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. 2002 Apr;128(1):155–162. doi: 10.1046/j.1365-2249.2002.01799.x

Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals

G LI PIRA *, D FENOGLIO *, L BOTTONE *, P TERRANOVA *, E PONTALI †, F CAROLI ‡, M SERI ‡, J-C CAILLIEZ §, G KOOPMAN , R ACCOLLA §§, F DEL GALDO **, G ABBATE **, R DE PALMA **, F MANCA *
PMCID: PMC1906359  PMID: 11982603

Abstract

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.

Keywords: CD4 cells, HIV, opportunistic infections, Pneumocystis carinii, T cell receptor, spectratyping

INTRODUCTION

The decline of CD4 lymphocytes in HIV infection [1,2] becomes clinically relevant when CD4 cells specific for opportunistic pathogens fall below a crucial threshold and thus no longer keep under control the relevant pathogen [3]. Diverse functions of specific CD4 cells are needed to control the different pathogens. A Th1 type function (e.g. characterized by production of IFN-γ upon antigen activation) facilitates the phagocytic and killer function of macrophages and it is relevant in mycobacterial and fungal infections [4]. A T helper function for antibody production and for expansion of specific CTL is critical to control latent virus reactivation [5,6].

Restoration of CD4 counts (endogenous reconstitution) can be obtained in responsive patients by treatment with antiretroviral therapy (ART) [7–12], or by reinfusion of in vitro expanded, polyclonal or antigen-specific CD4 cells (exogenous reconstitution) in ART non-responders [13,14]. Therefore, the analysis of the CD4 repertoires specific for relevant opportunistic pathogens should be evaluated further at the clonal level to understand better the specific immune status and its evolution.

We analysed the repertoire of CD4 T cells specific for Pneumocystis carinii because it causes severe pneumonia in AIDS [15], and specific CD4 cells are essential for protection [16,17]. In order to dissect the clonal make-up of the P. carinii-specific CD4 repertoire in control individuals and in HIV-infected, asymptomatic donors, P. carinii-specific CD4 T cell lines were generated and analysed for TCR BV gene usage and for TCR CDR3 region length heterogeneity by spectratyping [18].

MATERIALS AND METHODS

Media and antigens

RPMI 1640 (Flow, Irvine, Scotland, UK) containing penicillin, streptomycin, 2-mercaptoethanol and l-glutamine and 5% human AB serum [19] was used for proliferation assays and for generation and expansion of T cell lines. Human recombinant IL2 (Proleukin) was kindly provided by Eurocetus, Milan, Italy and used at 30 U/ml final concentration. P. carinii was prepared from homogenized lungs of immunosuppressed rats by gradient fractionation, as described previously in detail [20]. Candida albicans,Cryptococcus neoformans and Aspergillus fumigatus were grown in RPMI 1640 medium for 2 days. All pathogens were washed twice in PBS and autoclaved [14]. The pathogens were used at 2 × 106 bodies/ml final concentration in culture. This concentration, determined in preliminary titrations, was optimal for PBMC stimulation.

Proliferation assay

PBMC were obtained from a Ficoll-Hypaque gradient of heparinized venous blood, as described previously in detail [21]. PBMC at 2 × 106/ml medium were seeded at 200 ¼l per well in a flat-bottomed microtitre plate containing 20 ¼l antigens at 10-fold concentration. The wells were pulsed with 0·5 ¼Ci3H]thymidine (5 Ci/mmole specific activity, Amersham, Amersham, UK) on day 4 and harvested on day 5. The dry filters were counted in a Canberra Packard Matrix 9600 beta counter without scintillation fluid. Results are shown as mean values of duplicate wells and are expressed as kcpm (cpm/1000).

Generation of specific T cell lines

Antigen-specific CD4 cell lines were generated as described previously in detail [19,22,23]. Briefly, PBMC at 10 × 106/ml were pulsed with P. carinii at 2 × 106 bodies/ml final concentration for 4 h. The cell suspension was diluted to 2 × 106/ml and plated in a 24-well cluster plate (Costar, Cambridge, MA, USA) at 1·5 ml per well. Four days later IL2 was added to the wells, which were split according to cell growth. Expanded T cells were stimulated further after 3 weeks by plating 5 × 105 T cells with 106 autologous, P. carinii pulsed, 3000 cGy irradiated, fresh PBMC as a source of APC in the presence of IL2. The wells were split according to cell growth and at least four stimulation cycles were performed to select antigen specific CD4 cells. At this stage the lines contained >98% CD4 cells. The P. carinii reactive lines were conventional CD4 T cell lines according to MHC restriction and specificity. In fact, they were stimulated only by autologous or by MHC class II compatible APC and did not respond to other fungal antigens such as candida, cryptococcus or aspergillus. In terms of frequency of antigen-responsive cells, it can be estimated close to 100% after four restimulation cycles. Clones generated by limiting dilution following PHA stimulation from CD4 T cell lines produced according to this protocol exhibit antigen specificity when challenged with antigen and autologous APC. This was observed with T cell lines specific for different antigens, including tetanus toxoid, purified protein derivative (PPD) and C. albicans (data not shown). In addition, P. carinii exhibited no mitogenic activity for CD4 T cell lines specific for unrelated antigens such as tetanus toxoid, PPD and HIV gp120 (data not shown).

TCR BV gene family usage by RT-PCR and spectratyping

Two weeks after the last stimulation cycle, 5 × 106 CD4 T cells were washed and pelleted for RNA extraction and subsequent analysis. Details on RNA extraction, retrotranscription, amplification [24–27] and spectratyping are given elsewhere [18,24,28–31]. Briefly, retrotranscribed cDNA was amplified with a panel of BV specific primers [25,27]. The amplified products, separated on an agarose gel, were quantified as pixel intensity of the ethidium bromide stained bands with a Foto Analyst II system (FotoDyne, Inc., Hartland, WI, USA). For spectratyping, cDNAs were titrated for TCR message in order to amplify the same amount of starting material and amplified in a single PCR tube containing two primers specific for two different TCR BV families and a fluorescent primer specific for the TCR BC region [31,32]. PCR products were run and analysed on a sequencing gels in a fluorescence-based DNA sequencer (377, Applied Biosystem, Foster City, CA, USA). By analysis with Genescan software (Applied Biosystem) the bands are shown as peaks that can be quantified. An amplified TCR-BV family migrates as a series of bands corresponding to different CDR3 lengths and in normal conditions a TCR BV gene family shows a pseudo-Gaussian distribution. Alterations in distribution and/or intensity of peaks are expression of repertoire perturbation. PBMC from normal donors and from seropositives were cultured with PHA and IL2 for 2 weeks, collected and processed as the T cell lines as controls of activated polyclonal T cells.

RESULTS

Proliferative response of PBMC to P. carinii

For this study we selected two HIV seropositives (PG and RP, asymptomatic stage CDC A1 and CD4 counts >500, age 34 and 44 years), who became non-responders to P. carinii in a proliferation assay although they were responsive to other fungal antigens, as shown in Fig. 1. Loss of response was defined because LSI was higher than 8 in two previous assays (12 and 6 months) and dropped to <2 in two consecutive assays, 1 month apart. These two patients were identified after screening 36 asymptomatics who exhibited positive proliferative responses to P. carinii (LSI range 4–14). Since a large majority of the population (>95%) shows proliferative response to P. carinii[33–35], the inability of the two selected patients to respond to P. carinii was attributed to loss of a pre-existing function rather than to lack of priming. An example of an asymptomatic patient responding to P. carinii1 is also shown (GLB, right panel, Fig. 1) Therefore, in order to discriminate between vertical and horizontal clonal depletion of the P. carinii-specific repertoire, CD4 T cell lines were generated and analysed for clonal diversity.

Fig. 1.

Fig. 1

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PBMC proliferative response. Proliferative response of PBMC from three asymptomatic HIV seropositives (stage A1) to several opportunistic fungi (Pc, Pneumocystis carinii; Crypto, Cryptococcus neoformans; Asp, Aspergillus fumigatus, Ca, Candida albicans). Proliferation was negative in the presence of P. carinii, with LSI (lymphocyte stimulation index) <2 in patients PG and RP. This lack of response to P. carinii in these two patients followed two positive tests (LSI > 8) that were performed 12 and 6 months before this assay. The negative response was confirmed 1 month later in both patients. The T cell lines were generated at the stage of negative response. Patient GLB (right panel) is shown here as representative of P. carinii responders.

Proliferative response of P. carinii-specific CD4 T cell lines

The asymptomatic state of the two patients (absence of opportunistic infections and of clinically overt P. carinii infection, before the study and during the 1-year follow-up after generation of T cell lines) suggested that residual, but undetectable, P. carinii-specific CD4 cells were present and able to keep the pathogen at bay. Specific CD4 cells have been demonstrated to be essential control to P. carinii infection [16,17]. This was indeed the case, since P. carinii-specific CD4 T cell lines were generated successfully from peripheral CD4 T lymphocytes of these patients (PG and RP) stimulated in vitro with antigen pulsed, autologous presenting cells, in spite of the absence of proliferative response (Fig. 2, lower panels). A T cell line was also generated from a P. carinii-responding, HIV infected donor (GLB). The upper panels of Fig. 2 show proliferation of P. carinii-specific CD4 lines generated from four normal donors as controls. The generation of CD4 T cell lines makes available a selected population of antigen specific CD4 cells that can be examined for clonal heterogeneity.

Fig. 2.

Fig. 2

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Proliferative response of P. carinii-specific CD4 T cell lines. Antigen-driven proliferative responses of P. carinii-specific CD4 T cell lines from four normal controls (upper panels) and from the three asymptomatic patients (lower panels). Proliferation in the absence (−) and in the presence (+) of P. carinii was assessed after four restimulation cycles.

TCR BV gene usage by P. carinii-specific CD4 T cell lines

As shown in Fig. 3, upper panels, the P. carinii-specific lines from normal controls exhibited a remarkable clonal heterogeneity, as defined by usage of numerous TCR BV gene families. This is no surprise, as P. carinii is a complex pathogen that contains a large number of antigenic proteins [36,37], each one encompassing a variety of epitopes recognized by diverse CD4 clones. A similar clonal heterogeneity was exhibited by the T cell lines derived from the three patients (Fig. 3, lower panels), irrespective of their P. carinii responder status.

Fig. 3.

Fig. 3

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TCR BV gene family usage by P. carinii-specific CD4 T cell lines. RNA extracted from antigen specific T cell lines and retrotranscribed into cDNA was used in an RT-PCR assay to analyse usage of different TCR BV gene families. The upper panels show the profiles of four lines generated from normal donors. Numerous TCR BV families are expressed, indicating clonal heterogeneity of the P. carinii-specific CD4 response in normal donors. The lower panels show that the P. carinii-specific CD4 T cell lines from the three seropositive donors (two P. carinii non-responders and one P. carinii responder) exhibit a similar clonal diversity according to expressed TCR BV gene families.

Spectratyping of P. carinii-specific CD4 T cell lines

The information on TCR BV gene family usage does not mean that clonal heterogeneity is maintained in patients with the same breadth as in normal controls. Numerous specific clones using the same TCR BV gene family may be present in the lines from normal controls, whereas the lines from the patients may carry only one or few clones for each TCR BV gene family. By looking at the different lengths of the hypervariable CDR3 regions of several TCR BV gene families well-expressed in the T cell lines, we could dissect their clonal make-up further.

As shown in Fig. 4 (upper panels), spectratyping was performed on several well-expressed TCR BV gene families by the P. carinii-specific T cell lines from the patients. Even though this does not correlate directly with the actual frequency of cells using a given TCR BV gene family (because of different amplification efficiency of the family specific primers), primers designed for comparable efficiency were used for spectratyping [38–40]. This analysis showed a limited size distribution, with single peaks or with one dominant peak flanked by minor peaks in each TCR BV gene family. This limited repertoire within well-expressed TCR BV gene families was compared to the repertoire of T cell lines generated from HIV-negative donors. In this case (Fig. 4, lower panels) similar profiles were observed, with single or dominant peaks in the BV gene families analysed. As a positive control for polyclonality, the heterogenous distribution of CDR3 sizes of peripheral lymphocytes with the expected Gaussian profiles [18] was observed (data not shown).

Fig. 4.

Fig. 4

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Spectratyping of P. carinii-specific CD4 T cell lines. The spectratype profiles were performed on selected BV gene families well expressed in P. carinii-specific T cell lines from HIV seropositives (upper panels) and from normal HIV seronegative controls (lower panels). Single or dominant peaks are evident in these profiles. Spectratypes were also performed on PBMC blasts from normal controls. As expected, profiles with pseudo-Gaussian distribution of the peaks were evident in these cases, reflecting the polyclonal nature of peripheral T cells (data not shown). Similar pseudo-Gaussian profiles (data not shown) were obtained by spectratyping PBMC derived PHA blasts from the patients.

DISCUSSION

A visual aid to illustrate the concept of vertical and horizontal depletion we have investigated in this study is provided in Fig. 5. In the case of vertical depletion, the residual clones may not have the appropriate fine specificities or the appropriate functions (e.g. Th1 function) to provide an optimal protection. Therefore, such a repertoire, even if re-expanded endogenously (after ART) or exogenously (after infusion of the same cells expanded in vitro), may be inadequate for protection. In fact, it may carry over the clonal holes [41] unless ex novo generated naive lymphocytes appear with the desired specificity and repopulate the repertoire after antigen-driven selection [42]. Persistent repertoire perturbations have been reported according to spectratype analysis, but these studies investigated the polyclonal CD4 cell population as a whole, irrespective of antigen specificity [43–46]. The alternative – but not mutually exclusive – model of horizontal depletion envisages the loss of a relevant fraction of cells from each of the clones accounting for the specific repertoire. This could be a more favourable case. CD4 reconstitution may result in recovery of a fully heterogeneous repertoire (that mirrors the pristine repertoire) with optimal protective function, as enough cells were spared for each clone.

Fig. 5.

Fig. 5

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A hypothetical model for horizontal and vertical depletion of an antigen-specific CD4 T cell repertoire. (a) illustrates a normal CD4 T cell repertoire specific for a model antigen. Each bar represents one clone and the height of the bar indicates the arbitrary cell number accounting for each clonal progeny. (b) All of the clones have lost a fraction (90%) of their cells as a consequence of horizontal depletion, but a few cells are spared for each clone. (c) Most clones (90%) were lost as a whole, but one residual clone is left with its intact progeny, to illustrate vertical depletion. Even though in both cases the percentage reduction in CD4 numbers may be similar (90% reduction of CD4 cells in this example), the modes are different, but not mutually exclusive. If a 100% recovery of CD4 cells occurs as shown in the bottom panels, reconstitution of the horizontally depleted repertoire results in recovery of clonal heterogeneity (d), while this is not the case for vertical depletion (e).

Our data indicate that in asymptomatic HIV seropositives, even in the absence of a proliferative response to the ubiquitous pathogen P. carinii, residual specific CD4 cells are preserved below the detection threshold. In vivo they suffice to prevent outbreak of P. carinii infection, as in immunocompetent individuals, and in vitro they are able to give rise to antigen-specific CD4 T cell lines. Most importantly, the clonal heterogeneity of the P. carinii-specific repertoires was similar in normal controls and in seropositive asymptomatic individuals.

The profiles with single or dominant peaks were similar in the two groups, suggesting a very limited clonal diversity of individual TCR BV gene families in P. carinii-specific CD4 cells in all cases. Although single peaks do not prove monoclonality, we may face one clone when a TCR BV gene family shows a single peak in the assays performed on antigen specific CD4 T cell lines [47,48]. This is supported further by previous evidence with T cell lines specific for HIV antigens, in which single peaks correspond to monoclonal T cell lines defined by CDR3 region sequencing [24]. This assumption does not apply to spectratyping of polyclonal lymphocytes, where individual peaks in the spectratype assay encompass a variety of diverse clones differing in CDR3 region sequence, but not in length [47,49]. In order to define the clonal composition of the peaks more effectively, additional experiments are in progress. Preliminary results of single-strand conformational polymorphism (SSCP) analysis [31] suggest that a single sequence or a very limited number of sequences (two to four) can be observed within TCR BV gene families exhibiting one peak in these experiments. Furthermore, no differences could be detected when T cell lines from HIV seropositives and from normal controls were compared for clonality by SSCP (in prep.).

The same study is currently being extended to patients in more advanced stages – with or without P. carinii symptomatic infection – to ascertain whether clonal deletions become detectable during disease progression. CD4 cell loss according to horizontal depletion does not exclude vertical depletion. In addition, CD4 responses to other relevant opportunistic pathogens such as C. albicans, toxoplasma, CMV and mycobacteria will be examined from this perspective by using the antigen-specific CD4 T cell lines we are currently generating from HIV seropositives.

The spectratype analysis to assess clonal heterogeneity of specific CD4 T cell responses to relevant opportunistic pathogens, as reported here, may be performed in principle with simplified techniques (e.g. on positively selected antigen specific cells instead of established T cell lines) on patients at different disease stages. Theoretically this may avoid the possible bias introduced by long-term culture on clonal composition, but only highly efficient selection methods (very close to 100%) can be used. This is probably not the case with the available methods, in which carry-over of non-specific cells can occur. Clearly, if a bias is introduced by in vitro culture it should affect lines generated from both controls and patients. Therefore comparison between lines can be equally informative. It should also be stressed that in the case of non-responders due to low precursor frequency, only in vitro selected and expanded cells provide enough material for molecular analysis of repertoire composition. This will help monitor CD4 depletion or CD4 reconstitution, in addition to standard CD4 counts and to evaluation of clonal heterogeneity of polyclonal CD4 cells [44–46]. If a residual population of clonally heterogeneous, specific CD4 lymphocytes can be demonstrated, it may represent a springboard for functional reconstitution after ART. These assays may provide more rational information, in addition to CD4 counts, for maintenance or withdrawal of chemoprophylaxis for P. carinii infection [50], whose control is mediated by CD4 T cells. In this study [50] it was confirmed that immunoreconstitution after ART is also possible due to residual P. carinii-specific T cells that are lost in a reversible fashion. Moreover, the definition of clonal heterogeneity is valuable to check specific T cell lines produced for adoptive therapy, in order to ascertain that the cells to be reinfused in the progressor are as close as possible to the normal protective repertoire.

The clonal analysis of the CD4 response to HIV antigens (in the presence or in the absence of vaccination) may complement other data showing that HIV specific CD4 responses associate with a more favourable disease progression [51]. In addition, pathogen-specific CD4 T cells selected and expanded in vitro, as we have described here, can be targets for gene therapy protocols to introduce HIV resistance genes in view of their reinfusion for adoptive immunoreconstitution [52].

Acknowledgments

This work was supported by grants from the Italian National Health Institute (AIDS project and Tuberculosis project), from the National Research Council (CNR Target Project on Biotechnology) and from the European Union (FAIR-CT-97–3046, BHH4-CT97-2055, QLK2-CT-2000–01040, QLK2-CT-2000–01321).

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