stimulated NK cells from *3DL1*+*B\*27* individuals to that from carriers of *3DL1+Bw4*. NK cells from *\*h/\*y+B\*57* carriers have previously been shown to have superior tri-functional potential compared to *3DL1*+*Bw6* and \*h/\*y+*Bw4* subjects (Boulet et al. 2010; Kamya et al. 2011). Since *HLA-B\*57* is a *Bw4* allele we excluded carriers of either *HLA-B\*57* or *HLA-B\*27* alleles from the *3DL1+Bw4* group. Although the tri-functional potential of NK cells from 13 *3DL1+Bw4* subjects was lower (1.78% [0.00%, 5.71%]) than that from *3DL1+B\*27* individuals the difference did not achieve statistical significance (p=0.2644;

*3DL1+Bw4* **(n=13)**

**Age1** 50.7 ± 12.83 40.1 ± 9.5 44.9 ± 10.0 45.9 ± 11.0

**CD42** 677 ± 198 619 ± 306 655 ± 211 904 ± 363 **CD82** 989 ± 606 887 ± 494 963 ± 533 907 ± 292 **Log10 VL** 2.53 ± 1.2 2.95 ± 1.0 2.57 ± 0.86 2.21 ± 0.95

*3DL1+Bw6* **(n=14)**

*\*h/\*y+B\*57* **(n=12)**

incubator. All stimulation data shown is from cells that generated a positive result in the PMA and ionomycin stimulation condition.

Cells were stained for viability using the Aqua LIVE/DEAD® fixable dead cell stain kit (Invitrogen, Burlington, Ontario, Canada) following manufacturer's instructions. Cells were then stained for cell surface markers with anti-CD56-APC, anti-CD16-Pacific Blue (BD Biosciences), anti-CD3-ECD and CD158e-PE (ie: Z27-PE, Beckman Coulter, Mississauga, Ontario, Canada) for 30 min. After washing with phosphate buffered saline (PBS) containing 1% FBS (Wisent), cells were fixed and permeabilized using the Fix and Perm Kit (Invitrogen) and stained for intracellular cytokines using anti-IFN--Alexa 700 and anti-TNF--PE-Cy7 (BD Biosciences). Cells were washed and fixed with 1% paraformaldehyde solution (Fisher Scientific, Ottawa Ontario, Canada) and kept in the dark at 4oC until acquisition.

#### **2.5 Flow cytometry analysis**

Between 400,000 and 500,000 events were acquired per sample using an LSRII flow cytometer (BD Biosciences). Analysis for NK cell activation was performed using FlowJo software version 9.1 (Tree Star, San Carlos, CA). The functional profiles of stimulated NK cells were determined using a gating strategy where NK cells were defined as CD3-CD56+/- CD16+/-. Boolean gating was used to identify seven NK cell functional profiles, i.e. trifunctional NK cells (CD107a+ IFN-+ TNF-+), bi-functional NK cells (any combination of two of these functions) and mono-functional NK cells (any single one of these functions). All results for the frequency of individual functional subsets were background corrected by subtracting the frequency of positive NK cells in the unstimulated subset. Corrected results were used to generate the percent contribution of each functional subset to the total NK cells response to K562. Results reported as subset frequency or percent contribution of a subset to the total K562 response showed a high level of correlation with each other (Boulet et al. 2010).

#### **2.6 Statistical analysis**

GraphPad Instat 3.05 and GraphPad Prism 5.04 were used for statistical analyses and graphical presentations. A Kruskal-Wallis test was used to assess the significance of between group differences in age, duration of infection at the time point tested, CD4 counts, CD8 counts and VL. Mann-Whitney *U* tests were used to test the significance of between group differences in the percent contribution of an NK cell functional subset to the total NK cell response. A Spearman correlation test was used to test the significance of the trend towards declining tri-functional potential with 3DL1/HLA-B genotype combinations. A *p* value of <0.05 was considered significant.

#### **3. Results**

#### **3.1 Study population**

Table 2 provides information on the average and standard deviation for age, duration of infection, CD4 count, CD8 count and log10VL at the time point tested for NK functional potential for SP classified as *3DL1+B\*27*, *3DL1+Bw4*, *3DL1+Bw6* and *\*h/\*y+B\*57* described in Table 1. No between-group differences were seen for any of these parameters (Krukal-Wallis test).

HIV Infection in the Era of Highly Active 198 Antiretroviral Treatment and Some of Its Associated Complications

incubator. All stimulation data shown is from cells that generated a positive result in the

Cells were stained for viability using the Aqua LIVE/DEAD® fixable dead cell stain kit (Invitrogen, Burlington, Ontario, Canada) following manufacturer's instructions. Cells were then stained for cell surface markers with anti-CD56-APC, anti-CD16-Pacific Blue (BD Biosciences), anti-CD3-ECD and CD158e-PE (ie: Z27-PE, Beckman Coulter, Mississauga, Ontario, Canada) for 30 min. After washing with phosphate buffered saline (PBS) containing 1% FBS (Wisent), cells were fixed and permeabilized using the Fix and Perm Kit (Invitrogen) and stained for intracellular cytokines using anti-IFN--Alexa 700 and anti-TNF--PE-Cy7 (BD Biosciences). Cells were washed and fixed with 1% paraformaldehyde solution (Fisher Scientific, Ottawa Ontario, Canada) and kept in the dark at 4oC until

Between 400,000 and 500,000 events were acquired per sample using an LSRII flow cytometer (BD Biosciences). Analysis for NK cell activation was performed using FlowJo software version 9.1 (Tree Star, San Carlos, CA). The functional profiles of stimulated NK

CD16+/-. Boolean gating was used to identify seven NK cell functional profiles, i.e. trifunctional NK cells (CD107a+ IFN-+ TNF-+), bi-functional NK cells (any combination of two of these functions) and mono-functional NK cells (any single one of these functions). All results for the frequency of individual functional subsets were background corrected by subtracting the frequency of positive NK cells in the unstimulated subset. Corrected results were used to generate the percent contribution of each functional subset to the total NK cells response to K562. Results reported as subset frequency or percent contribution of a subset to the total K562 response showed a high level of correlation with each other (Boulet et al.

GraphPad Instat 3.05 and GraphPad Prism 5.04 were used for statistical analyses and graphical presentations. A Kruskal-Wallis test was used to assess the significance of between group differences in age, duration of infection at the time point tested, CD4 counts, CD8 counts and VL. Mann-Whitney *U* tests were used to test the significance of between group differences in the percent contribution of an NK cell functional subset to the total NK cell response. A Spearman correlation test was used to test the significance of the trend towards declining tri-functional potential with 3DL1/HLA-B genotype combinations. A *p*

Table 2 provides information on the average and standard deviation for age, duration of infection, CD4 count, CD8 count and log10VL at the time point tested for NK functional potential for SP classified as *3DL1+B\*27*, *3DL1+Bw4*, *3DL1+Bw6* and *\*h/\*y+B\*57* described in Table 1. No between-group differences were seen for any of these parameters (Krukal-Wallis

CD56+/-

cells were determined using a gating strategy where NK cells were defined as CD3-

PMA and ionomycin stimulation condition.

acquisition.

2010).

**3. Results**

test).

**3.1 Study population**

**2.6 Statistical analysis**

value of <0.05 was considered significant.

**2.5 Flow cytometry analysis**

Table 2. Summary of study population characteristics. I In years, 2 Cells/mm3, 3 Means ± standard deviation.

#### **3.2 NK cells from** *3DL1+B\*27* **carriers have a higher functional potential than those from** *3DL1+Bw6*

To investigate whether NK cells from HIV-infected SPs carrying the *3DL1+B\*27* genotype for an NK receptor/HLA-B ligand pair differ in their NK cell functional potential compared to *Bw6 hmz* with no ligand for 3DL1 receptors, we measured the frequency of NK cells expressing CD107a and secreting IFN- and TNF- from K562-stimulated NK cells using eight colour multi-parametric flow cytometry. In this analysis CD107a expressing NK cells were the sum of the mono-, bi- and tri-functional functional subsets expressing CD107a. This was also the case for IFN-γ and TNF-α secreting NK cells. As shown in Figure 1A the median (range) frequency of NK cells from *3DL1*+*B\*27* versus *3DL1+Bw6* carriers expressing CD107a was 6.92% (3.01%, 17.15%) versus 4.27% (0.74%, 13.32%), secreting IFN-γ was 8.91% (4.25%, 21.52%) versus 5.17% (0.55%, 25.92%) and secreting TNF-α was 0.92% (0.11%, 3.67%) versus 0.23% (0.01%, 3.06) (p<0.05 for all comparisons; Mann-Whitney test). Therefore, the frequency of NK cells with any of these functions was significantly greater when NK cells were from *3DL1*+*B\*27* individuals than from *3DL1+Bw6*.

Since we simultaneously measured these three functions following K562 stimulation we were able to assess the frequency of seven possible NK cell functional profiles and their percent contribution to the total K562 response. Figure 1B shows the percent contribution of each NK cell functional profile in PBMC from 12 *3DL1+B\*27* and 14 *3DL1+Bw6* subjects. Of the seven possible NK cell functional profiles, only the percent contribution of tri-functional NK cells to the total K562 response was significantly higher in the *3DL1+B\*27* group versus *3DL1+Bw6* SPs (2.99% [0.00%, 6.43%]) and (0.52% [0.00%, 7.36%]) for 3DL1*+B\*27* and *3DL1+Bw6*, respectively, p=0.019; Mann-Whitney *U* test). Based on these results we concentrated on the tri-functional NK subset in subsequent analyses.

We next compared the K562 stimulated NK cells from *3DL1*+*B\*27* individuals to that from carriers of *3DL1+Bw4*. NK cells from *\*h/\*y+B\*57* carriers have previously been shown to have superior tri-functional potential compared to *3DL1*+*Bw6* and \*h/\*y+*Bw4* subjects (Boulet et al. 2010; Kamya et al. 2011). Since *HLA-B\*57* is a *Bw4* allele we excluded carriers of either *HLA-B\*57* or *HLA-B\*27* alleles from the *3DL1+Bw4* group. Although the tri-functional potential of NK cells from 13 *3DL1+Bw4* subjects was lower (1.78% [0.00%, 5.71%]) than that from *3DL1+B\*27* individuals the difference did not achieve statistical significance (p=0.2644; Mann-Whitney *U* test) (**Figure 2A**).

Natural Killer Cells from HIV Infected Slow Progressors Who Carry

the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated... 201

Fig. 2. Comparisons of the tri-functional potential of NK cells from study subjects with defined *KIR/HLA* combinations. Scatter plots show percent contribution of tri-functional NK cells to the total K562 stimulated response in individuals who are *3DL1+B\*27, 3DL1+Bw4*  and *3DL1+Bw6* carriers (A), and *\*h/\*y+B\*27* and *\*l/\*x+B\*27* carriers (B)*.* Each data point represents results from a separate individual. The data point represented by an empty square and triangle refer to individuals that carry both *B\*27* and *B\*57*. The bar through each scatter plot indicates the median frequency for the group. A Mann-Whitney U test was used

to assess the significance of between-group differences.

Fig. 1. NK cells from individuals carrying the 3DL1+HLA B\*27 NK receptor-ligand pair have an increased functional potential. The frequency of NK cells that secrete IFN-γ (left), express CD107a (middle) or secrete TNF-α (right) in 3DL1 *hmz* individuals who carry *HLA-B\*27* or *HLA-Bw6*. Each data point represents a separate individual. The bar through each scatter plot indicates the median frequency for the group. A Mann-Whitney U test was used to assess the significance of between-group comparisons (A). The percent contribution of seven different functional profiles to the total NK cell response to K562 *HLA*-devoid cells from individuals carrying *3DL1+B\*27* (n=12) and *3DL1+Bw6* (n=14). Below the *x*-axis, dots refer to the presence of each functional marker (CD107a, IFN-γ and TNF-α) in that profile. The height of each bar represents the median for the group and the height of the error bar the interquartile range for that group. An asterisk (\*) over the line linking two bars indicates that the contribution of that functional subset of the NK cell response was significantly different in the two study populations. A Mann-Whitney U test was used to assess the significance of between-group comparisons and p<0.05 was considered significant (B).

HIV Infection in the Era of Highly Active 200 Antiretroviral Treatment and Some of Its Associated Complications

Fig. 1. NK cells from individuals carrying the 3DL1+HLA B\*27 NK receptor-ligand pair have an increased functional potential. The frequency of NK cells that secrete IFN-γ (left), express CD107a (middle) or secrete TNF-α (right) in 3DL1 *hmz* individuals who carry *HLA-B\*27* or *HLA-Bw6*. Each data point represents a separate individual. The bar through each scatter plot indicates the median frequency for the group. A Mann-Whitney U test was used to assess the significance of between-group comparisons (A). The percent contribution of seven different functional profiles to the total NK cell response to K562 *HLA*-devoid cells from individuals carrying *3DL1+B\*27* (n=12) and *3DL1+Bw6* (n=14). Below the *x*-axis, dots refer to the presence of each functional marker (CD107a, IFN-γ and TNF-α) in that profile. The height of each bar represents the median for the group and the height of the error bar the interquartile range for that group. An asterisk (\*) over the line linking two bars indicates that the contribution of that functional subset of the NK cell response was significantly different in the two study populations. A Mann-Whitney U test was used to assess the significance of between-group comparisons and p<0.05 was considered significant (B).

Fig. 2. Comparisons of the tri-functional potential of NK cells from study subjects with defined *KIR/HLA* combinations. Scatter plots show percent contribution of tri-functional NK cells to the total K562 stimulated response in individuals who are *3DL1+B\*27, 3DL1+Bw4*  and *3DL1+Bw6* carriers (A), and *\*h/\*y+B\*27* and *\*l/\*x+B\*27* carriers (B)*.* Each data point represents results from a separate individual. The data point represented by an empty square and triangle refer to individuals that carry both *B\*27* and *B\*57*. The bar through each scatter plot indicates the median frequency for the group. A Mann-Whitney U test was used to assess the significance of between-group differences.

Natural Killer Cells from HIV Infected Slow Progressors Who Carry

the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated... 203

Fig. 3. Comparisons of the tri-functional potential of NK cells from individuals carrying *3DL1+B\*27, \*h/\*y+HLA-B\*57* and *3DL1+Bw4.* Scatter plots show percent contribution of trifunctional NK cells to the total K562 stimulated response in individuals who are *3DL1+B\*27* and *\*h/\*y+B\*57* carriers (A) or *\*h/\*y+B\*57, 3DL1+B\*27* and *3DL1+Bw4* carriers (B)*.* Each data point represents results from a separate individual. The bar through each scatter plot indicates the median frequency for the group. A Mann-Whitney U test was used to assess the significance of between-group differences (A). A Spearman's correlation test was used to assess the significance of a trend towards decreasing functional potential in carriers of these

We previously showed that B\*57 is superior to most other Bw4 alleles in educating NK cells through its interaction with 3DL1 NK receptors for functional potential as measured by responses to missing self on K562 cells (Boulet et al 2010; Kamya et al. 2011). These results suggested that B\*57*,* an allele associated with slow time to AIDS and VL control, may contribute to viral control not only through its interaction with HIV epitopes recognized by CD8+ T cell but also through its interaction with 3DL1 inhibitory receptors on NK cells.  We therefore sought to determine whether B\*27 was also able to educate NK cells for potent

genotype combinations (B).

*3DL1 hmz* genotypes can be classified as either *\*h/\*y* or *\*l/\*x* depending on the 3DL1 alleles expressed. Previous work showed that NK cells from *\*h/\*y+B\*57* subjects had a significantly higher tri-functional potential than those from *\*l/\*x+B\*57* subjects. We therefore questioned whether NK cells from the 7 *\*h/\*y+B\*27* and 5 *\*l/\*x+B27* individuals differed from each other in their tri-functional potential. The median (range) NK tri-functional potential was 2.56% (0.00%, 6.18%) and 3.57% (0.47%, 6.43%) for NK cells from carriers of the *\*h/\*y+B\*27* and *\*l/\*x+B\*27* genotypes, respectively (p=0.53; Mann-Whitney test), a difference that was not statistically significant (Figure 2B). Therefore, HLA-B\*27 appears to be able to interact with NK receptor alleles in either the 3DL1 \*h/\*y and \*l/\*x genotype categories to educate NK cells for equivalent NK functional potential.

#### **3.3 NK cells from** *3DL1+B\*27 and \*h/\*y+B\*57* **carriers have a similar tri-functional potential**

Next we questioned whether the level of tri-functional potential of NK cells from *3DL1*+*B\*27* carriers was of a similar magnitude to that seen in NK cells from *\*h/\*y*+*B\*57* carriers. For this analysis we excluded the 2 *3DL1*+*B\*27* individuals who also expressed a *B\*57* allele and compared NK tri-functional potential in this group to that in 12 *\*h/\*y+B\*57* carriers. As seen in Figure 3A, although the tri-functional potential of NK cells from *\*h/\*y+B\*57* carriers was higher than that from *3DL1+B\*27* carriers this difference did not achieve statistical significance (p=0.09; Mann-Whitney test).

Pair-wise comparisons of the tri-functional potential of NK cells from carriers of *\*h/\*y+B\*57*, *3DL1+B\*27* and *3DL1+Bw4* revealed that only the comparison of *\*h/\*y+B\*57* and *3DL1+Bw4* was statistically significant. NK cells from each of these groups had higher tri-functional potential than those from the *3DL1+Bw6* group (not shown). Small group sizes and large variability within groups likely contributed to the lack of statistical significance in the tri-functional potential between the *\*h/\*y+B\*57* and *3DL1+B\*27* and the *3DL1+B\*27* and *3DL1+Bw4* groups. We performed another analysis testing for the significance of a trend towards declining NK functional potential among subjects with these *3DL1/HLA-B* genotypes. The rationale for this came from epidemiological studies showing that the effect of *B\*57* on time to AIDS and VL control was greater in the presence of *\*h/\*y* than *\*l/\*x 3DL1* genotypes and greater than that of *B\*27* on these outcomes in the presence of either *3DL1* genotype (Martin et al. 2007). When the trifunctional potential of NK cells from these 3 *3DL1/HLA-B* genotypes were assessed. Using a test of trend we observed that this measure decreased as follows: *\*h/\*y+B\*57* > *3DL1+B\*27 > 3DL1+Bw4* (r=-0.40, p=0.01; Spearman's correlation test). Together these results suggest that there is a hierarchy in the impact on NK cell education of HLA-B variants where B\*57 is the most potent in the context of *\*h/\*y* genotypes followed by B\*27 in the context of *3DL1* genotypes. Furthermore the impact of these alleles is superior to that of other Bw4 alleles co-expressed with *3DL1* genotypes.

#### **4. Discussion**

We have presented results showing that NK cells from carriers of the *3DL1*+*B\*27* KIR/HLA genotype combination have tri-functional responses to missing self that are 1) significantly higher than those from *3DL1+Bw6* and 2) have a tri-functional potential that falls between that of carriers of *\*h/\*y+B\*57* and *3DL1+Bw4*.

HIV Infection in the Era of Highly Active 202 Antiretroviral Treatment and Some of Its Associated Complications

*3DL1 hmz* genotypes can be classified as either *\*h/\*y* or *\*l/\*x* depending on the 3DL1 alleles expressed. Previous work showed that NK cells from *\*h/\*y+B\*57* subjects had a significantly higher tri-functional potential than those from *\*l/\*x+B\*57* subjects. We therefore questioned whether NK cells from the 7 *\*h/\*y+B\*27* and 5 *\*l/\*x+B27* individuals differed from each other in their tri-functional potential. The median (range) NK tri-functional potential was 2.56% (0.00%, 6.18%) and 3.57% (0.47%, 6.43%) for NK cells from carriers of the *\*h/\*y+B\*27* and *\*l/\*x+B\*27* genotypes, respectively (p=0.53; Mann-Whitney test), a difference that was not statistically significant (Figure 2B). Therefore, HLA-B\*27 appears to be able to interact with NK receptor alleles in either the 3DL1 \*h/\*y and \*l/\*x genotype categories to educate NK

**3.3 NK cells from** *3DL1+B\*27 and \*h/\*y+B\*57* **carriers have a similar tri-functional**

Next we questioned whether the level of tri-functional potential of NK cells from *3DL1*+*B\*27* carriers was of a similar magnitude to that seen in NK cells from *\*h/\*y*+*B\*57* carriers. For this analysis we excluded the 2 *3DL1*+*B\*27* individuals who also expressed a *B\*57* allele and compared NK tri-functional potential in this group to that in 12 *\*h/\*y+B\*57* carriers. As seen in Figure 3A, although the tri-functional potential of NK cells from *\*h/\*y+B\*57* carriers was higher than that from *3DL1+B\*27* carriers this difference did not achieve statistical

Pair-wise comparisons of the tri-functional potential of NK cells from carriers of *\*h/\*y+B\*57*, *3DL1+B\*27* and *3DL1+Bw4* revealed that only the comparison of *\*h/\*y+B\*57* and *3DL1+Bw4* was statistically significant. NK cells from each of these groups had higher tri-functional potential than those from the *3DL1+Bw6* group (not shown). Small group sizes and large variability within groups likely contributed to the lack of statistical significance in the tri-functional potential between the *\*h/\*y+B\*57* and *3DL1+B\*27* and the *3DL1+B\*27* and *3DL1+Bw4* groups. We performed another analysis testing for the significance of a trend towards declining NK functional potential among subjects with these *3DL1/HLA-B* genotypes. The rationale for this came from epidemiological studies showing that the effect of *B\*57* on time to AIDS and VL control was greater in the presence of *\*h/\*y* than *\*l/\*x 3DL1* genotypes and greater than that of *B\*27* on these outcomes in the presence of either *3DL1* genotype (Martin et al. 2007). When the trifunctional potential of NK cells from these 3 *3DL1/HLA-B* genotypes were assessed. Using a test of trend we observed that this measure decreased as follows: *\*h/\*y+B\*57* > *3DL1+B\*27 > 3DL1+Bw4* (r=-0.40, p=0.01; Spearman's correlation test). Together these results suggest that there is a hierarchy in the impact on NK cell education of HLA-B variants where B\*57 is the most potent in the context of *\*h/\*y* genotypes followed by B\*27 in the context of *3DL1* genotypes. Furthermore the impact of these alleles is superior to

We have presented results showing that NK cells from carriers of the *3DL1*+*B\*27* KIR/HLA genotype combination have tri-functional responses to missing self that are 1) significantly higher than those from *3DL1+Bw6* and 2) have a tri-functional potential that falls between

cells for equivalent NK functional potential.

significance (p=0.09; Mann-Whitney test).

that of other Bw4 alleles co-expressed with *3DL1* genotypes.

that of carriers of *\*h/\*y+B\*57* and *3DL1+Bw4*.

**potential**

**4. Discussion**

Fig. 3. Comparisons of the tri-functional potential of NK cells from individuals carrying *3DL1+B\*27, \*h/\*y+HLA-B\*57* and *3DL1+Bw4.* Scatter plots show percent contribution of trifunctional NK cells to the total K562 stimulated response in individuals who are *3DL1+B\*27* and *\*h/\*y+B\*57* carriers (A) or *\*h/\*y+B\*57, 3DL1+B\*27* and *3DL1+Bw4* carriers (B)*.* Each data point represents results from a separate individual. The bar through each scatter plot indicates the median frequency for the group. A Mann-Whitney U test was used to assess the significance of between-group differences (A). A Spearman's correlation test was used to assess the significance of a trend towards decreasing functional potential in carriers of these genotype combinations (B).

We previously showed that B\*57 is superior to most other Bw4 alleles in educating NK cells through its interaction with 3DL1 NK receptors for functional potential as measured by responses to missing self on K562 cells (Boulet et al 2010; Kamya et al. 2011). These results suggested that B\*57*,* an allele associated with slow time to AIDS and VL control, may contribute to viral control not only through its interaction with HIV epitopes recognized by CD8+ T cell but also through its interaction with 3DL1 inhibitory receptors on NK cells.  We therefore sought to determine whether B\*27 was also able to educate NK cells for potent

Natural Killer Cells from HIV Infected Slow Progressors Who Carry

more modest than that of B\*57.

warrants further investigation.

the Protective HLA-B\*27 Allele and Inhibitory KIR3DL1 Receptors Have Elevated... 205

enhanced in the presence of *3DL1\*h/\*y* compared to *3DL1\*l/\*x*. Overall, the effect of *B\*27* on these outcomes was more moderate than that of *B\*57* in the presence of *3DL1 \*h/\*y*. Compared to *Bw6 hmz* the *B\*27-80T* allele *B\*2705* was more protective in the presence of *3DL1\*l/\*x* than in the presence of *3DL1\*h/\*y* (Martin et al. 2007). All but one of the *3DL1+B\*27* group of subjects in our study population expressed *B\*27-80T* alleles (Carrington, Martin, and van Bergen 2008). Furthermore, there is also evidence that B\*2705 has a greater affinity for one or more of the KIR3DL1\*l allotypes (Luque et al. 1996). Our findings that NK cells from *\*h/\*y+B\*27* and *\*l/\*x+B\*27* carriers have similar levels of trifunctional potential that may be more modest than of *\*h/\*y+B\*57 carriers* is in line with B\*27 being able to interact with receptors encoded by *3DL1\*h/\*y* and *\*l/\*x* genotypes in *3DL1+B\*27* carriers and this more effectively that B\*57 interacting with receptors encoded by *3DL1\*l/\*x* genotypes (Kamya et al. 2011)*.* There is also a trend towards NK cells from *3DL1+B\*27* carriers having higher function that those from *3DL1+Bw4* individuals. These results argue in favour of HLA-B\*27 and B\*57 being unique among HLA-Bw4 antigens in their impact on educating NK cells for subsequent activity, although the effect of B\*27 is