Effects of thymic selection of the T-cell repertoire on HLA class I-associated control of HIV infection (and HCV)

Effects of thymic selection of the T-cell repertoire on HLA class I-associated control of HIV infection (and HCV)


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Letter

Nature advance online publication 5 May 2010 | doi:10.1038/nature08997; Received 13 October 2009; Accepted 11 March 2010; Published online 5 May 2010

Andrej Kosmrlj1,2,9, Elizabeth L. Read1,3,4,9, Ying Qi5, Todd M. Allen1, Marcus Altfeld1, Steven G. Deeks6, Florencia Pereyra1, Mary Carrington1,5, Bruce D. Walker1,7 & Arup K. Chakraborty1,3,4,8
1. Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts 02114, USA 2. Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
4. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
5. Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702, USA 6. University of California, San Francisco, California 94110, USA
7. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA
8. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, US1

"Superior control of viral load due to the greater precursor frequency and cross-reactivity of those T-cell repertoires restricted by HLA molecules that bind to few self peptides (for example, HLA-B57) should also confer protection against diseases caused by other fast-mutating viruses. Indeed, HLA-B57 is protective against hepatitis C virus (HCV)24, another highly mutable viral disease in which CD8+ T cells are important. Also, HLA-B8, which binds a greater diversity of self peptides, is associated with faster disease progression in HCV25 and HIV13. Thus, the correlation between the diversity of peptides presented in the thymus during T-cell development and control or progression of disease may be general.....Our results shed light on another intriguing observation; acutely infected patients with low viral loads (and protective HLAs) tend to target an immunodominant epitope that makes a larger relative contribution to the total CTL response as compared to individuals presenting with higher levels of viraemia30. This is counterintuitive"

ABSTRACT
Without therapy, most people infected with human immunodeficiency virus (HIV) ultimately progress to AIDS. Rare individuals ('elite controllers') maintain very low levels of HIV RNA without therapy, thereby making disease progression and transmission unlikely. Certain HLA class I alleles are markedly enriched in elite controllers, with the highest association observed for HLA-B57 (ref. 1). Because HLA molecules present viral peptides that activate CD8+ T cells, an immune-mediated mechanism is probably responsible for superior control of HIV. Here we describe how the peptide-binding characteristics of HLA-B57 molecules affect thymic development such that, compared to other HLA-restricted T cells, a larger fraction of the naive repertoire of B57-restricted clones recognizes a viral epitope, and these T cells are more cross-reactive to mutants of targeted epitopes. Our calculations predict that such a T-cell repertoire imposes strong immune pressure on immunodominant HIV epitopes and emergent mutants, thereby promoting efficient control of the virus. Supporting these predictions, in a large cohort of HLA-typed individuals, our experiments show that the relative ability of HLA-B alleles to control HIV correlates with their peptide-binding characteristics that affect thymic development. Our results provide a conceptual framework that unifies diverse empirical observations, and have implications for vaccination strategies.

HIV infection leads to acute high level viraemia, which is subsequently reduced to a set-point viral load. Without therapy, most patients experience a subsequent increase in viral load, and ultimately the development of AIDS. Viraemia levels and time to disease vary widely, and the differences correlate with the expression of different HLA class I molecules (reviewed in ref. 2). Effector CD8+ T cells (CTLs) are implicated in viral control because T-cell antigen receptors (TCRs) on CD8+ T cells recognize complexes of viral peptides and class I HLA molecules presented on the surface of infected cells, and depletion of CD8+ T cells leads to increased viraemia in animal models of HIV infection3. We describe a feature of the HLA-B57-restricted CD8+ T-cell repertoire that contributes to enhanced control of viraemia.

Algorithms4 based on experimental data predict whether a particular peptide will bind to a given HLA molecule5. We tested four predictive algorithms against available experimental data on peptide binding to diverse HLA molecules and found that, in most cases, they are highly accurate (Supplementary Fig. 1 and Supplementary Table 1). For example, predictions using the best algorithm for HLA-B*5701 were 97% accurate. Using these algorithms, we computed the fraction of peptides derived from the human proteome6 that bind to various HLA molecules. Of the ∼107 unique peptide sequences, only 70,000 are predicted to bind to HLA-B*5701, and 180,000 bind to HLA-B*0701 (an allele that is not protective against HIV). Essentially identical results were obtained for randomly generated peptides (data not shown). The protective allele in macaques, Mamu-B*17, also binds fewer self peptides than other Mamu molecules for which data are available (Mamu-B*17 binds 4, 6 and 13 times fewer self peptides than Mamu-A*11, Mamu-A*01 and Mamu-A*02, respectively; Supplementary Table 1).

The intrinsic differences in self-peptide binding among HLA molecules are important during T-cell repertoire development. Immature T cells are exposed to diverse host-derived peptide-HLA complexes presented in the thymus. As fewer self peptides are able to bind to HLA-B*5701 (and Mamu-B*17) molecules, a smaller diversity of self-peptide TCR contact sequences will be encountered by HLA-B*5701/Mamu-B*17-restricted T cells in the thymus (Supplementary Discussion 1).

The diversity of self peptides presented in the thymus shapes the characteristics of the mature T-cell repertoire. Experiments7, 8 and theoretical studies9, 10 show that T cells that develop in mice with only one type of peptide in the thymus are more cross-reactive to point mutants of peptide epitopes that they recognize than T cells from mice that express diverse self peptides. Thus, by encountering fewer self peptides during thymic development, HLA-B57-restricted CD8+ T cells should be more cross-reactive to point mutants of targeted viral peptides.

We carried out in silico thymic selection experiments to test this hypothesis. We chose an HLA-dependent number of thymic self peptides, each with amino acids of the TCR contact residues picked according to the frequency with which they appear in the human proteome6, 9. A diverse set of immature CD8+ T cells (thymocytes) was generated by choosing the sequences of their peptide contact residues in the same way, and by varying the TCR-HLA interactions. A thymocyte emerges from the thymus as a mature CD8+ T cell if its TCR binds to at least one self-peptide-major histocompatibility complex (pMHC; human MHC is called HLA) molecule with an affinity that exceeds the positive selection threshold, and does not interact with any pMHC more strongly than the negative selection threshold. Using a computational model9, 10 in the class of 'string models'11, we assessed the affinity of TCR-self-peptide-HLA complexes (Methods) to determine which T cells survive positive and negative selection, and become a part of the mature repertoire. Our qualitative results are independent of the parameters used to determine these interaction strengths (Supplementary Figs 2 and 3)9, 10.

The mature T cells that emerged from these in silico thymic selection experiments were then computationally challenged by a viral peptide (that is, not seen in the thymus) bound to the same HLA type. T cells that recognize this peptide-HLA complex were obtained by assessing whether the interaction strength exceeded the negative selection threshold (shown to be equal to the recognition threshold in mouse models12); qualitative results are invariant if the recognition threshold is not much weaker than that corresponding to negative selection (Supplementary Fig. 3). Cross-reactivity of these T cells was then determined in silico by mutating each TCR contact residue of the peptide to the other 19 possibilities. Sites on the viral peptide were called 'important contacts' if half the mutations therein abrogated recognition by T cells that target this epitope. The frequency of the number of important contacts in viral peptides that determine T-cell recognition was obtained by repeating this procedure 1,000 times with different choices of thymocytes and self and foreign peptides.

Our calculations predict that a T-cell repertoire restricted by an HLA molecule such as HLA-B*5701, which presents fewer self peptides in the thymus, has a higher frequency of occurrence of T cells that recognize viral peptides through smaller numbers of important contacts (Fig. 1a). In contrast, the frequency of occurrence of T cells that recognize viral peptides through many important contacts is larger for repertoires restricted by HLA alleles that present a greater diversity of self peptides in the thymus (data not shown for >four contacts). Mutations at sites different from the important contacts do not affect binding strength substantially. Therefore, when the interaction between peptide-HLA and TCR is mediated by fewer important contacts, a larger number of possible point mutations of the peptide do not affect peptide recognition, thereby making the T cells more cross-reactive to mutants that arise. Thus, the HLA-B57-restricted T-cell repertoire is expected to be more cross-reactive to mutants of targeted viral peptides than repertoires restricted by HLA alleles that present a greater diversity of self peptides.

Our computational models give this qualitative mechanistic insight, but do not provide quantitative estimates of the extent of this enhanced cross-reactivity of T cells. However, compelling experimental data13 has shown that the effect revealed by our studies is important in humans. Peripheral blood mononuclear cells from patients expressing HLA-B57 contained CTLs that were more cross-reactive to various HIV epitopes and their point mutants than those of HLA-B8-positive patients. HLA-B8 is associated with rapid progression to disease13, and the most accurate algorithm for peptide binding suggests that the HLA-B8 molecule binds a greater diversity of self peptides than HLA-B57 (Supplementary Fig. 4 and Supplementary Table 1). Other experimental studies also show that patients expressing HLA-B57 cross-recognize point mutants of the dominant epitope and use more public TCRs14, 15.

Next, we computed interaction strengths between diverse viral peptides and members of T-cell repertoires restricted by HLA molecules that present differing numbers of self peptides in the thymus. This allowed us to obtain the probability with which a randomly picked T-cell clone and viral peptide will interact sufficiently strongly for recognition to occur. The results (Fig. 1b) indicate that a typical CD8+ T cell restricted by an HLA molecule such as HLA-B*5701, which presents fewer peptides in the thymus, has a higher probability of recognizing a viral epitope compared to a T cell restricted by other HLA molecules. Thus, more HLA-B*5701-restricted T cell clones are likely to recognize a viral epitope, making effective precursor frequencies higher in an HLA-B*5701-restricted repertoire (a strong predictor of response magnitude16). A greater precursor frequency for viral epitopes in the naive repertoire restricted by HLA-B57 is indicated by experimental results showing that HLA-B*5701 contributes the most to acute-phase CTL responses of all HLA alleles tested17.

The results in Fig. 1 stem from the constraint that thymocytes must avoid being negatively selected by each self-peptide-HLA complex encountered during development in the thymus. T cells expressing TCRs with peptide contact residues composed of amino acids that interact strongly with other amino acids (for example, charged residues, flexible side chains) have a high probability of binding to a self peptide strongly. The greater the diversity of self peptides presented in the thymus, the higher the chance that a TCR with such peptide contact residues will encounter a self peptide with which strong interactions will result in negative selection. Thus, as the diversity of self peptides presented in the thymus increases, the peptide contact residues of TCRs in the mature T-cell repertoire are increasingly enriched in weakly interacting amino acids (Supplementary Fig. 5). T cells bearing TCRs with weakly interacting peptide contact residues recognize viral peptides by means of several moderate interactions, making many contacts important for recognition. In contrast, TCRs with peptide contact residues containing strongly interacting amino acids are more likely to recognize viral peptides through a few important contacts mediated by these residues, making recognition cross-reactive to mutations at other peptide sites. These mechanistic insights are supported by experimental results7, 9 (Supplementary Discussion 2).

By studying a model of host-pathogen dynamics that builds on past models of host-HIV interactions18, 19, 20, we explored the consequences of the HLA-B57-restricted CD8+ T-cell repertoire having a higher precursor frequency for viral peptides and being more cross-reactive to point mutants of targeted epitopes on the control of HIV. Because of the importance of immune control exerted by CD8+ T cells17, 21, we focused on the interaction between a mutating virus quasispecies and epitope-directed, variably cross-reactive, host CTL responses.

The essential features of the model are depicted in Fig. 2a (details in Methods). The virus is modelled as a number of epitopes consisting of strings of amino acids, and new viral strains (point mutations of epitopes), which differ in replicative fitness, arise over the course of infection. For each individual, an HLA-dependent CD8+ T-cell repertoire was chosen. To mimic the results obtained from our thymic selection calculations (Fig. 1b), more or less cross-reactive repertoires were chosen (Supplementary Fig. 6) to represent HLA-B57-restricted T cells and those restricted by other HLAs, respectively. Infection rates were limited by target CD4+ T cells, and CTL contraction and memory were included. Other dynamic models were studied, including one that does not incorporate target cell limitation or CTL contraction. Our qualitative results about the effects of cross-reactivity are robust to variations in parameters and model assumptions (Supplementary Figs 7-16).

We find that individuals with a more cross-reactive CTL repertoire control viral loads better during the acute phase of the infection (Fig. 2b). This is in agreement with findings in simian immunodeficiency virus (SIV)-infected rhesus macaques22, where the number of cross-reactive TCR clones negatively correlates with viral load. Our simulations show that a larger number of CTL clones in a more cross-reactive T-cell repertoire recognize epitopes from the infecting viral strain (Fig. 2c). This is because the predicted higher precursor frequency for viral epitopes (Fig. 1b) leads to a greater response magnitude (as in mouse models16). This conclusion is supported by data showing that in people with a protective HLA allele, the initial T-cell response to HIV is dominated by T cells restricted by the protective HLA and not those restricted by other HLAs expressed17. Our simulations also show that enhanced cross-reactivity of the T-cell repertoire leads to greater immune pressure on the emergent viral mutants by individuals expressing HLA-B57 compared to those with T cells restricted by HLA molecules that bind more types of self peptides. The stronger immune pressure on infecting and emerging viral strains results in superior control of viral load. Thus, we predict that HIV-infected individuals with HLA alleles that bind fewer self peptides are more likely to control viral loads to low values. To test this prediction, we studied two large HLA-typed cohorts: 1,110 controllers with less than 2,000 HIV particles ml-1 and 628 progressors (or non-controllers) with viral loads exceeding 104 ml-1 (Methods). From these data, we obtained the odds ratio (OR) for individual HLA alleles. People with HLA alleles associated with an OR value greater or less than one are more likely to be progressors or controllers, respectively. We focused on HLA-B alleles because they are associated with control of HIV23. Of 40 HLA-B alleles that were studied, significant results (P value <0.05) were obtained for five HLA-B alleles (Supplementary Table 2) and peptide-binding data are available for four of them. In support of our predictions, those HLA-B alleles associated with higher OR values also bind more self peptides (Fig. 3).

Superior control of viral load due to the greater precursor frequency and cross-reactivity of those T-cell repertoires restricted by HLA molecules that bind to few self peptides (for example, HLA-B57) should also confer protection against diseases caused by other fast-mutating viruses. Indeed, HLA-B57 is protective against hepatitis C virus (HCV)24, another highly mutable viral disease in which CD8+ T cells are important. Also, HLA-B8, which binds a greater diversity of self peptides, is associated with faster disease progression in HCV25 and HIV13. Thus, the correlation between the diversity of peptides presented in the thymus during T-cell development and control or progression of disease may be general.

Undoubtedly, many complex factors influence the relationship between HLA type and disease outcome. The effect of the new factor we have identified should be greatest for HLA molecules that bind relatively few (for example, HLA-B57) or many (for example, HLA-B7, -B35, -B8) self peptides. The strong association of HLA-B27-which binds an intermediate number of self peptides (twice as many as HLA-B57)-with viral control indicates that, in this case, the effects of T cell cross-reactivity are reinforced by this molecule binding HIV epitopes that are subject to very strong structural constraints.

Our results also point to a mechanistic explanation for as yet unexplained associations between HLA alleles that confer protection against HIV and autoimmune diseases. T cells restricted by HLA alleles that bind to few self peptides are subject to less stringent negative selection in the thymus, and should therefore be more prone to recognizing self peptides. Indeed, HLA-B57 has been associated with autoimmune psoriasis26 and hypersensitivity reactions27. Enhanced cross-reactivity of HLA-B27-restricted T cells and other unique properties of this molecule (misfolding, homodimers28) probably contribute to the enhanced risk of autoimmunity associated with this allele29.

Our results shed light on another intriguing observation; acutely infected patients with low viral loads (and protective HLAs) tend to target an immunodominant epitope that makes a larger relative contribution to the total CTL response as compared to individuals presenting with higher levels of viraemia30. This is counterintuitive as the most protective responses appear most focused, rather than broadly distributed over many epitopes. We calculated how viral load correlates with the number of CTLs responding to the immunodominant epitope divided by the total number of CTLs activated by the virus (a quantity analogous to relative contribution30). Mirroring experimental data, HLA alleles that restrict a more cross-reactive repertoire and are more protective also make a larger relative contribution (Supplementary Fig. 13). This result unifies the idea of both a broad and a focused response. The more cross-reactive repertoire targets more epitopes and emergent mutants, but a larger number of clones also recognize the dominant epitope (Fig. 2c). Cross-reactive T cells are rare in people with HLA alleles that present more self peptides in the thymus than the B57 allele, but they do exist. Our results suggest that a T-cell vaccine for a diverse population must aim to activate these rare cross-reactive T cells that also target epitopes from a conserved region of the HIV genome (like HLA-B57 Gag epitopes). This will enable robust responses to infecting and mutant strains until a strain with low replicative fitness emerges, enhancing control of viral load.

Methods Summary

Predictive algorithm tools for peptide binding to HLA and Mamu molecules were obtained from the Immune Epitope Database (IEDB)4 and were used to predict the fraction of bound peptide derived from the human and macaque proteomes6. Accuracies of these tools were tested on experimental data obtained from the IEDB4. To assess the effects of thymic selection on TCRs restricted by different MHC molecules (HLA or Mamu), we used a computational model of thymic selection described in Methods (and previously9, 10).

To explore host-pathogen dynamics, we constructed a small model of the HIV virus with distinct epitopes and sequence diversity, based in part on past work18, 19, 20. We carried out numerical simulations of ordinary differential equation models, shown schematically in Fig. 2a and Supplementary Fig. 7. Parameters and their justification are given in Supplementary Tables 3 and 4 and in the Supplementary Methods. To explore cross-reactivity, we varied the distribution of pairwise-interaction free energies of TCR-pMHC contacts. Our goal was not to obtain precise numbers, but to examine the qualitative effects of variation in repertoire cross-reactivity on virus control. Qualitative results are robust to variations in parameters and assumptions (Supplementary Figs 8-16).

HLA-typed cohorts of people of diverse races were divided into HIV controllers and HIV non-controllers, and analysed for HLA association with the ability to control HIV. The results (Fig. 3 and Supplementary Table 2) were adjusted for the effects of HLA-B*0702, HLA-B*3501, HLA-B*2705 and HLA-B*5701. Full methods accompany this paper.