Human Leukocyte Antigen Class I Alleles Research Articles

Aplastic Anemia Patients Have Diverse T Cell Repertoires and Flexible T Cell Responses Similar to Healthy Individuals

Acquired aplastic anemia (AA) is a bone marrow failure disease caused by T cell-mediated autoimmune attack on hematopoietic stem and progenitor cells (HSPCs). Identifying autoreactive T cells in AA has been challenging. Advances in T cell receptor (TCR) sequencing and the discovery of Human Leukocyte Antigen Class I (HLA-I) risk alleles linked to autoantigen presentation in AA have rekindled interest in exploring T cell repertoires in AA. We hypothesized that autoreactive CD8 + T cells in AA may have convergent TCR structures allowing them to recognize AA autoantigens presented by specific HLA-I risk alleles. To test our hypothesis, we analyzed TCR repertoires and HLA-restricted CD8 + T cell reactivity in AA patients. Using TCR Vβ (TRB) sequencing, we analyzed 83 patient samples obtained at AA diagnosis or relapse. High-risk alleles HLA-B*14:02 and B*40:02 were present in 26% and 10% of patients, respectively. HLA-DR*15 was present in 24%. Patient samples were largely sorted bone marrow (BM) CD8 + T lymphocytes. Patient TRB repertoires were compared to those in 709 control samples, largely of unsorted peripheral blood (PB). To account for sampling differences, we downsampled sequences to the minimum number of clones in the matching tissue. Normalized clone size distributions were visualized on rank plots (Fig 1). TRB repertoires in both patients and controls were diverse. Highly expanded clones (>5%) were rare in either group. Patients had 1,415,069 unique CDR3 amino acid sequences (CDR3AA), of which 106,446 (7.5%) were shared by ≥2 patients. Among the 106,446 shared CDR3AA, 95.4% were found in controls, and 4.3% TCRs had reported specificity to exogenous (mostly viral) or endogenous antigens in VDJdb. 4,920 (4.6%) of the shared CDR3AA were detected only in patients (median 2 patients (2-25)). Of these, 190 exhibited TCR convergence in ≥1 patient, such that the same CDR3AA was encoded by ≥2 distinct nucleotide sequences. Clustering 4,920 patient-associated CDR3AA using GLIPH (Grouping of lymphocyte interactions by paratope hotspots) identified 43 TCR antigen convergence groups. CDR3AA in each cluster were not significantly expanded in the majority of patients and were not enriched for the high-risk alleles HLA-B*14:02 or B*40:02. Our data suggest that most CDR3AA in AA are private (unique to individual patients). Shared autoreactive CDR3AA may exist in patients but are likely to also be present in healthy individuals. As the next step towards identifying autoreactive T cells, we focused on AA patients with known HLA-I risk alleles. We tested their CD8 + T cells for reactivity with endogenously expressed antigens by stimulating T cells with hematopoietic cell lines engineered to express single HLA-I alleles matching those of the patients (Fig 2). TRB sequencing showed increased clonality and significant expansion of a subset of clonotypes after T cell stimulation. Interferon γ enzyme-linked immunospot assays confirmed the expanded T cells' responsiveness to antigens presented by HLA alleles used for T cell stimulation. Additionally, these expanded T cells demonstrated variable reactivity to cells expressing other HLA alleles from the same patient. Among the 1% of CDR3AA clonotypes most highly expanded with single HLA-I alleles (63-3000-fold), we identified several that were expanded in an HLA-restricted manner. However, several highly expanded CDR3AA were highly expanded with multiple HLA alleles. Potential reasons for expansion of some CDR3AA after stimulation with cells expressing different HLA alleles include 1) binding of the same CDR3AA to different antigens presented by distinct HLA alleles, 2) expansion in response to the same antigens presented by distinct HLA alleles, or 3) expansion in response to non-HLA antigens. In conclusion, our data show diverse and mostly private TRB repertoires in AA patients. Our in vitro findings suggest that much like T cell responses in non-AA individuals, T cell responses in AA are diverse and exhibit flexibility with respect to activation in response to various endogenous antigens in the context of different self HLA-I alleles. Given the diversity of TCR responses observed in AA patients, the tissue specificity of autoimmune attack and established association with risk HLA-I alleles in AA remain a mystery and likely require HSPC-specific autoantigen expression and efficient autoantigen presentation by individual patients' HLA-I alleles.

Pervasiveness of HLA allele-specific expression loss across tumor types

BackgroundEfficient presentation of mutant peptide fragments by the human leukocyte antigen class I (HLA-I) genes is necessary for immune-mediated killing of cancer cells. According to recent reports, patient HLA-I genotypes can impact the efficacy of cancer immunotherapy, and the somatic loss of HLA-I heterozygosity has been established as a factor in immune evasion. While global deregulated expression of HLA-I has also been reported in different tumor types, the role of HLA-I allele-specific expression loss — that is, the preferential RNA expression loss of specific HLA-I alleles — has not been fully characterized in cancer.MethodsHere, we use RNA and whole-exome sequencing data to quantify HLA-I allele-specific expression (ASE) in cancer using our novel method arcasHLA-quant.ResultsWe show that HLA-I ASE loss in at least one of the three HLA-I genes is a pervasive phenomenon across TCGA tumor types. In pancreatic adenocarcinoma, tumor-specific HLA-I ASE loss is associated with decreased overall survival specifically in the basal-like subtype, a finding that we validated in an independent cohort through laser-capture microdissection. Additionally, we show that HLA-I ASE loss is associated with poor immunotherapy outcomes in metastatic melanoma through retrospective analyses.ConclusionsTogether, our results highlight the prevalence of HLA-I ASE loss and provide initial evidence of its clinical significance in cancer prognosis and immunotherapy treatment.

High frequency of CD8 escape mutations in elite controllers as new obstacle for HIV cure

ABSTRACT Accumulation of mutations in epitopes of cytolytic-T-lymphocytes immune response (CTL) in HIV-reservoir seems to be one of the reasons for shock-and-kill strategy failure. Ten non-controller patients on successful cART (TX) and seven elite controllers (EC) were included. HIV-Gag gene from purified resting memory CD4+ T-cells was sequenced by Next-Generation-Sequencing. HLA class-I alleles were typed to predict optimal HIV-Gag CTL epitopes. For each subject, the frequency of mutated epitopes in the HIV-Gag gene, the proportion of them considered as CTL-escape variants as well as their effect on antigen recognition by HLA were assessed. The proportion (%) of mutated HIV-Gag CTL epitopes in the reservoir was high and similar in EC and TX (86%[50–100] and 57%[48–82] respectively, p=0.315). Many of them were predicted to negatively impact antigen recognition. Moreover, the proportion of mutated epitopes considered to be CTL-escape variants was also similar in TX and EC (77%[49–92] vs. 50%[33–75] respectively, p=0.117). Thus, the most relevant finding of our study was the high and similar proportions of HIV-Gag CTL-escape mutations in the reservoir of both HIV-noncontroller patients with cART (TX) and patients with spontaneous HIV-control (EC). Our findings suggest that escape mutations of CTL-response may be another obstacle to eliminate the HIV reservoir and constitute a proof of concept that challenges HIV cure strategies focused on the reactivation of reservoirs. Due to the small sample size that could impact the robustness of the study, further studies with larger cohorts of elite controller patients are needed to confirm these results.

Genome editing of peptide loading reveals risks of immunotherapeutic targeting of TAP1 independent peptides

Current immunotherapeutic strategies for cancer aim to target HLA class I (HLA-I) presented neo-epitopes. A subgroup of neo-epitopes has been proposed to be presented only in the absence of the TAP transporter. We here identified a novel TAP-independent antigen that was surprisingly effectively presented by TAP-sufficient cells. We engineered a library of HLA-I pathway CRISPR/Cas9-mediated knockout cells on a single genetic background allowing for an optimal comparison of individual component contributions to antigen presentation. Sequence-verified knockouts for HLA-I (-A, -B and -C), B2M, Calnexin, α-Glucosidase II, Calreticulin, ERAP1, TAP1, Tapasin and ERp57 were created. The HLA-I surface expression of these cells depended on both the targeted gene and the detected HLA-I allele, as expected. To investigate the presentation of single antigens, we targeted the knockout library with a panel of peptide-specific T cell clones. Surprisingly, T cells specific for a peptide derived from the endogenously expressed SSR1 protein recognized TAP1-deficient cells at a similar efficiency as wild type cells. These T cells were derived from a patient during graft-versus-host-disease, suggesting that they contributed to the destruction of healthy TAP-sufficient cells. Further experiments demonstrated the functional presentation of the SSR1 antigen by various healthy cells, underscoring that TAP-independent antigens can contribute to unwanted T cell reactivity. In conclusion, we generated and validated a novel cellular toolset that allows comparative research in the HLA-I antigen processing and presentation pathway. Using this toolset we found a TAP-independent peptide that is functionally presented on healthy cells. Our data underscore potential pitfalls of targeting TAP-independent antigens as immunotherapeutic approach

Unconventional modes of peptide-HLA-I presentation change the rules of TCR engagement.

The intracellular proteome of virtually every nucleated cell in the body is continuously presented at the cell surface via the human leukocyte antigen class I (HLA-I) antigen processing pathway. This pathway classically involves proteasomal degradation of intracellular proteins into short peptides that can be presented by HLA-I molecules for interrogation by T-cell receptors (TCRs) expressed on the surface of CD8+ T cells. During the initiation of a T-cell immune response, the TCR acts as the T cell's primary sensor, using flexible loops to mould around the surface of the pHLA-I molecule to identify foreign or dysregulated antigens. Recent findings demonstrate that pHLA-I molecules can also be highly flexible and dynamic, altering their shape according to minor polymorphisms between different HLA-I alleles, or interactions with different peptides. These flexible presentation modes have important biological consequences that can, for example, explain why some HLA-I alleles offer greater protection against HIV, or why some cancer vaccine approaches have been ineffective. This review explores how these recent findings redefine the rules for peptide presentation by HLA-I molecules and extend our understanding of the molecular mechanisms that govern TCR-mediated antigen discrimination.

Negative trade-off between neoantigen repertoire breadth and the specificity of HLA-I molecules shapes antitumor immunity.

Human leukocyte antigen class I (HLA-I) genes shape our immune response against pathogens and cancer. Certain HLA-I variants can bind a wider range of peptides than others, a feature that could be favorable against a range of viral diseases. However, the implications of this phenomenon on cancer immune response are unknown. Here we quantified peptide repertoire breadth (or promiscuity) of a representative set of HLA-I alleles and found that patients with cancer who were carrying HLA-I alleles with high peptide-binding promiscuity have significantly worse prognosis after immune checkpoint inhibition. This can be explained by a reduced capacity of the immune system to discriminate tumor neopeptides from self-peptides when patients carry highly promiscuous HLA-I variants, shifting the regulation of tumor-infiltrating T cells from activation to tolerance. In summary, HLA-I peptide-binding specificity shapes neopeptide immunogenicity and the self-immunopeptidome repertoire in an antagonistic manner, and could underlie a negative trade-off between antitumor immunity and genetic susceptibility to viral infections.

Abstract 1907: Negative trade-off between neoantigen repertoire breadth and the specificity of HLA-I molecules shapes antitumor immunity

Abstract The human leukocyte antigen class I (HLA-I) genes shape our immune response against pathogens and cancer. Certain HLA-I variants can bind a much wider range of peptides than others, a feature that could be favorable against a range of viral diseases. However, the implications of this phenomenon on cancer immune response is unknown. In this paper, we quantified peptide repertoire breadth (or promiscuity) of a representative set of HLA-I alleles, and found that cancer patients that carry HLA-I alleles with high peptide binding promiscuity are characterized by significantly worse prognosis after immune checkpoint inhibitor treatment. This trend can be explained by a reduced capacity of promiscuous HLA-I molecules to discriminate between human self and tumor peptides, yielding a shift in regulation of T-cells in the tumor microenvironment from activation to tolerance. In summary, HLA-I peptide binding specificity shapes neopeptide immunogenicity and the self-immunopeptidome repertoire in an antagonistic manner. It could also underlie a negative trade-off between antitumour immunity and the genetic susceptibility to viral infections. Citation Format: Benjamin Tamas Papp, Máté Manczinger, Gergő Balogh, Balázs Koncz, Leó Asztalos, Lajos Kemény, Balázs Papp, Csaba Pál. Negative trade-off between neoantigen repertoire breadth and the specificity of HLA-I molecules shapes antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1907.

Therapeutic Potential of HLA-I Polyreactive mAbs Mimicking the HLA-I Polyreactivity and Immunoregulatory Functions of IVIg

HLA class-I (HLA-I) polyreactive monoclonal antibodies (mAbs) reacting to all HLA-I alleles were developed by immunizing mice with HLA-E monomeric, α-heavy chain (αHC) open conformers (OCs). Two mAbs (TFL-006 and TFL-007) were bound to the αHC’s coated on a solid matrix. The binding was inhibited by the peptide 117AYDGKDY123, present in all alleles of the six HLA-I isoforms but masked by β2-microglobulin (β2-m) in intact HLA-I trimers (closed conformers, CCs). IVIg preparations administered to lower anti-HLA Abs in pre-and post-transplant patients have also shown HLA-I polyreactivity. We hypothesized that the mAbs that mimic IVIg HLA-I polyreactivity might also possess the immunomodulatory capabilities of IVIg. We tested the relative binding affinities of the mAbs and IVIg for both OCs and CCs and compared their effects on (a) the phytohemagglutinin (PHA)-activation T-cells; (b) the production of anti-HLA-II antibody (Ab) by B-memory cells and anti-HLA-I Ab by immortalized B-cells; and (c) the upregulation of CD4+, CD25+, and Fox P3+ T-regs. The mAbs bound only to OC, whereas IVIg bound to both CC and OC. The mAbs suppressed blastogenesis and proliferation of PHA-activated T-cells and anti-HLA Ab production by B-cells and expanded T-regs better than IVIg. We conclude that a humanized version of the TFL-mAbs could be an ideal, therapeutic IVIg-mimetic.

RBM-MHC: A Semi-Supervised Machine-Learning Method for Sample-Specific Prediction of Antigen Presentation by HLA-I Alleles.

SummaryThe recent increase of immunopeptidomics data, obtained by mass spectrometry or binding assays, opens up possibilities for investigating endogenous antigen presentation by the highly polymorphic human leukocyte antigen class I (HLA-I) protein. State-of-the-art methods predict with high accuracy presentation by HLA alleles that are well represented in databases at the time of release but have a poorer performance for rarer and less characterized alleles. Here, we introduce a method based on Restricted Boltzmann Machines (RBMs) for prediction of antigens presented on the Major Histocompatibility Complex (MHC) encoded by HLA genes—RBM-MHC. RBM-MHC can be trained on custom and newly available samples with no or a small amount of HLA annotations. RBM-MHC ensures improved predictions for rare alleles and matches state-of-the-art performance for well-characterized alleles while being less data demanding. RBM-MHC is shown to be a flexible and easily interpretable method that can be used as a predictor of cancer neoantigens and viral epitopes, as a tool for feature discovery, and to reconstruct peptide motifs presented on specific HLA molecules.

Probable HLA-mediated immunoediting of JAK2 V617F-driven oncogenesis

Human leukocyte antigen class I (HLA-I) genotype has been found to influence cancer development through the presentation of mutational neoepitopes. However, our understanding of its effect on the development of myeloproliferative neoplasms (MPNs) remains limited. We aimed to elucidate the putative protective role of HLA-I alleles in the development of JAK2 V617F-driven MPNs using a population genetics approach. The variability of the HLA-I genotype had no effect on the presence of JAK2 V617F mutation. However, three alleles were found to be inversely correlated with the presence of JAK2 V617F mutation: HLA-A*02:01 (p = 0.036), HLA-B*35:01 (p = 0.017), and HLA-C*15:02 (p = 0.033). The HLA-B*35:01 allele was predicted to bind to a 9-mer peptide derived from JAK2 V617F mutant protein. Gene expression analysis revealed a lower expression of HLA-A and -B in MPN CD34+ cells compared with normal CD34+ cells, which was modulated by ruxolitinib and interferon-α treatment. In summary, we provide robust evidence that specific HLA-I molecules restrict JAK2 V617F-driven oncogenesis. JAK2 V617F+ stem cells evade immune surveillance through downregulation of the HLA-I expression. Therefore, the presence of specific HLA-I alleles might be a predictive marker for response to certain immunotherapies upregulating HLA-I expression. Finally, our findings have implications in the development of mutational neoepitope-based vaccines in MPNs.

Evolutionary divergence of HLA class I genotype impacts efficacy of cancer immunotherapy.

Functional diversity of the highly polymorphic human leukocyte antigen class I (HLA-I) genes underlies successful immunologic control of both infectious disease and cancer. The divergent allele advantage hypothesis dictates that an HLA-I genotype with two alleles with sequences that are more divergent enables presentation of more diverse immunopeptidomes1-3. However, the effect of sequence divergence between HLA-I alleles-a quantifiable measure of HLA-I evolution-on the efficacy of immune checkpoint inhibitor (ICI) treatment for cancer remains unknown. In the present study the germline HLA-I evolutionary divergence (HED) of patients with cancer treated with ICIs was determined by quantifying the physiochemical sequence divergence between HLA-I alleles of each patient's genotype. HED was a strong determinant of survival after treatment with ICIs. Even among patients fully heterozygous at HLA-I, patients with an HED in the upper quartile respond better to ICIs than patients with a low HED. Furthermore, HED strongly impacts the diversity of tumor, viral and self-immunopeptidomes and intratumoral T cell receptor clonality. Similar to tumor mutation burden, HED is a fundamental metric of diversity at the major histocompatibility complex-peptide complex, which dictates ICI efficacy. The data link divergent HLA allele advantage to immunotherapy efficacy and unveil how ICI response relies on the evolved efficiency of HLA-mediated immunity.

Differential HLA allele frequency in Mycobacterium africanum vs Mycobacterium tuberculosis in Mali.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC), however, the distribution and frequency of MTBC lineages and sublineages vary in different parts of the globe. Mycobacterium africanum, a member of MTBC is responsible for a large percentage of TB cases in West Africa, however, it is rarely identified outside of this part of the World. Whether or not differential HLA polymorphism (an important host factor) is contributing to the geographic restriction of M. africanum to West Africa is unknown. Here, we conducted a cohort study in Mali of newly diagnosed individuals with active pulmonary TB and normal healthy controls. The MTBC isolates were spoligotyped to determine the TB study groups (M. tuberculosis sensu stricto LAM10 and M. africanum), and HLA typing was performed on peripheral blood. Unlike previous reports on other populations, we found that HLA class-I alleles were significantly associated with active TB disease in this population. HLA-B alleles (B*07:02, B*08:01, B*14:02, B*15:03, B*15:10, B*18:01, B*42:01, B*42:02, B*51:01 and B*81:01) were significantly associated with M. africanum (40%-45%) and M. tuberculosis (75%) compared with healthy controls. Many HLA-A alleles (A*02:05, A*34:02, A*66:01 and A*68:02) were also associated with both TB groups (65%-70%). However, many class II HLA-DR variants were found to be associated with M. tuberculosis but not M. africanum with the exception of the DRB1*03:01, which was associated with both groups. The differential HLA distribution observed in this study might be at least partially responsible for the geographical restriction of M. africanum infections to West Africa.

Chikungunya virus: genomic microevolution in Eastern India and its in-silico epitope prediction.

This is the first study reporting whole genome sequences of two CHIKV strains (KJ679577 and KJ679578) isolated from Eastern Indian patients sera during 2010-2011 outbreak, both of which were of ECSA genotype, but from different subgroups: Indian Ocean outbreak and ECSA subtypes. Furthermore, viral sequences were analyzed using different in-silico approaches to identify potential genetic variations that might have functional implications on various aspects of virus replication, viral protein functionality, immunogenicity and transmission. Epitope prediction analysis revealed 70.9% increase in number of MHC Class-II interacting epitopes of KJ679578 and 25-28% increase in Class-I interacting epitopes of KJ679577 and KJ679578 compared to that of EF027141 (CHIKV of Asian genotype circulating in India during 1973, after which CHIKV infection disappeared from India for three decades). CHIKV peptides DLAKLAFKRSSKYDLECAQIPVHMKSDA and KVVLCGDPKQCGFFNMMQMKYNYNHNI were predicted to interact with maximum number of HLA Class-I (68 and 76.5%, respectively) and Class-II (47 and 100%, respectively) alleles present within Indian population with allele frequency of > 0.1 and were also recognized as predicted B-cell epitopes with BCPred score between 0.766 and 0.961 and with antigenicity ranging from 0.52 to 1.69; thus these peptides might be used to induce T- and B-cell-mediated immunity against CHIKV. Thus, the present study might help to bridge the gap between virus microevolution and its implication in host immunity by taking into account viral genetic and conformational changes. Predicted epitopes might be used as promising targets for peptide-based vaccine development and rapid diagnostics against CHIKV infection.

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8+ T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8+ T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8+ T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8+ T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8+ T cell activation, mediated by the binding of empty HLA-I to CD8.

KIR3DL1 alleles and their epistatic interactions with human leukocyte antigen class I influence resistance and susceptibility to HIV-1 acquisition in the Pumwani sex worker cohort.

To determine the associations of KIR3DL1/S1(3DL1/S1) and its epistatic interactions with human leukocyte antigen class I (HLA-I) alleles with resistance and susceptibility to HIV-1. Despite repeated exposure to HIV-1, a subset of women enrolled in the Pumwani sex worker cohort remain HIV uninfected. Previous studies have shown that specific HLA class I and II alleles were associated with this natural immunity. In this study, we investigated the association of 3DL1/S1 and its epistatic interactions with HLA-I, with resistance or susceptibility to HIV-1 acquisition. We used a sequence-based typing method to genotype 3DL1/S1 of 641 women in this cohort. The association of 3DL1/S1 and its epistatic interactions with HLA-I were analyzed using SPSS statistics software. 3DL1041 is enriched in the HIV-1-resistant women [P = 0.009, Pc = 0.0468, odds ratio (OR): 3.359, 95% confidence interval (CI): 1.39-8.32], whereas, 3DL1020 was associated with susceptibility to HIV-1 infection before correction for multiple comparisons (P = 0.029, Pc = 0.0858, OR: 0.316, 95%CI: 0.10-1.04). Epistatic interactions between several 3DL1 alleles and specific HLA-I alleles were observed. Among them the cocarriage of 3DL1041 with Bw4 (P = 1E - 05, Pc = 0.0015, OR: 13.33, 95%CI: 3.43-51.9), or Bw6 (P = 0.008, Pc = 0.272, OR: 3.92, 95%CI: 1.51-10.17), increased the odds of remaining HIV-1 uninfected. Further, 3DL1041+/Bw4+ women who entered the cohort HIV negative remained uninfected (P = 0.032, Pc = 0.0858). Cocarriage of 3DL101501 with C02 : 10 (P = 2.73E - 07, Pc = 7.0954E - 06), B15 : 03 (P = 3.21E - 04, Pc = 0.0042), A24 supertype (P = 8.89E - 04, Pc = 0.0077), or A23 : 01 (P = 0.0036, Pc = 0.0236) was associated with increased susceptibility to seroconversion. The effects of interactions between 3DL1 and HLA-I alleles on resistance/susceptibility to HIV-1 infection suggest that innate immunity plays an important role in HIV-1 acquisition and should be studied and explored for HIV prevention.

Role of genetic heterogeneity in determining the epidemiological severity of H1N1 influenza.

Genetic differences contribute to variations in the immune response mounted by different individuals to a pathogen. Such differential response can influence the spread of infectious disease, indicating why such diseases impact some populations more than others. Here, we study the impact of population-level genetic heterogeneity on the epidemic spread of different strains of H1N1 influenza. For a population with known HLA class-I allele frequency and for a given H1N1 viral strain, we classify individuals into sub-populations according to their level of susceptibility to infection. Our core hypothesis is that the susceptibility of a given individual to a disease such as H1N1 influenza is inversely proportional to the number of high affinity viral epitopes the individual can present. This number can be extracted from the HLA genetic profile of the individual. We use ethnicity-specific HLA class-I allele frequency data, together with genome sequences of various H1N1 viral strains, to obtain susceptibility sub-populations for 61 ethnicities and 81 viral strains isolated in 2009, as well as 85 strains isolated in other years. We incorporate these data into a multi-compartment SIR model to analyse the epidemic dynamics for these (ethnicity, viral strain) epidemic pairs. Our results show that HLA allele profiles which lead to a large spread in individual susceptibility values can act as a protective barrier against the spread of influenza. We predict that populations skewed such that a small number of highly susceptible individuals coexist with a large number of less susceptible ones, should exhibit smaller outbreaks than populations with the same average susceptibility but distributed more uniformly across individuals. Our model tracks some well-known qualitative trends of influenza spread worldwide, suggesting that HLA genetic diversity plays a crucial role in determining the spreading potential of different influenza viral strains across populations.

Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies allostery regulating HLA specificity.

The precise identification of Human Leukocyte Antigen class I (HLA-I) binding motifs plays a central role in our ability to understand and predict (neo-)antigen presentation in infectious diseases and cancer. Here, by exploiting co-occurrence of HLA-I alleles across ten newly generated as well as forty public HLA peptidomics datasets comprising more than 115,000 unique peptides, we show that we can rapidly and accurately identify many HLA-I binding motifs and map them to their corresponding alleles without any a priori knowledge of HLA-I binding specificity. Our approach recapitulates and refines known motifs for 43 of the most frequent alleles, uncovers new motifs for 9 alleles that up to now had less than five known ligands and provides a scalable framework to incorporate additional HLA peptidomics studies in the future. The refined motifs improve neo-antigen and cancer testis antigen predictions, indicating that unbiased HLA peptidomics data are ideal for in silico predictions of neo-antigens from tumor exome sequencing data. The new motifs further reveal distant modulation of the binding specificity at P2 for some HLA-I alleles by residues in the HLA-I binding site but outside of the B-pocket and we unravel the underlying mechanisms by protein structure analysis, mutagenesis and in vitro binding assays.

Dynamics and Correlates of CD8 T-Cell Counts in Africans with Primary Human Immunodeficiency Virus Type 1 Infection.

Longitudinal data from 497 HIV-1 seroconverters allowed us to systematically evaluate the dynamics and correlates of CD8+ T-cell counts during untreated primary HIV-1 infection in eastern and southern Africans. Our findings suggest that individuals with certain HLA-I alleles, including A*03 (exclusively A*03:01), persistently maintain relatively high CD8 counts following HIV-1 infection, a finding which may offer an intriguing explanation for the recently reported, negative association of A*03 with HIV-1-specific, broadly neutralizing antibody responses. In future studies, attention to HLA-I genotyping data may benefit in-depth understanding of both cellular and humoral immunity, as well as the intrinsic balances of these types of immunity, especially in settings where there is emerging evidence of antagonism between the two arms of adaptive immunity.

Adoptively Transferred CMV-Specific T-Cells Responsive to Immunodominant CMVpp65 Epitopes Extend Durable in-Vivo Suppression of Clonogenic CMVpp65 [+] Human Colon Carcinoma Cells in Comparison to T-Cells Responsive to Subdominant Epitopes

Adoptively transferred transplant donor or third party donor derived CMV-specific T-cells (CMV-CTLs) can effectively prevent and treat CMV disease in HSCT recipients. T-cells (TC) respond to specific viral epitopes when presented by HLA class-I and class-II alleles on infected cells. We have infused third party CTLs from HLA partially matched donors that were chosen based on in-vitro CTL activity against epitopes presented by HLA alleles shared by the recipient, and this approach has afforded a 60% response rate among patients treated for CMV viremia and/or disease. A recurring feature of TC generated in-vitro or directly selected in-vivo is the striking preponderance of TC specific for 1-2 immunodominant epitopes presented by specific HLA alleles. However, the functional activity of such immunodominant CMV-CTLs as compared to CTLs directed against subdominant CMV epitopes has not been evaluated. To directly compare the activity of CMV-CTLs directed against immunodominant and subdominant epitopes, we developed an in-vivo model using human colon carcinoma cells transduced with CMVpp65 as a surrogate system. Human colon carcinoma cells co-expressing HLA A0201 and A2402 (cocapp65), and a melanoma cell line lacking expression of these HLA alleles (melpp65) were each transduced to also express CMVpp65 and a GFP-firefly luciferase transgene. Subcutaneous (s.c.) inoculation of 1 x 105 tumor cellsinto NOD/Scid-IL2Rgc-KO/J mice (NSG) provided consistent engraftment. CMV-CTLs responsive to either the immunodominant NLV epitope presented by HLA A0201 (A2-NLV) or the subdominant QYD epitope presented by HLA A2402 (A24-QYD) were generated from 2 donors, each co-inheriting HLA A0201 and A2402, using NIH 3T3 artificial antigen presenting cells, each expressing a single HLA class-I allele (A0201 or A2402), B7.1, LFA-3 and ICAM1.Groups of 5-6 NSG mice, each bearing 2 established tumors: cocapp65 and melpp65 (control), were intravenously injected with 2-4 x 106 of tetramer+ A2-NLV or A24-QYD specific CMV-CTLs per mouse. Control animals either did not receive any TC, or received HLA B0801- LTM specific CMV-CTLs, and IL-2 was given 2 x/week to all groups. Tumor growth was monitored for 6-8 weeks by bioluminescent imaging.Both A2-NLV and A24-QYD CMV-CTLs significantly suppressed growth of cocapp65 tumors in all treated animals with infusion of equivalent doses of epitope specific tet [+] T-cells (p = ns). No tumor suppression was observed in control animals, and there was no evidence of GvHD in CTL treated animals. A2-NLV CTLs eradicated cocapp65 tumor in 4/10 treated animals, while A24-QYD CTLs did not completely eradicate the tumor in any treated animal. In all A2-NLV treated animals, the cocapp65 remained suppressed until end of study (6 wks), while the melpp65 continued to grow. Treatment with A24-QYD CTLs induced tumor suppression after a time lag, as evidenced by initial cocapp65 growth for 10-14 days followed by suppression for 4-5 weeks and then stabilization of tumor size. In subsequent studies with animals bearing single cocapp65 xenografts, and infused with either A2-NLV or A24-QYD CTLs, the tumors remained suppressed for upto 8 weeks after A2-NLV CTL infusion, while recurrent tumor growth was observed 5 weeks after A24-QYD CTL infusion. Studies detailing the relative accumulation of CTLs within tumor tissue, as well as engraftment of CTLs in these animals are in progress. Phenotypic analysis of the infused epitope specific TC demonstrated 91-95% TEM with 5-9% TCM, with a highly restricted oligoclonal TCRVβ repertoire.This model provides a platform for direct comparative evaluation of the in-vivo cytotoxic activity of epitope specific TC. These studies demonstrate that CMV-CTLs responsive to both immunodominant as well as subdominant epitopes, that are generated using AAPC from the same donor co-inheriting the presenting HLA alleles, can suppress the growth of clonogenic human carcinoma cells co-expressing an immunogenic viral antigen in-vivo at equivalent doses of antigen specific TC. However, less robust antigen specific cytotoxic activity was demonstrated by the subdominant A24-QYD CMV CTLs, which maybe reflective of reduced in-vivo proliferation, effector function or persistence of such TC. Experiments of define these variables contributing to disparities in the in-vivo CTL activity are in progress. DisclosuresHasan:Atara Biotherapeutics: Research Funding. O'Reilly:Atara Biotherapeutics: Research Funding.

Influence of Age and HLA Alleles on the CMV-Specific Cell-Mediated Immunity Among CMV-Seropositive Kidney Transplant Candidates

Influence of Age and HLA Alleles on the CMV-Specific Cell-Mediated Immunity Among CMV-Seropositive Kidney Transplant Candidates