Oligoclonal T cells are infiltrating the brains of children with AIDS: sequence analysis reveals high proportions of identical β-chain T-cell receptor transcripts

Abstract

We have recently described the presence of perivascular CD3+ CD45RO+ T cells infiltrating the brains of children with AIDS. To determine whether these infiltrates contain oligoclonal populations of T cells, we amplified by PCR β-chain T-cell receptor (TCR) transcripts from autopsy brains of four paediatric patients with AIDS. The amplified transcripts were cloned and sequenced. Sequence analysis of the β-chain TCR transcripts from all four patients revealed multiple identical copies of TCR β-chain transcripts, suggesting the presence of oligoclonal populations of T-cells. These TCR transcripts were novel. The presence of oligoclonal populations of T cells in the brains of these four paediatric patients with AIDS suggests that these T cells have undergone antigen-driven proliferation and clonal expansion very likely in situ, in the brains of these AIDS patients, in response to viral or self-antigens. Although the specificity of the clonally expanded β-chain TCR transcripts remains to be elucidated, none of the β-chain TCR transcripts identified in this study were identical to those specific for HIV-1 antigens that are currently reported in the GENBANK/EMBL databases. Certain common CDR3 motifs were observed in brain-infiltrating T cells within and between certain patients. Large proportions (24 of 61; 39%) of β-chain TCR clones from one patient (NP95-73) and 2 of 27 (7%) of another patient (NP95-184-O) exhibited substantial CDR3 homology to myelin basic protein (MBP)-specific TCR derived from normal donors or TCR expressed in the brain of patients with multiple sclerosis (MS) or with viral encephalitis. These two patients (NP95-73 and NP95-184-O) also shared HLA class II with the normal donors and the MS patients who expressed these homologous TCR. Pathologic examination at autopsy of the brains revealed the presence of myelin pallor only in patient NP95-73. T-cell clones identified in the brain of patients NP95-73 and NP95-184-O may recognize MBP or another CNS self antigen and this recognition may be restricted by either DRB1*15 or DQB1*0602 specificities.

Introduction

Neurological impairments associated with HIV-1 infection of the CNS have been designated AIDS-dementia complex in adults and HIV-1-associated progressive encephalopathy (PE) in children. PE is observed in up to 30–50% of children with AIDS [1–6] and it is clinically associated with cognitive, motor and behavioural symptoms [4], and with infection of the CNS with HIV-1 [7–9]. Up to 40% of HIV-1-infected children exhibit HIV-1 encephalitis, characterized by foci of inflammatory cells, microglial nodules, multinucleated giant cells, axonal and myelin pathology and astrocytosis [4,8,9]. Most HIV-1-infected children do not develop either opportunistic infections or neoplasms in the CNS [5].

Evidence demonstrating that the CNS is infected by HIV-1 at the time of primary infection [2,10] has been provided by studies of autopsy brains of adult HIV-1-infected asymptomatic individuals who died accidentally of unnatural causes [2,3,11], and from an individual who was iatrogenically infected [10]. Infection of the brain by HIV-1 induces a T-cell response in the CNS, resulting in mononuclear cell infiltration (T-cells and to a lesser degree monocyte/macrophages), vasculitis and leptomeningitis, activation of microglia, enhancement of class II HLA expression, and cytokine production [2]. Cerebral vasculitis has been documented in HIV-1-infected individuals in the absence of infection other than HIV-1 [12,13]. Although vasculitis is, in general, not observed in the brains of adult AIDS patients with late stage disease [2,3,10], it very likely precedes vasculopathy which is found in the CNS of many patients with AIDS at autopsy [2,3] and is characterized by calcium mineralization and by fibrous thickening of the walls of blood vessels.

Although not prevalent in adult patients with AIDS, angiocentric mononuclear cell infiltrates have been found at autopsy in the brains of up to 40% of HIV-1-infected children [4–6,14–18]. Sharer et al. [14] reported vascular or perivascular mononuclear cell inflammatory infiltrates in small or medium size blood vessels in the brains of 5 out of 11 children with AIDS (ages 4 months to 11 years), obtained by autopsy [14]. These infiltrating angiocentric mononuclear cells were comprised primarily of lymphocytes. In contrast, in inflammatory lesions in HIV-1 encephalitis, the infiltrating mononuclear cells are primarily of monocyte/macrophage lineage [14]. Katsetos et al. [18] reported angiocentric (perivascular and in certain cases also transmural) mononuclear cell infiltrates in brain tissue specimens collected at autopsy from five of six children with AIDS. Immunohistochemical studies demonstrated that these angiocentric infiltrates mostly consisted of CD3+ CD8+ CD45RO+ T cells and of CD68+ monocyte/macrophages. CD3+ transvascular (including endothelial) infiltrates (CD8+/CD45RO+) were also seen in certain cases [18]. In contrast to the perivascular space, the neuropil contained only scant CD3+CD8+ cells. These infiltrating T cells may be responsible for HIV-1-associated CNS vasculitis/vasculopathy, for endothelial-cell injury and the opening of the blood–brain barrier in children with AIDS.

Very limited information is available about the role of T cells in CNS endothelial injury in HIV-1-infected patients. Perivascular and/or transmural infiltrating T cells may be the result of antigen-driven clonal expansions of T cells, or they may be comprised of polyclonal populations of T cells randomly infiltrating the CNS due to the destruction of the blood—brain barrier. To test the hypothesis that T cells infiltrating the perivascular space of CNS vessels of children with AIDS contain antigen-driven oligoclonal populations of T cells, we amplified by PCR, cloned and sequenced β-chain TCR transcripts from autopsy brain specimens from children with AIDS. Sequence analysis revealed substantial proportions of identical β-chain TCR transcripts, demonstrating the presence of oligoclonal T cells.

Materials and methods

Patient specimens

Brain specimens from children from both sexes who died of AIDS were obtained at autopsy and were snap frozen in isopentane cooled by liquid nitrogen. The postmortem intervals ranged from 4 to 19 h. The blocks were cryopreserved at −80 °C until use. Pathology of these specimens, clinical characteristics and the treatment status of the patients are shown in Table 1. These studies were reviewed and approved by the Institutional Review Board of Temple University Hospital. In all cases consent was obtained for autopsy.

Table 1.

Patient Information

Patient	Age/sex	Clinical Data	Neuropathology	Frozen block	Pathology of block*
NP95-73	7y, 9 m/Male	Recurrent systemic bacterial and fungal infections, FTT, respiratory failure, normal neuro exam; Rx'd with ZDV and ddC	BW = 1100 g; disseminated MGN, white matter pallor	G: Pons (basis)	MGN
NP89-213	6 m/Male	PCP; cerebral atrophy on scans, probable PE	BW = 600 g; HIV-1 encephalitis, moderate, with vascular inflammation, mild ventricular enlargement	N: Pons	MGN, MGC,vascular inflammation
NP95-184-O	8y, 6 m/ Male	Cardiomyopathy, disseminated MAC, poor weight gain; PE, cerebral atrophy on scans, seizures, spastic quadriparesis; Rx'd with ZDV	BW = 992 g; mild ventricular enlargement, old and recent infarcts, vascular inflammation, calcifications in basal ganglia, multifocal necrotizing leukoencephalopathy in pons, with calcifications	O: Pons (basis)	Multifocal necrotizing leuko- encephalopathy,with calcifications
NP94-34	8y, 6 m/ Female	Disseminated MAC, FTT, cardiomyopathy; Rx’d with  ZDV and ddC; normal neuro exam	BW = 1100 g; calcifications in basal ganglia, vascular inflammation, with macrophages, rare MGC	Y: Corpus callosum and cingulate gyrus	A few cells about vessels, including macrophages and a rare MGC

y, years; m, months; BW, brain weight (fresh, unfixed); MGN, microglial nodules; PE, progressive encephalopathy; PCP, Pneumocystis carinii pneumonia; MGC, multinucleated giant cells; MAC, Mycobacterium avium complex; ZDV, zidovudine (AZT); FTT, failure to thrive; ddC, dideoxycytidine (zalcitabine);

^*pathology of paraffin block taken adjacent to frozen block that was used for the TCR transcript studies.

Peripheral blood from normal donors

Peripheral blood from healthy normal donors (HIV-1-negative and free of hepatitis C virus (HVC); and anti-HVC antibody) was obtained using an informed consent approved by the IRB of Temple University Hospital. Peripheral blood mononuclear cells (PBMC) were prepared by centrifugation on a Ficoll-Hypaque density cushion, following established methods. PBMC were collected from the interface and were washed twice before preparation of RNA.

Preparation of RNA

Brain tissue (100 mg) from pons (basis) (patients NP95-73 and NP95-184-O), pons (patient NP89-213), or corpus callosum and cingulate gyrus (patient NP94-34) (Table 1) was homogenized in Stratagene denaturing solution containing guanidinium thiocyanate (Stratagene, La Jolla, CA, USA) and was used for RNA isolation (yield of 20–50 µg), as previously described [19]. The pathology of paraffin blocks taken adjacent to frozen blocks that were used in preparation of RNA in these studies is described in Table 1. Total RNA was isolated by the guanidinium thiocyanate phenol-chloroform single-step extraction method, following the procedure recommended by the manufacturer (Stratagene). Phenol extraction was performed at least twice on all samples. The purity of the isolated RNA was checked by visualization of the ribosomal RNA 28 s and 18 s after agarose gel electrophoresis.

Synthesis of cDNA

Total RNA (5–10 µg) was used for double-stranded cDNA synthesis as described [20–23]. The first strand was synthesized (in a 20 µl reaction volume) using SuperScript RTase (Gibco-BRL Life Technologies, Gaithersburg, MD, USA) and primed with either a NotI-oligo(dT)₁₅ or NotI-hCβ primer (5′-TGCGGCCGCAGTATCTGGAGTC-3′; NotI: TGCGGC CGC (hCβ = human constant region β-chain). The mixture was incubated at 42°C for 1 h. The second strand cDNA was synthesized in a reaction volume of 160 µl by adding directly to the first strand synthesis 5 U of E.coli DNA ligase, 40 U of E.coli DNA polymerase, 1·5 U of RNAseH, 0·19 mM dNTP, and 3·8 µM DTT in the second strand buffer. The mixture was incubated for 2 h at 16°C. 10 U of T4 polymerase was added to the mixture for 45 min at 16°C. The product was extracted with equal volume of phenol-chloroform (1 : 1) and precipitated with 0·5 Vol of NH₄OAc (4 M) and 2·5 Vol of 100% ETOH. The pellet was washed once with 70% ETOH and resuspended in 10 µl of sterile water.

Adaptor ligation and NotI digestion

A nonpalindromic double-stranded adaptor comprised of the nucleotide (5′-AATTCGAACCCCTTCGAGAATGCT-3′) and its complementary nucleotide (5′-pCGCATTCTC GAAGGGGTTCG-3′) was ligated onto the 5′-and 3′ blunt ends of the cDNA, using 1·4 U of T4 DNA ligase (Gibco-BRL), by overnight incubation at 14°C. This adaptor is a modification of the one that we have described previously [20–23]. The adaptor was removed from the 3′ end of the cDNA by digestion for 3 h with 7·5 U of NotI restriction nuclease (Gibco-BRL) in a 50 µl volume. The NotI nuclease digested cDNA was further purified by using a G-50 spin column, by centrifugation for 5 min at 1100 g, according to the procedure provided by the manufacturer (5 prime to 3 prime, Boulder, CO, USA).

Amplification by the nonpalindromic adaptor-PCR (NPA-PCR)/Vβ-specific PCR

First cycle of amplification by NPA-PCR

NPA-PCR was carried out essentially as previously described [20–26], with minor modifications (see above). The NotI-digested cDNA (1/5–1/10 volume), purified by centrifugation using a G-50 spin column, was denatured at 94°C for 3 min in a reaction volume of 100 µl and amplified by 35 cycles of PCR, using Taq polymerase (Promega, Madison, WI, USA) at 94 °C for 1 min, 45 °C for 1 min and a final extension at 72 °C for 10 min. The 5′-amplification primer was the nonpalidromic adaptor. A human Cβ oligonucleotide, designated hCβ3 (5′-CAGGCAGTATCTGGAGTCAT TGA-3′) was used as the 3′ amplification primer and it is located 5′-to the hCβ primer used for cDNA synthesis. hCβ3 was located in the Cβ region starting at nucleotide 208 (Table 2). The product was purified using a G-50 spin column.

Table 2.

Human Vβ family primers used for amplification.

NPA-PCR
5′ end primer:	5′-AATTCGAACCCCTTCGAGAATGCT-3′
3′ end primer:
hCβ3	5′-CAGGCAGTATCTGGAGTCATTGA-3′
Vβ-specific amplifications 5′ end primers:
Vβ1	CCGCACAACAGTTCCCTGACTTGC
Vβ2	GGCCACATACGAGCAAGGCGTCGA
Vβ3	GTCTCTAGAGAGAAGAAGGAGCGC
Vβ4	TTCCCATCAGCCGCCCAAACCTAA
Vβ5	ATACTTCAGTGAGACACAGAGA
Vβ6	TCTCAGGTGTGATCCAAATTCGGG
Vβ7	CCTGAATGCCCCAACAGCTCTCTC
Vβ8	CCATGATGCGGGGACTGGAGTTGC
Vβ9	TTCCCTGGAGCTTGGTGACTCTGC
Vβ10	CCACGGAGTCAGGGGACACAGCAC
Vβ11	TGCCAGGCCCTCACATACCTCTCA
Vβ12	TGTCACCAGACTGGGAACCACCAC
Vβ13	CACTGCGGTGTACCCAGGATATGA
Vβ14	GGGCTCGGCTTAAGGCAGACCTAC
Vβ15	CAGGCACAGGCTAAATTCTCCCTG
Vβ16	GCCTGCAGAACTGGAGGATTCTGG
Vβ17	CTGCTGAATTTCCCAAAGAGGGCC
Vβ18	TGCCCCAGAATCTCTCAGCCTCCA
Vβ19	TCCTCTCACTGTGACATCGGCCA
Vβ20	TCTCAATGCCCCAAGAACGCACCC
Vβ21	GATTCACAGTTGCCTAAGGA
Vβ22	AAGTGATCTTGCGCTGTGTCCCCA
Vβ23	GCAGGGTCCAGGTCAGGACCCCCA
Vβ24	CCCAGTTTGGAAAGCCAGTGACCC
3′ end primer:
hCβ2	5′-ACCAGCACTCAGCTCCACGTGGTC-3′

Second cycle of amplification by individual Vβ-specific PCR

A second cycle of Vβ-specific amplification was carried out as described [24] using as a template β-chain TCR cDNA (5–10 µl) previously amplified by NPA-PCR, as described in the previous paragraph. Oligonucleotides from each of 24 Vβ families (Vβ1 to Vβ24) [27], were used as a 5′ end amplification primer in 24 separate amplification reactions (Table 2). A human Cβ primer designated hcβ2 (5′-ACCAG CACTCAGCTCCACGTGGTC-3′) was used as a-3′ amplification primer and it was located 5′ of the hcβ3 primer mentioned above (nested design). This oligonucleotide was located in the Cβ region starting at nucleotide 113. The reaction mixture (50 µl) was denatured by heating at 94 °C for 3 min, and amplified by 35 cycles of PCR, using Taq polymerase (Promega), at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 10 min

Cloning

Ten µl from the products of each of the 24 PCR amplifications were mixed and 2 µl of the mixture were ligated with 50 ng of vector pCR2·1 (Invitrogen, San Diego, CA, USA), using 2 U of T4 ligase (Gibco-BRL) in a volume of 10 µl. DH5α competent cells (Gibco-BRL) were transformed with ligated PCR products, and white colonies were picked up. In addition, TCR transcripts from patient NP95-73 amplified by either NPA-PCR/Vβ5·1-specific PCR or NPA-PCR/Vβ22·1-specific PCR were ligated, each one separately, with the pCR2·1 vector. DH5α competent cells were transformed with Vβ5·1- or Vβ22·1-ligated PCR products and white colonies were picked up.

The maximum theoretical number of potentially unique β-chain TCR transcripts has been estimated to be in the range of 10¹²[28]. Theoretically, the probability of randomly finding two identical copies of a single β-chain TCR transcript within an independent sample population is negligible due to the large number of unique TCR transcripts. During transformation of DH5α competent cells, the plasmid/cell mixture was subjected to heat shock (at 42 °C for 45 s) followed by incubation on ice for 2 min and growth for 1 h in SOC media at 37 °C before plating. Under ideal conditions (log phase), E. coli has a doubling time of 20 min that would result in two doublings after 60 min [29]. After heat shock, though, the DH5α cells need to recover and do not immediately enter log phase. However the unlikely possibility for a few of the transformed cells to double before plating does exist. This may result in the presence of two E. coli cells with identical TCR inserts. Therefore, identical TCR sequences from two different colonies (a doublet) may indicate a clonal expansion or could be a result from a singly transfected E. coli cell that doubled before plating. In the studies presented here we have sequenced 39 β-chain TCR transcripts from normal PBMC after either NPA-PCR/Vβ5·1-specific PCR, or NPA-PCR/Vβ22·1-specific PCR amplification and cloning (Table 5). All these PBMC β-chain TCR transcripts were unique when compared to each other. In another study [25], we sequenced 150 β-chain TCR transcripts from normal PBMC after either NPA-PCR or Vβ-specific PCR amplification. All TCR transcripts were unique when compared to each other, with the exception of one transcript (Vβ12·1 Dβ1·1 Jβ1·6), which appeared in duplicate. In another study [26], we have sequenced 58 β-chain TCR transcripts from the peripheral blood of normal donors. All of these transcripts were unique when compared to each other demonstrating the presence of polyclonal populations of T cells. All these results taken together demonstrate that possible doubling of a singly transfected E. coli cell before plating is rather infrequent. In contrast, sequence analysis of TCR transcripts from the brain of children with AIDS (this report), or other pathological specimens [24–26] yielded several examples of two identical copies of a single β-chain TCR transcript. These transcripts may represent clonal expansions. The odds for a triplet (3 identical transcripts) to be due to this heat shock artifact are negligible.

Table 5.

CDR3 sequences of TCR transcripts from PBMC from a normal donor.

Clone	Variable	N-Diverse-N	Joining
Determined after amplification by NPA-PCR/Vβ5·1-specific PCR, cloning and sequencing
	C A S S	P R G G V	N T E A	Vβ5·1 Dβ2·1 Jβ1·1
1	TGC GCC AGC AGC	CCT CGG GGG GGA GTG	AAC ACT GAA GCT	(1 of 20)
	C A S S	S L E F S	E A F	Vβ5·1 Dβ2·1 Jβ1·1
2	TGC GCC AGC AGC	TCC CTT GAA TTC AGT	GAA GCT TTC	(1 of 20)
	C A S S	P E Q G R	N E K L	Vβ5·1 Dβ1·1 Dβ1·4
3	TGC GCC AGC AGC	CCT GAA CAA GGG AGG	AAT GAA AAA CTG	(1 of 20)
	C A S S	S T G T G I	E K L F	Vβ5·1 Dβ2·1 Jβ1·4
4	TGC GCC AGC AGC	TCG ACC GGG ACA GGG ATC	GAA AAA CTG TTT	(1 of 20)
	C A S S	L S T G G	T L H F	Vβ5·1 Dβ1·1 Jβ1·6
5	TGC GCC AGC AGC	TTG AGC ACA GGG GGG	ACC CTC CAC TTT	(1 of 20)
	C A S S	I Q G G	A Y N S	Vβ5·1 Dβ2·1 Jβ1·6
6	TGC GCC AGC AGC	ATC CAG GGG GGC	GCC TAT AAT TCA	(1 of 20)
	C A S S	P R A G S P G	E Q F F	Vβ5·1 Dβ2·1 Jβ2·1
7	TGC GCC AGC AGC	CCC CGG GCG GGA AGC CCA GGG	GAG CAG TTC TTC	(1 of 20)
	C A S S	L L S	N T G E	Vβ5·1 Dβ2·1 Jβ2·2
8	TGC GCC AGC AGC	CTA CTG AGT	AAC ACC GGG GAG	(1 of 20)
	C A S S	Y E S S G G G I I Y S	N T G E	Vβ5·1 Dβ2·1 Jβ2·2
9	TGC GCC AGC AGC	TAC GAA TCT AGC GGG GGG GGG ATA ATT TAC TCG	AAC ACC GGG GAG	(1 of 20)
	C A S S	L T V A	D T Q	Vβ5·1 Dβ2·1 Jβ2·3
10	TGC GCC AGC AGC	TTA ACG GTG GCA	GAT ACG CAG	(1 of 20)
	C A S S	F L A G G K E	T Q Y F	Vβ5·1 Dβ2·1 Jβ2·3
11	TGC GCC AGC AGC	TTT CTA GCG GGA GGG AAA GAA	ACG CAG TAT TTT	(1 of 20)
	C A S S	L G G	S T D T	Vβ5·1 Dβ2·1 Jβ2·3
12	TGC GCC AGC AGC	TTG GGG GGC	AGC ACA GAT ACG	(1 of 20)
	C A S S	R Q A G G S	K N I Q	Vβ5·1 Dβ2·1 Jβ2·4
13	TGC GCC AGC AGC	CGT CAA GCA GGG GGC TCT	AAA AAC ATT CAG	(1 of 20)
	C A S S	L T R E Y E E	E T Q Y	Vβ5·1 Dβ2·1 Jβ2·5
14	TGC GCC AGC AGC	TTG ACT CGG GAA TAT GAG GAG	GAG ACC CAG TAC	(1 of 20)
	C A S S	S P G I	S G A N	Vβ5·1 Dβ2·1 Jβ2·6
15	TGC GCC AGC AGC	TCC CCC GGG ATT	TCT GGG GCC AAC	(1 of 20)
	C A S S	R D E A W	E Q Y F	Vβ5·1 Dβ2·1 Jβ2·7
16	TGC GCC AGC AGC	AGG GAC GAG GCC TGG	GAG CAG TAC TTC	(1 of 20)
	C A S S	S G T S G T	E Q Y	Vβ5·1 Dβ2·1 Jβ2·7
17	TGC GCC AGC AGC	TCT GGG ACT AGC GGG ACC	GAG CAG TAC	(1 of 20)
	C A S S	L N G Q G G	T G E L	Vβ5·1 Dβ1·1 Jβ2·2
18	TGC GCC AGC AGC	TTG AAC GGA CAG GGG GGA	ACC GGG GAG CTG	(1 of 20)
	C A S S	L L N	T G E	Vβ5·1 Dβ2·1 Jβ2·2
19	TGC GCC AGC AGC	TTG TTA AAC	ACC GGG GAG	(1 of 20)
	C A S S	F G T G	T G A N	Vβ5·1 Dβ1·1 Jβ2·6
20	TGC GCC AGC AGC	TTC GGG ACA GGG	ACT GGG GCC AAC	(1 of 20)
Determined after amplification by NPA-PCR/Vβ22·1-specific PCR, cloning and squencing
	F C A S	H G P T	N T E A	Vβ5·1 Dβ2·1 Jβ1·1
1	TTC TGT GCC AGT	CAT GGA CCC ACG	AAC ACT GAA GCT	(1 of 19)
	C A S R	P G F S G V	A F F	Vβ5·1 Dβ2·1 Jβ1·1
2	TGT GCC AGC AGA	CCA GGG TTC TCG GGT GTA	GCT TTC TTT	(1 of 19)
	C A S S	E A F W T G	T E A	Vβ5·1 Dβ2·1 Jβ1·1
3	TGT GCC AGC AGT	GAA GCG TTT TGG ACA GGC	ACT GAA GCT	(1 of 19)
	C A S S	R Q I	T E A F	Vβ5·1 Dβ2·1 Jβ1·1
4	TGT GCC AGC AGT	CGA CAG ATT	ACT GAA GCT TTC	(1 of 19)
	C A S R	Q D M	N T E A	Vβ5·1 Dβ2·1 Jβ1·1
5	TGT GCC AGC AGA	CAG GAC ATG	AAC ACT GAA GCT	(1 of 19)
	C A S S	P G Q G F	G Y T F	Vβ5·1 Dβ2·1 Jβ1·1
6	TGT GCC AGC AGT	CCG GGA CAG GGC TTC	GGC TAC ACC TTC	(1 of 19)
	C A S R	F W	G N T I	Vβ5·1 Dβ2·1 Jβ1·1
7	TGT GCC AGC AGA	TTC TGG	GGA AAC ACC ATA	(1 of 19)
	C A S S	P G S	E K L	Vβ5·1 Dβ2·1 Jβ1·1
8	TGT GCC AGC AGT	CCA GGG AGT	GAA AAA CTG	(1 of 19)
	C A S S	E E G G L P	Q P Q	Vβ5·1 Dβ2·1 Jβ1·1
9	TGT GCC AGC AGT	GAA GAG GGG GGG TTG CCT	CAG CCC CAG	(1 of 19)
	C A S S	E L G R E	Q P Q H	Vβ5·1 Dβ2·1 Jβ1·1
10	TGT GCC AGC AGT	GAA TTG GGC AGG GAG	CAG CCC CAG CAT	(1 of 19)
	C A S S	I S W P R G	S P L H	Vβ5·1 Dβ2·1 Jβ1·1
11	TGT GCC AGC AGC	ATT TCG TGG CCC CGA GGG	TCA CCC CTC CAC	(1 of 19)
	C A S S	E R T G V	S P L H	Vβ5·1 Dβ2·1 Jβ1·1
12	TGT GCC AGC AGT	GAA CGG ACA GGG GTG	TCA CCC CTC CAC	(1 of 19)
	C A S S	S P G Q E	N N E Q	Vβ5·1 Dβ2·1 Jβ1·1
13	TGT GCC AGC AGT	TCA CCG GGA CAG GAG	AAC AAT GAG CAG	(1 of 19)
	C A S R	G F R V R Q	A Y N E	Vβ5·1 Dβ2·1 Jβ1·1
14	TGT GCC AGC AGG	GGG TTC AGG GTA CGG CAG	GCC TAC AAT GAG	(1 of 19)
	C A S S	A L L G	T D T	Vβ5·1 Dβ2·1 Jβ1·1
15	TGT GCC AGC AGT	GCA TTA TTG GGC	ACA GAT ACG	(1 of 19)
	C A S S	E Q G G S N	Q Y F G	Vβ5·1 Dβ2·1 Jβ1·1
16	TGT GCC AGC AGT	GAA CAG GGT GGC TCC AAT	CAG TAT TTT GGC	(1 of 19)
	C A S S	D R V L	A K N I	Vβ5·1 Dβ2·1 Jβ1·1
17	TGT GCC AGC AGT	GAC AGG GTA CTA	GCC AAA AAC ATT	(1 of 19)
	C A S S	D	Q E T Q	Vβ5·1 Dβ2·1 Jβ1·1
18	TGT GCC AGC AGT	GAC	CAG TAT TTT GGC	(1 of 19)
	C A S S	E A A R T G	T Y E G	Vβ5·1 Dβ2·1 Jβ1·1
19	TGT GCC AGC AGT	GAA GCG GCC CGG ACA GGA	ACC TAC GAG CAG	(1 of 19)

Sequencing

Plasmids were isolated using the Perfect Prep Kit, according to the procedure provided by the manufacturer (5 prime to 3 prime). Sequencing was carried out by the dideoxy chain termination method, using Sequenase 2·0 (USB, Cleveland, OH, USA).

Computer analysis of DNA sequences

The nucleic acid and the deduced amino acid sequence obtained were compared to those in the GEnBANK/EMBL/SWISS PROT DataBases using FASTA and BLAST Software.

Histocompatibility testing

DNA was extracted from specimens NP95-73 and NP95-184-O and used for DNA-based typing of A, DRB1 and DQB1 loci. The method of sequence-specific primers (SSP-PCR) [30,31] was employed, using ARMS technology (U.S. patent 5595890 under license from AstraZeneca, Wilmington, DE, USA). Low resolution A, DRB1 typing was performed by ‘One Lambda’ SSP products. Depending on the low resolution results of DRB1 typing, appropriate kits were chosen (Genovision Inc.) for DRB1 subtyping. DQB1 was typed by the ‘One Lambda’ SSP kit for high resolution typing.

Statistical analysis

To determine whether the clonal expansions of β-chain TCR transcripts from brain specimens from paediatric patients with AIDS were statistically significant, we used binomial distribution [32] to calculate exact probabilities, P, of multiple copies of the same transcript appearing against the alternative hypothesis of a single transcript appearing out of n transcripts where the number n is based on the number of transcripts examined using the same methodology when examining β-chain TCR transcripts from PBMC from normal donors.

Results

Sequence analysis of β-chain TCR transcripts from brain specimens from patient NP95-73 (7 years and 9 months old, male), after NPA-PCR/Vβ-specific PCR amplification and cloning is shown in Table 3. Three possible clonal expansions were identified:

• (a) clone 1·1, Vβ5·1 Dβ2·1 Jβ2·4; CDR3: CASSLELAKN; accounted for 3 of 24 (13%) transcripts.

• (b) clone 19·1, Vβ22·1 Dβ2·1 Jβ2·1; CDR3: CASSEELAGGSYNEQ; accounted for 2 of 24 (8%) transcripts.

• (c) clone 9·1, Vβ21·1 Dβ2·1 Jβ2·2; CDR3: CASSFGTTGELD; accounted for 2 of 24 (8%) transcripts (Table 3).

All remaining 17 β-chain TCR transcripts were unique when compared to each other, suggesting that they have been derived from polyclonal populations of T cells. To confirm that the transcripts that were expressed in Table 3 in triplicate or duplicate represent indeed clonal expansions, we carried out Vβ-specific PCR amplification of β-chain TCR transcripts that were previously amplified by NPA-PCR (NPA-PCR/Vβ-specific PCR).

Table 3.

CDR3 Sequences of NPA-PCR/Vβ-specific PCR amplified β-chain TCR transcripts from the brain of a pediatric patient with AIDS (NP95-73).

Clone	Variable				N-Diverse-N			Joining
	C	A	S	S	L	E	L					A	K	N	I	Q	Vβ5·1 Dβ2·1 Jβ2·4
1·1	TGC	GCC	AGC	AGC	TTG	GAA	TTA					GCC	AAA	AAC	ATT	CAG	(3 of 24)
C	A	S	S	E	E	L	A	G	G		S	Y	N	E	Q	Vβ22·1 Dβ2·1 Jβ2·1
19·1	TGT	GCC	AGC	AGT	GAG	GAG	CTA	GCG	GGA	GGA		TCC	TAC	AAT	GAG	CAG	(2 of 24)
C	A	S	S	F	G	T					T	G	E	L	F	Vβ21·1 Dβ2·1 Jβ2·2
9·1	TGT	GCC	AGC	AGC	TTT	GGA	ACT					ACC	GGG	GAG	CTG	TTT	(2 of 24)
C	A	S	S	A	L	W					K	L	F	F	G	Vβ22·1 Dβ2·1 Jβ1·4
18·1	TGT	GCC	AGC	AGT	GCT	TTG	TGG					AAA	CTG	TTT	TTT	GGC	(1 of 24)
C	A	S	S	L	T	A	G	G			N	T	G	E	L	Vβ5·1 Dβ2·1 Jβ2·2
4·1	TGC	GCC	AGC	AGC	TTG	ACG	GCG	GGG	GGG			AAC	ACC	GGG	GAG	CTG	(1 of 24)
C	A	S	S	L	D	S	Y				T	G	E	L	F	Vβ6·1 Dβ1·1 Jβ2·2
5·1	TGT	GCC	AGC	AGC	TTA	GAC	AGC	TAC				ACC	GGG	GAG	CTG	TTT	(1 of 24)
C	A	S	S	S	W	T	V	A			Y	E	Q	Y	F	Vβ6·6 Dβ1·1 Jβ2·7
6·1	TGT	GCC	AGC	AGC	TCC	TGG	ACA	GTC	GCC			TAC	GAG	CAG	TAC	TTC	(1 of 24)
R	C	A	S	S	S						G	A	N	V	L	Vβ6·3 Dβ2·1 Jβ2·6
24·1	CGC	TGT	GCC	AGC	TCG	AGT						GGG	GCC	AAC	GTC	CTG	(1 of 24)
C	A	S	S	L	G	I	P	E	N	A	Y	N	E	Q	F	Vβ6·3 Dβ2·1 Jβ2·1
7·1	TGT	GCC	AGC	AGC	TTA	GGG	ATC	CCG	GAG	AAT	GCG	TAC	AAT	GAG	CAG	TTC	(1 of 24)
C	A	S	S	F	S	S	G	R	P		G	E	L	F	F	Vβ14·1 Dβ2·1 Jβ2·2
8·1	TGT	GCC	AGC	AGT	TTT	TCC	TCA	GGG	CGC	CCC		GGG	GAG	CTG	TTT	TTT	(1 of 24)
C	A	S	S	L	E	A	G				Y	E	Q	Y	F	Vβ21·1 Dβ2·1 Jβ2·7
11·1	TGT	GCC	AGC	AGC	TTA	GAA	GCA	GGC				TAC	GAG	CAG	TAC	TTC	(1 of 24)
C	A	S	S	P	S	G	A	G	L		N	T	E	A	F	Vβ21·3 Dβ2·1 Jβ1·1
12·1	TGT	GCC	AGC	AGC	CCC	TCA	GGG	GCG	GGG	TTG		AAC	ACT	GAA	GCT	TTC	(1 of 24)
C	A	S	S	L	G	L					Y	N	E	Q	F	Vβ21·1 Dβ2·1 Jβ2·1
13·1	TGT	GCC	AGC	AGC	TTA	GGT	CTT					TAC	AAT	GAG	CAG	TTC	(1 of 24)
C	A	S	S	S	R	T	G	G	A	G	N	E	Q	F	F	Vβ21·1 Dβ1·1 Jβ2·1
14·1	TGT	GCC	AGC	AGC	TCC	CGG	ACA	GGG	GGC	GCT	GGC	AAT	GAG	CAG	TTC	TTC	(1 of 24)
C	A	S	S	S	P	T	L				L	W	G	Q	R	Vβ21·1 Dβ2·1 Jβ2·6
15·1	TGT	GCC	AGC	AGC	TCC	CCG	ACC	CTA				CTC	TGG	GGC	CAA	CGT	(1 of 24)
C	A	S	S	P	L	S	G	G	D	Y	Y	E	Q	Y	F	Vβ21·1 Dβ2·1 Jβ2·7
16·1	TGT	GCC	AGC	AGC	CCC	TTG	AGC	GGG	GGT	GAT	TAC	TAC	GAG	CAG	TAC	TTC	(1 of 24)
L	C	A	S	V	P	A	H				N	E	Q	F	F	Vβ21·1 Dβ2·1 Jβ2·1
17·1	CTC	TGT	GCC	AGC	GTC	CCT	GCC	CAC				AAT	GAG	CAG	TTC	TTC	(1 of 24)
C	A	S	S	L	T	V	S				S	Y	N	E	Q	Vβ23·1 Dβ2·1 Jβ2·1
21·1	TGT	GCC	AGC	AGC	TTG	ACT	GTG	AGC				TCC	TAC	AAT	GAG	CAG	(1 of 24)
C	A	T	S	D	G	T	V	G	E		P	Q	H	F	G	Vβ24·1 Dβ2·1 Jβ1·5
22·1	TGT	GCC	ACC	AGC	GAT	GGG	ACG	GTG	GGG	GAG		CCC	CAG	CAT	TTT	GGT	(1 of 24)
A	T	S	R	D	G	G	I	G	T		Y	N	E	Q	F	Vβ24·1 Dβ2·1 Jβ2·1
23·1	GCC	ACC	AGC	AGA	GAT	GGG	GGG	ATA	GGG	ACC		TAC	AAT	GAG	CAG	TTC	(1 of 24)

Confirmation that clone 1·1 was indeed clonally expanded was obtained using NPA-PCR/Vβ5·1-specific PCR amplification, followed by cloning and sequencing (Table 4). This clone accounted for 9 of 21 (43%) of the Vβ5·1 transcripts sequenced. PBMC from a normal donor were used as a methodological control and for statistical comparison. NPA-PCR/Vβ5·1-specific PCR amplification followed by cloning and sequencing of PBMC from a normal donor revealed unique sequences when compared to each other, typical of polyclonal populations of T cells (Table 5). Statistical analysis was performed using the binomial distribution [32] to determine the probability, p, of the fractions of Vβ5·1 TCR identical transcripts found in the brain of paediatric patient NP95-73 with AIDS (9 of 21 identical Vβ5·1 transcripts, Table 4) against the alternative hypothesis that all transcripts sequenced are present in a single copy only (that is to say they are unique when compared to each other) of unique Vβ5·1 TCR transcripts when using the same methodology from PBMC from normal donors (n = 20 in this experiment, Table 5). The clonal expansion of the Vβ5·1Dβ2·1Jβ2·4 transcript (clone 1·1,Table 4) was highly significant (P = 3 × 10⁻⁷, Table 4). The 95% confidence interval (C.I) [33] of the proportions of identical transcripts (9 (43%) of 21; Table 4) for clone 1·1 was 23%-67%.

Table 4.

CDR3 sequences ofβ-chain TCR transcripts amplified by NPA-PCR/Vβ-specific PCR from the brain of a pediatric patient with AIDS (NP95-73).

Clone	Variable	N-Diverse-N	Joining
NPA-PCR/Vβ5·1-specific PCR
	C A S S	L E L	A K N I Q	Vβ5·1 Dβ2·1 Jβ2·4
1·1	TGC GCC AGC AGC	TTG GAA TTA	GCC AAA AAC ATT CAG	(9 of 21; p = 3 × 10−7)
	C A S S	L E L	A K N I Q	Vβ5·1 Dβ2·1 Jβ2·4
10·2	TGC GCC AGC AGC	TTA GAA CTA	GCC AAA AAC ATT CAG	(1 of 21)
	C A S S	L T A G G Q	H R G A V	Vβ5·1 Dβ2·1 Jβ2·2
11·2	TGC GCC AGC AGC	TTG ACG GCG GGG GGG CAA	CAC CGG GGA GCT GTT	(4 of 21; p = 0·02)
	C A S S	L V G L R G	N T E A F	Vβ5·1 Dβ1·1 Jβ1·1
15·2	TGC GCC AGC AGC	TTG GTA GGG CTA CGT GGA	AAC ACT GAA GCT TTC	(2 of 21)
	C A S S	F S G L T	Y E Q Y F	Vβ5·1 Dβ2·1 Jβ2·7
17·2	TGC GCC AGC AGC	TTT TCG GGA CTG ACC	TAC GAG CAG TAC TTC	(1 of 21)
	C A S S	L E G	S Y E Q Y	Vβ5·1 Dβ2·1 Jβ2·7
18·2	TGC GCC AGC AGC	TTG GAG GGC	TCC TAC GAG CAG TAC	(1 of 21)
	C A S S	L F E L A G A G T	Y E Q Y F	Vβ5·1 Dβ2·1 Jβ2·7
19·2	TGC GCC AGC AGC	TTG TTC GAA CTA GCG GGG GCC GGA ACT	TAC GAG CAG TAC TTC	(1 of 21)
	C A S S	L V P Q A R	Y E Q Y F	Vβ5·1 Dβ1·1 Jβ2·7
20·2	TGC GCC AGC AGC	TTG GTC CCA CAG GCC CGC	TAC GAG CAG TAC TTC	(1 of 21)
	L C A S	P G A S	Y E Q Y F	Vβ5·1 Dβ2·1 Jβ2·7
21·2	CTT TGC GCC AGC	CCC GGG GCT TCC	TAC GAG CAG TAC TTC	(1 of 21)
NPA-PCR/Vβ22·1-specific PCR
	C C A S	E E L A G G	S Y N E Q	Vβ22·1 Dβ2·1 Jβ2·1
19·1	TGT GCC AGC AGT	GAG GAG CTA GCG GGA GGA	TCC TAC AAT GAG CAG	(5 of 16; P = 0·0009)
	C C A S	A L W	K L F F G	Vβ22·1 Dβ1·1 Jβ1·4
18·1	TGT GCC AGC AGT	GCT TTG TGG	AAA CTG TTT TTT GGC	(6 of 16; P = 9 × 10−5)
	F C A S	E G A S G I	Y N E Q F	Vβ22·1 Dβ2·1 Jβ2·1
12·3	TTC TGT GCC AGC	GAG GGG GCT AGC GGG ATC	TAC AAT GAG CAG TTC	(1 of 16)
	C C A S	E V R G G G A G H	E Q Y F G	Vβ22·1 Dβ2·1 Jβ2·7
13·3	TGT GCC AGC AGT	GAA GTC CGG GGG GGG GGG GCG GGC CAC	GAG CAG TAC TTC GGG	(1 of 16)
	F C A S	S P G F G	T L K L S	Vβ22·1 Dβ1·1 Jβ1·1
14·3	TTC TGT GCC AGC	TCT CCG GGC TTC GGG	ACA CTG AAG CTT TCT	(1 of 16)
	F C A S	I G	A K N I Q	Vβ22·1 Dβ2·1 Jβ2·4
15·3	TTC TGT GCC AGC	ATC GGA	GCC AAA AAC ATT CAG	(1 of 16)
	C C A S	E A E T A F	N S P L H	Vβ22·1 Dβ1·1 Jβ1·6
16·3	TGT GCC AGC AGT	GAA GCG GAG ACA GCC TTC	AAT TCA CCC CTC CAC	(1 of 16)

An additional clone 10·2 exhibited identical deduced amino acid sequence, but different nucleic acid CDR3 sequence, as follows: TTGGAATTA (clone 1·1) and TTAGGACTA (clone 10·2). Thus, the CASSLELAKNIQ transcript accounted for 10 of 21 (47%; C.I. 26%, 70%) (P = 2 × 10⁻⁸) Vβ5·1 transcripts sequenced.

Clone 11·2; Vβ5·1Dβ2·1Jβ2·2; CDR3: CASSLTAGGQHRGA; also represented a clonal expansion that was statistically significant (P = 0·02), and accounted for 4 of 21 (19%; C.I. 6%, 43%) transcripts sequenced (Table 4). This clone was not identified after amplification by NPA-PCR/Vβ-specific PCR (Table 3), however, a different clone (4·1) that used the same Vβ5·1 and the same N-D-N (CASSLTAGG), but different Jβ was found. The remaining 6 of 21 transcripts were unique when compared to each other.

Sequence analysis after NPA-PCR/Vβ22·1-specific amplification, followed by cloning, confirmed that the clonal expansion of clone 19·1 was statistically significant (P = 0·0009) (Table 4). For the NPA-PCR/Vβ22·1-specific PCR in Table 4, we compared the probability of the fraction of the identical transcripts (5 of 16) found in the Vβ22·1 family against the alternative hypothesis that all transcripts sequenced are present in a single copy only (that is to say they are unique when compared to each other). We obtained unique Vβ22·1 TCR transcripts when using the same methodology from PBMC from normal donors (n = 19 in this experiment, Table 5). This 19·1 clone, Vβ22·1Dβ2·1Jβ2·1; CDR3: CASSEELAGGSYN; accounted for 5 of 16 (31%; C. I. 12%, 56%) of the Vβ22·1 transcripts sequenced. A second clonal expansion that was statistically significant (P = 9 × 10⁻⁵) was clone 18·1, Vβ22·1Dβ2·1Jβ1·4; CDR3: CASSALWKLFFG; and accounted for 6 of 16 (38%; C.I. 16%, 64%) of the Vβ22·1 transcripts sequenced. A single copy of this clone was also found after amplification of β-chain TCR transcripts from the same brain specimen by NPA-PCR/Vβ-specific PCR, followed by cloning and sequencing (Table 3). The remaining five Vβ22·1 TCR transcripts were unique when compared to each other.

PBMC from normal donors were used as methodological control and for the purpose of statistical comparison (Table 5). Sequence analysis of 60 β-chain TCR transcripts from PBMC from normal donors, after NPA-PCR/Vβ5·1-specific PCR or after NPA-PCR/Vβ22·1-specific PCR (Table 5), or after NPA-PCR/Vβ13·6-specific PCR amplification and cloning (data not shown), revealed unique sequences when compared to each other, as anticipated for polyclonal populations of T cells, in agreement with our previous observations [20–23,25,26].

Sequence analysis of β-chain TCR transcripts, amplified by NPA-PCR/Vβ-specific PCR, from an autopsy brain specimen from patient NP89-213 (6 months old, male) is shown in Table 6. Clone 61·4, Vβ21·3Dβ1·1Jβ1·4; CDR3: CASSLTGTNEK; accounted for 11 of 22 (50%; C.I. 29%, 71%) of the β-chain TCR transcripts sequenced. Statistical analysis was performed using the binomial distribution [32], as described above, to determine the probability, P, of the fractions of identical transcripts found in this patient, against the alternative hypothesis that all transcripts sequenced are present in a single copy only (that is to say they are unique when compared to each other) of Vβ TCR transcripts when using the same methodology from PBMC from normal donors. Typically PBMC from normal donors contained unique transcripts when compared to each other [20,21,24–26]. In a typical experiment [25] 22 β-chain TCR transcripts were sequenced from PBMC from a normal donor after amplification by NPA-PCR and cloning, and contained unique transcripts when compared to each other. We used the results from such an experiment [25] as a control for the statistical analysis of the results shown in Table 6. The clonal expansion Vβ21·3Dβ1·1Jβ1·4 (clone 61·4; Table 6) was statistically significant (P = 7 × 10⁻¹⁰). A second clone (612·4), Vβ21·1Dβ2·1Jβ2·7; CDR3: LCASLLRGVRRAV; was found in duplicate copies (2 (9%) of 22).

Table 6. CDR3 Sequences of NPA-PCR/Vβ-specific PCR Amplified β-chain TCR Transcripts from the Brains of Pediatric Patients with AIDS (NP89-213, NP95-184-O, and NP94-34).

Clone	Variable	N-Diverse-N	Joining
Patient NP89-213
	C A S S	L T G	T N E K	Vβ21·3 Dβ1·1 Jβ1·4
61·4	TGT GCC AGC AGC	TTG ACA GGG	ACT AAT GAA AAA	(11 of 22; P = 7 × 10−10)
	L C A S	L L R G V R	R A V L	Vβ21·1 Dβ2·1 Jβ2·7
612·4	CTC TGT GCC AGT	CTA CTC CGA GGG GTA CGA	CGA GCA GTA CTT	(2 of 22)
	C A S S	L E L	A K N I	Vβ5·1 Dβ2·1 Jβ2·4
61·1	TGC GCC AGC AGC	TTG GAA TTA	GCC AAA AAC ATT	(1 of 22)
	C A S S	L V G L R G	N T E A	Vβ5·1 Dβ1·1 Jβ1·1
615·2	TGC GCC AGC AGC	TTG GTA GGG CTA CGT GGA	AAC ACT GAA GCT	(1 of 22)
	C A S S	L W V T G G	E Q F F	Vβ21·3 Dβ2·1 Jβ2·1
614·4	TGT GCC AGC AGC	TTA TGG GTG ACG GGT GGT	GAG CAG TTC TTC	(1 of 22)
	L C A S	K E G A	G E L F	Vβ5·1 Dβ2·1 Jβ2·2
617·4	CTT TGC GCC AGC	AAG GAG GGC GCC	GGG GAG CTG TTT	(1 of 22)
	C A S S	L S H R P A	T S S T	Vβ5·1 Dβ1·1 Jβ2·7
618·4	TGC GCC AGC AGC	TTG TCC CAC AGG CCC GCT	ACG AGC AGT ACT	(1 of 22)
	C A S S	E W G A A	Y N E Q	Vβ11·1 Dβ2·1 Jβ2·1
621·4	TGT GCC AGC AGT	GAA TGG GGA GCG GCC	TAC AAT GAG CAG	(1 of 22)
	C A S S	R G G R H	N E Q F	Vβ7·1 Dβ2·1 Jβ2·1
622·4	TGC GCC AGC AGC	CGG GGG GGA AGG CAC	AAT GAG CAG TTC	(1 of 22)
Patient NP95-184-O
	C A S S	L A S	Y T E A	Vβ3·1 Dβ1·1 Jβ1·1
75·1	TGT GCC AGC AGT	TTA GCC TCC	TAC ACT GAA GCT	(7 of 27; P = 0·0001)
	C A S S	L P D R G F S R	E T Q Y	Vβ5·1 Dβ1·1 Jβ2·5
75·2	TGC GCC AGC AGC	TTG CCG GAC AGG GGG TTT TCA AGG	GAG ACC CAG TAC	(9 of 27; P = 2 × 10−6)
	C A S S	L Y G	P Y N E	Vβ21·3 Dβ2·1 Jβ2·1
75·3	TGT GCC AGC AGC	TTA TAC GGG	CCC TAC AAT GAG	(3 of 27)
	C A S S	E R G T	N S P L	Vβ11·1 Dβ1·1 Jβ1·6
75·4	TGT GCC AGC AGT	GAA CGA GGC ACC	AAT TCA CCC CTC	(2 of 27)
	C A S S	G D S R	D E Q F	Vβ11·1 Dβ2·1 Jβ2·1
75·5	TGT GCC AGC AGT	GGG GAT TCT AGG	GAT GAG CAG TTC	(2 of 27)
	L C S V	E E T D	K Q Y F	Vβ4·1 Dβ1·1 Jβ2·7
75·6	CTC TGC AGC GTT	GAG GAG ACC GAC	AAG CAG TAC TTC	(2 of 27)
	L C S V	V T G D	G Y T F	Vβ4·1 Dβ1·1 Jβ1·2
75·7	CTC TGC AGC GTA	GTT ACA GGG GAC	GGC TAC ACC TTC	(1 of 27)
	L C A S	S S A	N Y G Y	Vβ8·1 Dβ1·1 Jβ1·2
75·8	CTC TGT GCC AGC	AGT TCT GCT	AAC TAT GGC TAC	(1 of 27)
Patient NP94-34
	C A S S	S G Q	A Y N S	Vβ21·3 Dβ1·1 Jβ1·6
86·1	TGT GCC AGC AGC	TCG GGA CAG	GCC TAT AAT TCA	(14 of 31; P = 2 × 10−11)
	C A S S	L D R D	N T G E	Vβ21·1 Dβ2·1 Jβ2·2
86·2	TGT GCC AGC AGC	TTA GAT CGG GAC	AAC ACC GGG GAG	(4 of 31; P = 0·04)
	F C A S	R F E R E L	G Q P Q	Vβ13·6 Dβ1·1 Jβ1·5
86·3	TTC TGT GCC AGC	AGG TTT GAG CGG GAA TTG	GGT CAG CCC CAG	(9 of 31; P = 6 × 10−16)
	C A S S	L A S	Y T E A	Vβ3·1 Dβ1·1 Jβ1·1
86·4	TGT GCC AGC AGT	TTA GCC TCC	TAC ACT GAA GCT	(1 of 31)
	C A S S	L Y S M	N T E A	Vβ8·1 Dβ1·1 Jβ1·1
86·5	TGT GCC AGC AGT	CTG TAC AGC ATG	AAC ACT GAA GCT	(1 of 31)
	C A S S	S T S L	N E K L	Vβ8·1 Dβ1·1 Jβ1·4
86·6	TGT GCC AGC AGC	TCA ACC AGC CTA	AAT GAA AAA CTG	(2 of 31)

Several clonal expansions, two of them statistically significant, were identified after amplification by NPA-PCR/Vβ-specific PCR, cloning and sequencing of β-chain TCR transcripts from an autopsy brain specimen from patient NP95-184-O (eight years and six months old, male) (Table 6). These transcripts included the following: (a) clone 75·1; Vβ3·1 Dβ2·1 Jβ1·1; CDR3: CASSLASYTEA; which accounted for 7 of 27 (26%; C.I. 12%, 47%) of β-chain transcripts sequenced and represented a statistically significant (P = 0·0001) expansion; (b) clone 75·2, Vβ5·1 Dβ1·1 Jβ2·5; CDR3: CASSLPDRGFSRETQY, which accounted for 9 of 27 (33%; C.I. 17%, 54%) of the β-chain transcripts sequenced and represented another statistically significant (P = 2 × 10⁻⁶) expansion; and (c) clone 75·3; Vβ21·3 Dβ2·1 Jβ2·1; CDR3: CASSLYGPYNE, which accounted for 3 of 27 (11%) transcripts sequenced (Table 6).

Several statistically significant clonal expansions of β-chain TCR transcripts were also found in autopsy brain material from patient NP94-34 (eight years and six months old, female) after NPA-PCR/Vβ-specific PCR amplification, followed by cloning and sequencing (Table 6). Clone 86·1; Vβ21·3Dβ1·1Jβ1·6; CDR3: CASSSGQAYNS, accounted for 14 of 31 (45%; C.I. 28%,64%) transcripts (P = 2 × 10⁻¹¹). Clone 86·2; Vβ22·2 Dβ2·1 Jβ2·2; CDR3: CASSLDRDNTGE, accounted for 4 of 31 (13%; C.I. 4%, 31%) transcripts (P = 0·04). Clone 86·3; Vβ13·6 Dβ2·1 Jβ1·5; CDR3: FCASRFERELGQPQ; accounted for 9 of 31 (29%; C.I. 15%, 48%) transcripts (P = 6 × 10⁻¹⁶)(Table 6). Clone 86·4; CDR3: CASSLASYTEA which appeared in a single copy in this patient NP94-34 (Table 6), was clonally expanded in patient NP95-184-O (Clone 75·1) where it accounted for 7 of 27 (26%; C.I. 12%, 47%) (P = 0·0001) sequenced transcripts (Table 6), very likely denoting a T-cell response to the same peptide/MHC epitope in these two patients.

Comparison of the nucleic acid and deduced amino acid sequences obtained here (Tables 3–6) to those in the GENBANK/EMBL/SWISS PROT databases revealed that these sequences were typical of β-chain TCR, have not been previously identified, and, therefore are novel. None of the TCR sequences reported here are identical to those TCR specific for HIV-1 antigens that are currently reported in the GENBANK/EMBL/SWISS PROT databases. Of course this does not rule out the recognition of HIV-1 antigens by the clonally expanded T cells, since their TCR sequences may have not been yet identified and reported to the GENBANK/EMBL/SWISS PROT databases. Certain important homologies have been found between the TCR clones sequenced here and those in the GENBANK/EMBL databases. The most important of these homologies are listed below (Table 7).

Table 7. Clones sharing homology with sequences in the GenBank/EMBL/SwissProt databases.

Patient	Clone	CDR3 sequence	Homologous CDR3	Source	Reference
95–73	1·1/10·2	CASSLELAKN	CASSLELGTE	MBP-specific T-cell clone	[34]
DRB1*1503,				DRA/DRB*0101
0301,DRB1*0602,0201			CASSLKLA CASSLALGTG	HLA-restricted tetanus toxoid-specific T-cell cloneSmall nuclear ribonucleoprotein reactive T-cell clone derived from a patient with connective tissue disease*	[35]
			CASSQEL	Pan troglodytes TCR clone	[36]
	19·1	CASSEELAGGSYNE	CASSLELAGYNE	MBP-specific T-cell cloneDRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401	[37]
			CASSLKLAGG	HLA-restricted tetanus toxoid-specific T-cell clone	[35]
			SSGNLAGG	Pan troglodytes TCR clone	[36]
			CASSPGLAGG	Patient with chronic encephalitis of Rasmussen	[38]
	8·1	CASSFSSGRPGELF	CASSTSGQPGELF	Human T-cell clone from a patient with viral encephalitis DRB1*1201,1501, DQB1*0301,0602, DPB1*0401,0501	[37]
			CASSYASGGP	Patient with chronic encephalitis of Rasmussen	[38]
	21·1	CASSLTVSSYNEQ	CASSLNVNSYNEQ	Human TCR clone isolated from a patient with MS	†
			CASSLTVGSYNEQ	MBP-specific T-cell clone isolated from a normal donor DR7/DRw53	[39]
			CASSLTRSSHNEQ	Mycolic acid-specific CD1b- restricted T cell clone	[40]
	24·1	RCASSSGANV	LCASSSGANV	Human MBP-specific T-cell clone DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401Human MBP-specific T-cell cloneDRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401	[37]
			CASSLGANV	Human MBP-specific T-cell clone	[37]
			LCASSQGANV	Human TCR clone	‡
			LCASSNSGANV	Pan troglodytes TCR clone	[36]
	18·2	CASSLEGSYEQY	CASSQEASYEQY	PBMC-derived human T-cell cloneDRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401	[37]
95–184-O	75·4	CASSERGTNSPL	CASSFLGYNSPL	T-cell clone isolated from human brain plaques from	[37]
DRB1*1503,				a patient with MS
0301,				DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401
DQB1*0602,0201			CASSLRLANSPL	T-cell clone isolated from human brain plaques from  a patient with MS DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401Human T-cell clone from a patient with viral encephalitisDRB1*1201,1501, DQB1*0301,0602, DPB1*0401,0501	[37]
			CASSLRLANSPL	Human T-cell clone from a patient with viral encephalitis DRB1*1201,1501, DQB1*0301,0602, DPB1*0401,0501	[37]

^*PL Talken, M-M Holyst, RW Hoffman. GENBANK Accession Number: AF018168.

^†Z Illes, T Yamamura, T Kondo, K Yokoyama, T Ohashi, T Tabira. GENBANK Accession Number: AB011251.

^‡JD Fraser. GENBANK Accession Number: U47744

Clone 1·1 (Table 3) and clone 10·2 (Table 4), both Vβ5·1 Dβ2·1 Jβ2·4, exhibited substantial CDR3 homology to the following TCR clones (Table 7):

• (a) CK10, a clone specific for myelin basic protein (MBP) [34];

• (b) AKG6, an HLA class II-restricted tetanus toxoid-specific T-cell clone [35];

• (c) AF018168·1, a T-cell clone reactive to small nuclear ribonucleoprotein and derived from a patient with connective tissue disease (P.L. Talken, M.-M. Holyst, R.W. Hoffman) (AF018168);

• (d) a Pan troglodytes TCR clone (U04597) [36] (Table 7).

Clone 19·1, Vβ22·1 Dβ2·1 Jβ2·1; (Tables 3 and 4), exhibited substantial CDR3 homology with the following TCR clones (Table 7):

• (a) KL-3(8), which is an MBP-specific TCR isolated from brain plaques from a patient with MS [37];

• (b) AKG6, a tetanus toxoid-specific class II-restricted T-cell clone [35]. It is possible that the identical amino acid between 19·1 and AKG6 are rather responsible for HLA restriction than for interactions with the peptide;

• (c) Pan troglodytes TCR clone U04605 [36];

• (d) Human TCR clone U55163 [38].

Clones (a–c) are different from clone 19·1 only by the two first amino acids of the NDN (Table 7).

Clone 8·1, Vβ14·1 Dβ2·1 Jβ2·2 (Table 3) exhibited substantial CDR3 homology with the human MBP-reactive T-cell clone SE(8) [37]. The same clone was homologous in the CDR3 to a TCR clone (conservative substitutions of Y for F and A for S) found in the brain of a patient with chronic encephalitis of Rasmussen [38] (Table 7).

Clone 21·1, Vβ23·1 Dβ2·1 Jβ2·1 (Table 3) is homologous in the CDR3 to the following clones:

• (a) a human TCR clone (conservative substitution of N for S), isolated from a patient with multiple sclerosis (Illes Z., T. Yamamura, T. Kondo, K. Yokoyama, T. Ohashi, T. Tabira (baa24999));

• (b) an MBP-specific T-cell clone isolated from a patient with MS [39];

• (c) a mycolic acid-specific CD1b-restricted T cell clone (conservative substitution of H for Y) [40] (Table 7).

Clone 24·1, Vβ6·3 Dβ2·1 Jβ2·6 (Table 3), has substantial CDR3 homology with MPB-specific T-cell clones found in brain plaques from a patient with MS: Clone KL-3(18) and clone KL-3(19) [37] (Table 7). Other CDR3 homologous clones include:

• (a) a human TCR clone (U47744);

• (b) a Pan troglodytes TCR clone (U04554) [36](Table 7).

Clone 18·2, Vβ5·1 Dβ2·1 Jβ2·7 (Table 4), exhibited CDR3 homology to the PBMC-derived human MBP reactive T-cell clone BM(5) [37](Table 7).

Clone 75·4, Vβ11·1 Dβ1·1 Jβ1·6 (Table 6), has partial sequence homology in the CDR3 with the following MBP-specific TCR transcripts:

• (a) clone KL-1(7), isolated from human brain plaques from a patient with MS [37];

• (b) clone KL-1(3), isolated from human brain plaques from a patient with MS [37];

• (c) clone SE(3), isolated from human inflammatory brain lesions from a patient with MS (conservative substitution of R for Q) [37];

• (d) clone SE(4), isolated from human inflammatory brain lesions from a patient with MS (conservative substitution of E for Q) [37] (Table 7).

Twenty-six of 141 β-chain TCR transcripts sequenced from these brain specimens exhibited substantial CDR3 homology to MBP-specific β-chain TCR clones. However, these TCR clones were restricted mainly to one patient (NP95-73), where 24 of 61 (39%) TCR clones exhibited homology to MBP-specific TCR. Pathologic examination of the brain of this patient at autopsy revealed myelin pallor, which was not observed in any one of the other patients (Table 1). Epitope spreading may be responsible for these T-cell responses. Such TCR were absent in patients NP89-213 and NP94-34 and accounted for only 2 of 27 of the β-chain TCR transcripts sequenced from patient NP95-184-O.

DNA based HLA typing of the two patients that expressed β-chain TCR transcripts with homology to those of T-cell clones either specific for MBP or found in the brain of patients with MS or viral encephalitis revealed the following: Patient NP95-73: HLA-A*29xx, 30XX, DRB1*1503,0301, DQB1*0602,0201; Patient NP95-184-O: HLA-A*01xx, 02XX, DRB1*1503,0301, DQB1*0602,0201. These two patients exhibited identical class II, and different class I HLA-A molecules. Clones 19·1, 8·1, 24·1 and 18·2 expressed in the brain of patient NP95-73 and clone 75·4 expressed in the brain of patient NP95-184-O not only exhibited CDR3 highly homologous to T-cell clones expressed in other patients/donors reported in the literature as either specific for MPB or expressed in the brain of patients with MS or viral encephalitis (Table 7), but also shared the DQB1*0602 antigen with those patients/donors. Additionally, these patients/donors are reported as DRB1*1501 (Table 7). Patients NP95-73 and NP95-184-O are DRB1*1503, which is only one amino acid different from DRB1*1503 at position DRbeta30 [DRB1*1501(Y30) versus DRB1*1503(H30)].

The amino acid Leu was found in the first position of the N-D-N sequence in 67 of 141 (48%) β-chain TCR transcripts sequenced from the brain of children with AIDS. Such transcripts accounted for 45% (28 of 61) of all transcripts sequenced from patient NP95-73 (Tables 3,4), for 60% (15 of 22) of those sequenced from patient NP89-213 (Table 6), for 70% (19 of 27) of those sequenced from patient NP95-184-O (Table 6), and for 19% (6 of 31) of those sequenced from the fourth patient NP94-34 (Table 6). TCR β-chain transcripts with Leu in the first N-D-N position accounted for 90% of the Vβ5·1 TCR transcripts sequenced from patient NP 95–73. However, Leu in the first N-D-N position was not found in any of the Vβ22·1 transcripts sequenced from this patient. Leu was also found in the first N-D-N position in 10 (17%) of 60 TCR β-chain transcripts from the peripheral blood from normal donors. Leu in the N-D-N sequence was followed by: (a) Thr (T) in two patients, NP95-73 and NP89-213, in 6 of 61 (10%) and 11 of 22 (50%) transcripts, respectively; whereas it was present in only 3 of 60 (5%) of TCR β-chain transcripts from normal donors; and (b) Ala (A) in three patients, NP95-73, NP95-184-O and NP94-34, in 17 of 61 (28%) and 1 of 22 (5%), 7 of 27 (26%) and 1 of 31 (3%), transcripts, respectively. Ala following a first position Leu in the N-D-N sequence was only found in 1 of 60 (2%) TCR β-chain transcripts from normal donors.

It may be argued that amplification of β-chain TCR transcripts from very few T cells by two PCR cycles may lead erroneously to results resembling those reported in Tables 3–6. We have demonstrated that this is not the case and that the results presented here do indeed reflect true oligoclonal expansions. We have carried out studies using methodological controls (PBMC from normal donors) to address this very question [26]. In these experiments [26], cell mixtures (a total of 1 × 10⁶ cells) comprised of various proportions of an ovarian tumour cell line (CAOV3) which does not express TCR transcripts and peripheral blood T cells from a normal donor were prepared containing 6% and 0·6% T lymphocytes. RNA was prepared from the mixtures as described above and 50 ng of RNA from each mixture was used for amplification by NPA-PCR, cloning, and sequencing of β-chain TCR transcripts. It is widely accepted that 50 ng of RNA has been derived from 50 000 cells. Therefore these two mixtures will contain β-chain TCR transcripts from 3000 and 300 T cells, respectively. Sequence analysis, after NPA-PCR amplification and cloning, revealed the presence of unique transcripts when compared with each other in these two mixtures, typical of polyclonal populations of T cells (data not shown) [26].

Using immunohistochemistry techniques [18] we determined the number of CD3+ T cells in the brain of a representative population of paediatric patients with AIDS versus the total number of cells on formalin-fixed paraffin-embedded sections [18]. The number of CD3+ T cells versus the total number of cells for these patients were as follows: patient 1: 104 (10%) of 1026; patient 2: 27 (3%) of 870; patient 3: 81 (8%) of 1047; patient 4: 281 (18%) of 1552; and patient 5: 24 (2%) of 1147. The range was from 2% to 18% with a mean of 8%. Since 50 ng of RNA, representing 5 × 10⁴ cells, were used for these experiments, 8% of this RNA has been derived from 4000 CD3+ T cells. Even in the case of the specimen with the lowest percentage of T cells (2%), 50 ng of RNA would still contain transcripts from 1000 CD3+ T cells. These numbers of T cells are higher than the numbers of T cells (lower end of the range) used in our control experiments (3000 and 300 T cells; see previous paragraph). These control experiments revealed the presence of unique β-chain TCR transcripts when compared to each other, typical of polyclonal populations of T cells, in PBMC from normal donors. Therefore, the clonal expansions of T cells observed in autopsy brain specimens from children with AIDS represent true oligoclonal expansions of T cells.

Similarly, three mixtures of cells from the ovarian tumour cell line (CAOV3) and peripheral blood T lymphocytes from a normal donor were prepared (total cells 1 × 10⁶) and contained 6%, 2·5%, and 0·6% T lymphocytes. RNA was prepared from these mixtures and 50 ng containing 3000, 1250, and 300 T cells were amplified by Vβ2-specific PCR, cloned and sequenced. Amplification of Vβ2+ TCR transcripts was selected as representative of a Vβ family, with Vβ2+ cells representing 8% of the T cells in the peripheral blood [26]. The proportions of Vβ2+ cells in the three mixtures above were 240, 100, and 24 cells, respectively. Sequence analysis after PCR amplification and cloning from the first two mixtures revealed unique transcripts when compared to each other, typical of polyclonal populations of T cells. Sequence analysis of the specimen containing transcripts from only 24 Vβ2+ T cells revealed a somewhat restricted pattern of expression. The brain specimens from paediatric patients with HIV-infection contained on the average 320 Vβ2+ cells or for the specimen with the lowest value, 80 Vβ2+ T cells. These cell numbers are in the range or above the range of those used in our control experiments, demonstrating that clonal expansions observed in experiments using this cell number range are genuine.

Discussion

To determine the clonality of T-cells infiltrating the perivascular space of blood vessels in the brains of children with AIDS, we amplified, cloned and sequenced β-chain TCR transcripts from autopsy brain specimens with angiocentric mononuclear cell infiltrates from four of these patients. Sequence analysis revealed substantial proportions of identical β-chain TCR transcripts, demonstrating the presence of oligoclonal T-cells infiltrating the CNS in all four patients examined, and in many cases, significantly expanded. Although the specificity of these CNS-infiltrating T cells is unknown, these T-cells may have undergone antigen-driven proliferation and clonal expansion in situ, in the brains of these paediatric AIDS patients in response to either viral or self-antigens.

Limited information is available in neuroAIDS, including paediatric AIDS, on whether T cells are present in the infected brain of these patients. Inflammation in the CNS of patients with HIV-1 infection or AIDS has been associated with the presence of cells of the monocyte/macrophage lineage and not of T cells [14]. Monocytes have been reported as the cell responsible for the introduction of the HIV into the brain (reviewed in [1]). We have reported [18] the presence of angiocentric CD3+CD45RO+ T-cell infiltrates in the brain of children with AIDS. These results have been extended by others who report the presence of T-cell infiltrates in the brain of patients with AIDS [41] or rhesus macaques infected with simian immunodeficiency virus (SIV) [42]. We report here for the first time clonal expansions of β-chain TCR transcripts in the autopsy brain of children with AIDS, demonstrating that the infiltrating T cells have undergone specific antigen-driven proliferation and clonal expansion. These infiltrating T cells are in their vast majority CD3+CD8+ [18] and they may recognize viral or self epitopes. Inflammation in the CNS in HIV-1 infected patients is very different than encephalitis induced by other neurotropic viruses, because of the profound immunodeficiency found in these HIV-1 infected patients. Additionally, the role of CD8+ T cells in controlling HIV-1 infection in the brain is poorly understood. AIDS patients receiving HAART therapy resulting, at least in part, in immunorestoration of their immune functions, develop encephalitis mediated by CD8+ T cells [43]. These findings indicate that these CD8+ T cells may recognize self-antigens [43].

T-cells infiltrating the blood vessels in the CNS of children with AIDS may indeed represent an immune response to HIV-1 and may be partially responsible for containing the infection. If this is true, it is not known which are the antigen-presenting cells (APC) and which are the HIV-1 antigens that elicit the response. Strong HLA class I-restricted HIV-1 specific CD8+ cytotoxic T lymphocyte (CTL) responses have been well documented in HIV-1 infected individuals (reviewed in [44–46]). The appearance of these CTL correlates well with the decline of viremia, and their loss is associated with disease progression (reviewed in [44–46]). Several peptides derived from either structural or regulatory HIV-1 antigens are recognized by T cells in association with HLA class I or class II (reviewed in [44–46]).

Alternatively, T cells infiltrating blood vessels in the CNS of paediatric patients with AIDS may respond to self-antigens, and thus may be autoimmune in nature. This hypothesis is supported by two major arguments: firstly, the viral loads in the CNS are very low during the inflammatory response and, secondly, the presence of the infiltrating T cells is not associated with the appearance of multinucleated giant cells. Price [47] has suggested that the extensive immune dysregulation that occurs during the initial phase of the HIV-1 infection and its progression may lead to autoimmune conditions. Hoffman [48] has proposed a model where the TCR plays a role both in HIV infection and in the auto-immunity that follows the infection. HIV-specific T cells are preferentially infected with the virus, and HIV, acting as an antigen, stimulates the expansion of the infectable pool of T cells. Later on during the course of the disease immunity against HIV cross-reacts with suppressor T cell clones, disrupting the function of the helper T cell repertoire and resulting in autoimmunity [48]. Several additional lines of evidence support an autoimmune T-cell response.

HIV-1-infected patients have also in the circulation autoreactive CTL, which recognize host, and not viral, peptides [49–51]. HIV-1-infected humans have circulating CTL that lyse uninfected CD4+ T cells. HIVgp120 activates CD4-specific T-cell responses against cryptic CD4 peptide epitopes by altering the processing of the CD4 molecule [49]. This mechanism may be responsible at least in part for the severe depletion of CD4+ T cells that is so commonly observed in patients with AIDS, suggesting that an autoimmune T-cell response is linked to HIV-1 infection [52]. Overexpression of cellular proteins in HIV-1-infected cells has been reported, and may be caused by Tat-mediated transactivation of cellular promoters [53]. For example, vinculin is overexpressed in T and B cells from HIV-1-infected patients, and many of these patients exhibited an antivinculin CTL response [50]. Additionally it has been suggested that immune responses to retrovirus-like particles, distinct from known exogenous retroviruses, and an immune response to endogenous retroviruses have been found in autoimmune diseases [54].

Autoantibodies against antigens expressed on T cells [55] and other cells, including brain cells [56], α-myosin [57], and erythropoietin [58], have been reported in HIV-infected patients [59]. Structural homologies between HIV-1 envelope protein and several molecules expressed on the cell surface, such as HLA-DR4, -DR2, certain V regions of α-, β- and γ-chains of the TCR, Fas protein, and others, may be responsible for this autoreactivity [55,57,58,60]. Also, peripheral neuropathies are commonly observed in HIV-1 infected patients [61] and may be related to autoimmune vasculitis [47,62].

In addition, HIV-1 specific T cells may recognize as viral, those host determinants, that cross react with the virus due to molecular mimicry. Molecular mimicry is defined as the sharing of common antigenic epitopes between host proteins and microorganisms [56,63–66]. Molecular mimicry is responsible for the development of several autoimmune diseases (reviewed in [56,66]). As previously discussed, extensive homologies between HIV-1 antigens and a number of host proteins exist which have the potential to result in extensive molecular mimicry [56,63,67,68].

Clonally expanded T cells specific for common herpes viruses, such as Epstein Barr virus (EBV) and cytomegalovirus (CMV), have been reported to be nonspecifically trapped in inflammatory sites located at the synoviun, the eye or the brain of patients with autoimmune diseases [69,70]. These reports have been focused primarily in the rheumatoid synovium. It could be argued that these antiviral T cells contribute to the clonal expansions reported here in the brain of paediatric patients with AIDS. Although the contribution of these antiviral T cells in the clonal expansions reported here cannot be excluded, several lines of evidence suggest that they are rather ‘bystanders’ in these inflammatory sites and their presence is coincidental and nonspecific, with the possible exception of the synovial membrane of patients with rheumatoid arthritis (RA), where molecular mimicry between viral and host epitopes may be responsible [71–73]. Activated or memory T cells express on their cell surface in high density the chemokine receptor CCR5 [74,75] and may be recruited to inflammatory sites by nonspecific means such as chemokines [76]. In addition, the proportions of T cells specific for a single epitope of an EBV lytic cycle protein [70,75] in the synovium of patients with rheumatologic diseases is small (4–15·5% of the T cells present) and cannot account for the high proportions of identical β-chain TCR transcripts found in this study in the brain of paediatric patients with AIDS. The proportions of these EBV-specific T cells have been determined using an HLA class I-peptide tetramer staining method which provides erroneously high estimates of these T cells [77]. Furthermore, EBV gene expression in the synovium has been demonstrated only in low proportions (less than 20%) or was absent in patients with rheumatic diseases other than RA [78,79]. In RA it was found in up to 50% of the patients [79]. Additional studies do not support the role of clonally expanded anti-EBV T cells in the pathogenesis of RA [80,81].

Nevertheless, a definite answer on whether the clonally expanded T cells in the brain of patients with AIDS recognize viral or self antigens will require identification of the antigen(s) recognized by the clonally expanded TCR. This is one of the targets of this research program. Such identification is an ambitious goal. Sequencing of the clonally expanded TCR transcripts in vivo is required in order to identify the antigen(s) that these T cells recognize. This can be achieved by expressing full-length copies of the clonally expanded TCR into appropriate T cells and by using these T cells to screen putative antigens or appropriate cDNA libraries expressed on appropriate antigen-presenting cells.

Several cell types in the CNS may be capable in serving as APCs. These include:

• (a) monocyte/macrophages, microglia and multinucleated giant cells. All these cells can be infected by HIV-1 and can present antigen to T cells;

• (b) cerebrovascular endothelial cells, expressing HLA class II, can effectively present antigen to T cells [82]. Furthermore, cerebrovascular endothelial cells can be infected with HIV-1 in vitro, very likely through a CD4/galactosylceramide-independent mechanism [83–85]. Certain HIV-1 strains appear to selectively infect cerebrovascular endothelial cells, although viral replication of HIV-1 in these cells appear to be minimal [85]. Peptides derived from these and other HIV-1 proteins may be presented to T cells by activated endothelial cells and may trigger a T cell response directed against blood vessels especially during the early stage of the HIV-1 infection;

• (c) brain microvascular smooth muscle cells (SMC) treated in vitro with IFN-γ are able to present antigen to CD4+ T cells [86];

• (d) Although HIV-1 can infect fetal neurones [87] and the virus is occasionally found in pyramidal neurones [88], neurones in adults do not appear to be infected by HIV-1 [87–91];

• (e) restricted replication of HIV-1 has been also found in astrocytes, and in particular in perivascular astrocytes [90,91].

The extraordinary ability of HIV-1 to mutate has led to speculation that it facilitates the selection of mutants that avoid CTL responses. Some viral mutations detected in asymptomatic HIV-1-infected patients may lead to a great reduction or abrogation of CTL activity [45,46]. Molecular studies revealed that CTLs recognizing certain viral epitopes are monoclonally expanded during primary HIV-1 infection [92,93] and disappear later perhaps due to clonal exhaustion [94], whereas, the viral epitopes that were recognized are still present. CTLs control viral load either by lysing the virus-infected cells [94,95] or by noncytolytic suppression of HIV-1 replication [89–91]. The latter is mediated in part via chemokines [96,97] or certain cytokines [98]. HIV-1-specific CTLs have been reported in the cerebrospinal fluid (CSF) of HIV-1-infected patients [99,100]. Whether CTLs exhibit a damaging effect on CNS either directly and/or through the production of cytokines is not clear at the present.

T cells in HIV-1 infection are under the influence of two opposing forces: (a) proliferation and clonal expansion in response to viral antigens; and (b) elimination directly by CTL [94,101], direct viral cytotoxicity [102], and apoptosis [103,104]. TCR sequencing of CD4-positive and CD4-negative peripheral blood T cells from HIV-infected patients demonstrated oligoclonal expansions only in the CD4-negative population. Longitudinal studies of PBL during primary HIV-1 infection revealed major early HIV-specific oligoclonal expansions of Vβ families which declined later on [101,105], perhaps because of high dose antigen-induced tolerance or clonal exhaustion. Longitudinal analysis of TCR usage by CTL clones which recognized an HLA-B14-restricted epitope of gp41 in one HIV-1-infected patient revealed a monoclonal expansion [106].

Although the oligoclonality of the anti-HIV-1 response in the peripheral blood of these patients has been studied, there is only limited information on the TCR transcripts utilized by T cells infiltrating organs. In spleen white pulps HIV-1-specific T cells are highly compartmentalized [107]. In lymph nodes [108] and lungs [109] certain Vβ families were overrepresented in CD8+ T cells. T cells in labial salivary glands in patients with the sicca syndrome of HIV-1 preferentially utilized Jβ1·2 [110]. There is no information about the specificity of T cells infiltrating the CNS of HIV-1-infected patients. Comparison of the nucleic acid and deduced amino acid sequences of β-chain TCR transcript obtained in this study (Tables 3–6) to those in the GENBANK/EMBL/SWISS PROT databases revealed that none of the sequences reported here are identical or substantially homologous to the TCR sequences of T-cells with anti-HIV activity already contributed to these banks.

Substantial proportions (24 of 61; 39%) of β-chain TCR transcripts from patient NP95-73 exhibited CDR3 homology to TCR transcripts expressed in MBP-specific T cells. Two of these TCR transcripts were clonally expanded (Tables 4–7). One β-chain TCR transcript (present in two copies) from patient NP95-184-O also exhibited CDR3 homology to TCR transcripts isolated from the brain of patients with MS or with viral encephalitis.

It is remarkable that clones expressed in the brain of patients NP95-73 and NP95-184-O not only exhibited CDR3 highly homologous to T-cell clones specific for MPB or expressed in the brain of patients with MS or with viral encephalitis, but also shared HLA class II with the donors/patients who expressed these T-cell clones (See Results and Table 7). It is therefore likely that T-cell clones identified in the brain of patients NP95-73 and NP95-184-O may recognize MBP or another CNS self antigen and that this recognition is restricted by either DRB1*15 or DQB1*0602 specificities. Patient NP95-73 was the only one from those studied here that exhibited myelin pallor in the brain (Table 1). Clonally expanded MBP-specific T cell clones may be responsible for myelin pathology in this patient. These MBP-specific T cell responses may be attributed to epitope spreading, a phenomenon that was originally described in autoimmune demyelinating diseases of the CNS [111–114]. It is defined as the generation of de novo immune responses to epitopes different than those that initiated the immune response. The acquired neoreactivity (epitope spreading), appears to be the result of endogenous priming to new self determinants [111] during the chronic inflammatory conditions that are associated with CNS demyelinating disease. Chronic inflammation may result in breaking tolerance to self antigens and may be responsible for epitope spreading. On the other hand, de novo priming of self reactive T cells to sequestered (auto)antigen epitopes, released as a result of an immune response, may also lead to epitope spreading, as demonstrated in CNS demyelinating diseases [111–114].

In conclusion, we report here the presence of oligoclonal T cells infiltrating the brain of children with AIDS. It is very likely that these T cells have undergone antigen-driven proliferation and significant clonal expansion in vivo, in situ, in the brain, in response to as yet unidentified antigens, either viral or host.