Impact of Aging, Cytomegalovirus Infection, and Long-Term Treatment for Human Immunodeficiency Virus on CD8+ T-Cell Subsets.

Both healthy aging and human immunodeficiency virus (HIV) infection lead to a progressive decline in naive CD8+ T-cell numbers and expansion of the CD8+ T-cell memory and effector compartments. HIV infection is therefore often considered a condition of premature aging. Total CD8+ T-cell numbers of HIV-infected individuals typically stay increased even after long-term (LT) combination antiretroviral treatment (cART), which is associated with an increased risk of non-AIDS morbidity and mortality. The causes of these persistent changes in the CD8+ T-cell pool remain debated. Here, we studied the impact of age, CMV infection, and LT successful cART on absolute cell numbers in different CD8+ T-cell subsets. While naïve CD8+ T-cell numbers in cART-treated individuals (N = 38) increased to healthy levels, central memory (CM), effector memory (EM), and effector CD8+ T-cell numbers remained higher than in (unselected) age-matched healthy controls (N = 107). Longitudinal analysis in a subset of patients showed that cART did result in a loss of memory CD8+ T-cells, mainly during the first year of cART, after which memory cell numbers remained relatively stable. As CMV infection is known to increase CD8+ T-cell numbers in healthy individuals, we studied whether any of the persistent changes in the CD8+ T-cell pools of cART-treated patients could be a direct reflection of the high CMV prevalence among HIV-infected individuals. We found that EM and effector CD8+ T-cell numbers in CMV+ healthy individuals (N = 87) were significantly higher than in CMV- (N = 170) healthy individuals. As a result, EM and effector CD8+ T-cell numbers in successfully cART-treated HIV-infected individuals did not deviate significantly from those of age-matched CMV+ healthy controls (N = 39). By contrast, CM T-cell numbers were quite similar in CMV+ and CMV- healthy individuals across all ages. The LT expansion of the CM CD8+ T-cell pool in cART-treated individuals could thus not be attributed directly to CMV and was also not related to residual HIV RNA or to the presence of HIV-specific CM T-cells. It remains to be investigated why the CM CD8+ T-cell subset shows seemingly irreversible changes despite years of effective treatment.

Immunomodulation by hyperimmunoglobulins after solid organ transplantation: Beyond prevention of viral infection.

Hyperimmunoglobulins are pharmaceutical formulations of human IgG which contain high titers of antibodies against specific viruses. They have been successfully used in solid organ transplantation (SOT) to prevent Cytomegalovirus (CMV) and Hepatitis B Virus (HBV) infection. The introduction of effective and cheaper antiviral drugs has resulted in decreasing usage of hyperimmunoglobulins in SOT. However, it may still be attractive to combine antiviral drug therapy with hyperimmunoglobulins after SOT, as there is some evidence that hyperimmunoglobulins, similar to high doses of intravenous immunoglobulins (IVIgs), might exert anti-inflammatory activity and thereby prevent immunological graft damage and improve graft and patient survival. In this review we discuss the existing clinical evidence for beneficial anti-inflammatory effects of hyperimmunoglobulins after cardiac, lung, kidney, and liver transplantation. Only a limited number of studies have addressed this issue, and these studies often included small patient cohorts and showed considerable variations in the type, intensity and duration of treatment regimens. Due to these limitations, it is difficult to draw firm conclusions. Retrospective studies consistently demonstrated that addition of CMV hyperimmunoglobulin (CMV-Ig) to antiviral drug prophylaxis after lung transplantation is associated with reduced rates of CMV disease and bronchiolitis obliterans syndrome (BOS), and improved patient survival. The doses of CMV-Ig administered after SOT are much lower than the minimal effective dose of IVIg used for anti-inflammatory therapy in auto-immune diseases. Therefore, it is questionable whether the reduced incidence of BOS is the result of 'direct' anti-inflammatory effects of CMV-Ig or is caused by a reduction of CMV infection, which is a risk factor for BOS. No or very limited evidence for better prevention of immunological graft damage by anti-CMV combination therapy is available for heart, kidney and liver transplant patients. In liver transplantation published evidence suggests that the high-doses of Hepatitis B virus hyperimmunoglobulin (HBIg) administered to prevent HBV-infection may reduce the risk of acute rejection, while combination therapy of HBIg and antiviral drugs in HBV-infected patients is consistently associated with better graft and patient survival compared to antiviral monotherapy. Well-designed prospective randomized studies with larger patient cohorts are needed to substantiate the current limited evidence for anti-inflammatory benefits of hyperimmunoglobulins besides prevention of CMV and HBV infection after SOT.

Quantification of naive and memory T-cell turnover during HIV-1 infection.

BACKGROUND: In HIV infection, the homeostasis of CD4 and CD8 T cells is dramatically disturbed, and several studies have pointed out that T-cell turnover rates are increased. To understand how the CD4 and CD8 T-cell pools are affected, it is important to have quantitative insights into the lifespans of the cells constituting the different T-lymphocyte populations. METHODS: We used long-term in-vivo H2O labeling and mathematical modeling to estimate the average lifespans of naive and memory CD4 and CD8 T cells in untreated (n = 4) and combination antiretroviral therapy-treated (n = 3) HIV-1-infected individuals. RESULTS: During untreated chronic HIV-1 infection, naive CD4 and CD8 T cells lived on average 618 and 271 days, whereas memory CD4 and CD8 T cells had average lifespans of 53 and 43 days, respectively. These lifespans were at least three-fold shorter than those in healthy controls (n = 5). In patients on effective combination antiretroviral therapy with total CD4 T-cell counts in the normal range, we found that naive CD4 and CD8 T-cell lifespans had not completely normalized and were still two-fold shortened. CONCLUSION: The average lifespan of both naive and memory CD4 and CD8 T cells decreased during untreated chronic HIV-1 infection. Although the turnover of the memory T-cell populations nearly normalized during effective treatment, the turnover of naive CD4 and CD8 T cells did not seem to normalize completely.

Cytomegalovirus-Induced Expression of CD244 after Liver Transplantation Is Associated with CD8+ T Cell Hyporesponsiveness to Alloantigen.

The chronic presence of viral Ags can induce T cell exhaustion, which is characterized by upregulation of coinhibitory receptors and loss of T cell function. We studied whether a similar phenomenon occurs after liver transplantation (LTx), when there is continuous exposure to alloantigen. Expression of coinhibitory receptors on circulating CD4(+) and CD8(+) T cells was analyzed longitudinally in 19 patients until 6 mo after LTx and cross-sectionally in 38 patients late (1-12 y) after LTx. Expression of the coinhibitory receptors CD160 and CD244 on circulating CD8(+) T cells was already higher 6 mo after LTx compared with pre-LTx, and the elevated expression was sustained late after LTx, with CD244 showing the more prominent increase. The strongest upregulation of CD244 on circulating CD8(+) T cells was observed in patients who experienced CMV infection after LTx. CMV infection also was associated with reduced CD8(+) T cell proliferation and cytotoxic degranulation in response to alloantigen late after LTx. Purified CD244(+)CD8(+) T cells from LTx patients showed lower proliferative responses to alloantigen, as well as to polyclonal stimulation, than did their CD244(-) counterparts. In addition, the CD244(+)CD8(+) T cell population contained the majority of CMV peptide-loaded MHC class I tetramer-binding cells. In conclusion, CMV infection after LTx, rather than persistence of alloantigen, induces the accumulation of dysfunctional CD244(+)CD8(+) T cells in the circulation that persist long-term, resulting in reduced frequencies of circulating alloreactive CD8(+) T cells. These results suggest that CMV infection restrains CD8(+) T cell alloresponses after LTx.

Differences in Anti-Inflammatory Actions of Intravenous Immunoglobulin between Mice and Men: More than Meets the Eye.

Intravenous immunoglobulin (IVIg) is a therapeutic preparation of polyspecific human IgGs purified from plasma pooled from thousands of individuals. When administered at a high dose, IVIg inhibits inflammation and has proven efficacy in the treatment of various autoimmune and systemic inflammatory diseases. Importantly, IVIg therapy can ameliorate both auto-antibody-mediated and T-cell mediated immune pathologies. In the last few decades, extensive research in murine disease models has resulted in the elucidation of two novel anti-inflammatory mechanisms-of-action of IVIg: induction of FcγRIIB expression by sialylated Fc, and stimulation of regulatory T cells. Whereas controversial findings in mice studies have recently inspired intense scientific debate regarding the validity of the sialylated Fc-FcγRIIB model, the most fundamental question is whether these anti-inflammatory mechanisms of IVIg are operational in humans treated with IVIg. In this review, we examine the evidence for the involvement of these anti-inflammatory mechanisms in the therapeutic effects of IVIg in humans. We demonstrate that although several elements of both immune-modulatory pathways of IVIg are activated in humans, incorrect extrapolations from mice to men have been made on the molecular and cellular components involved in these cascades that warrant for critical re-evaluation of these anti-inflammatory mechanisms of IVIg in humans.

Human graft-derived mesenchymal stromal cells potently suppress alloreactive T-cell responses.

After organ transplantation, recipient T cells contribute to graft rejection. Mesenchymal stromal cells from the bone marrow (BM-MSCs) are known to suppress allogeneic T-cell responses, suggesting a possible clinical application of MSCs in organ transplantation. Human liver grafts harbor resident populations of MSCs (L-MSCs). We aimed to determine the immunosuppressive effects of these graft-derived MSCs on allogeneic T-cell responses and to compare these with the effects of BM-MSCs. BM-MSCs were harvested from aspirates and L-MSCs from liver graft perfusates. We cultured them for 21 days and compared their suppressive effects with the effects of BM-MSCs on allogeneic T-cell responses. Proliferation, cytotoxic degranulation, and interferon-gamma production of alloreactive T cells were more potently suppressed by L-MSCs than BM-MSCs. Suppression was mediated by both cell-cell contact and secreted factors. In addition, L-MSCs showed ex vivo a higher expression of PD-L1 than BM-MSCs, which was associated with inhibition of T-cell proliferation and cytotoxic degranulation in vitro. Blocking PD-L1 partly abrogated the inhibition of cytotoxic degranulation by L-MSCs. In addition, blocking indoleamine 2,3-dioxygenase partly abrogated the inhibitive effects of L-MSCs, but not BM-MSCs, on T-cell proliferation. In conclusion, liver graft-derived MSC suppression of allogeneic T-cell responses is stronger than BM-MSCs, which may be related to in situ priming and mobilization from the graft. These graft-derived MSCs may therefore be relevant in transplantation by promoting allohyporesponsiveness.

Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation.

C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development.

Rotterdam: main port for organ transplantation research in the Netherlands.

This overview describes the full spectrum of current pre-clinical and clinical kidney-, liver-, heart- and lung transplantation research performed in Erasmus MC - University Medical Centre in Rotterdam, The Netherlands. An update is provided on the development of a large living donor kidney transplantation program and on optimization of kidney allocation, including the implementation of a domino kidney-donation program. Our current research efforts to optimize immunosuppressive regimens and find novel targets for immunosuppressive therapy, our recent studies on prevention of ischemia-reperfusion-induced graft injury, our newest findings on stimulation of tissue regeneration, our novel approaches to prevent rejection and viral infection, and our latest insights in the regulation of allograft rejection, are summarized.

Intravenous immunoglobulin treatment in humans suppresses dendritic cell function via stimulation of IL-4 and IL-13 production.

High-dose i.v. Ig (IVIg) is a prominent immunomodulatory therapy for various autoimmune and inflammatory diseases. Recent mice studies suggest that IVIg inhibits myeloid cell function by inducing a cascade of IL-33-Th2 cytokine production causing upregulation of the inhibitory FcγRIIb, as well as by modulating IFN-γ signaling. The purpose of our study was to explore whether and how these mechanisms are operational in IVIg-treated patients. We show that IVIg in patients results in increases in plasma levels of IL-33, IL-4, and IL-13 and that increments in IL-33 levels correlate with rises in plasma IL-4 and IL-13 levels. Strikingly, no upregulation of FcγRIIb expression was found, but instead a decreased expression of the activating FcγRIIa on circulating myeloid dendritic cells (mDCs) after high-dose, but not after low-dose, IVIg treatment. In addition, expression of the signaling IFN-γR2 subunit of the IFN-γR on mDCs was downregulated upon high-dose IVIg therapy. In vitro experiments suggest that the modulation of FcγRs and IFN-γR2 on mDCs is mediated by IL-4 and IL-13, which functionally suppress the responsiveness of mDCs to immune complexes or IFN-γ. Human lymph nodes and macrophages were identified as potential sources of IL-33 during IVIg treatment. Interestingly, stimulation of IL-33 production in human macrophages by IVIg was not mediated by dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). In conclusion, high-dose IVIg treatment inhibits inflammatory responsiveness of mDCs in humans by Th2 cytokine-mediated downregulation of FcγRIIa and IFN-γR2 and not by upregulation of FcγRIIb. Our results suggest that this cascade is initiated by stimulation of IL-33 production that seems DC-SIGN independent.

SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner.

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD+-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis.

Prevention of immunoglobulin G immobilization eliminates artifactual stimulation of dendritic cell maturation by intravenous immunoglobulin in vitro.

Intravenous immunoglobulin (IVIg), a therapeutic preparation containing pooled human immunoglobulin (Ig) G, has been suggested to inhibit differentiation and maturation of dendritic cells (DCs); however, controversies exist on this issue. We aimed to reinvestigate the effects of IVIg on human DC maturation and cytokine production, and to determine whether an artifactual determinant is involved in the observed effects. Human monocyte-derived DCs or freshly isolated blood myeloid DCs were cultured in the presence of IVIg in vitro, and the expression of maturation markers CD80, CD86, CD83, and Human Leukocyte Antigen-DR were determined by flow cytometry, whereas production of interleukin (IL)-12 and IL-10 was measured by enzyme-linked immunosorbent assay, and T-cell stimulatory capacity was determined in cocultures with allogeneic CD4(+) T cells. Interestingly, we observed that IVIg did not inhibit, but instead stimulated, spontaneous maturation and T-cell stimulatory ability of human DCs, while leaving lipopolysaccharide-induced DC maturation and cytokine production unaffected. Strikingly, prevention of IVIg binding to culture plate surface, or blocking of the activating Fcγ receptor IIa on DC, abrogated the stimulatory effect of IVIg on costimulatory molecule expression and on T-cell stimulatory capacity of DCs, suggesting that IVIg activates DCs on IgG adsorption to the plastic surface. This study warrants for careful study design when performing cell culture studies with IVIg to prevent artifactual effects, and shows that IVIg does not modulate directly costimulatory molecule expression, cytokine production, or allogeneic T-cell stimulatory capacity of human DCs.

T-cell inhibitory capacity of hyperimmunoglobulins is influenced by the production process.

Intravenous immunoglobulin (IVIg) preparations are widely used for anti-inflammatory therapy of autoimmune and systemic inflammatory diseases. Hyperimmunoglobulins enriched in neutralizing antibodies against viruses can, in addition to their virus-neutralizing activity, also exert immunomodulatory activity. Previously, we observed that Cytotect®, an anti-CMV hyperimmunoglobulin, was less effective in suppressing human T-cell responses in vitro compared to Hepatect® CP, an anti-HBV hyperimmunoglobulin. We hypothesized that the poor immunomodulatory activity of Cytotect® results from treatment with ß-propiolactone during the manufacturing process. The manufacturer of these hyperimmunoglobulins has now introduced a new anti-CMV hyperimmunoglobulin, called Cytotect® CP, in which ß-propiolactone treatment is omitted. Here we show that Cytotect® CP inhibits PHA-driven T-cell proliferation and cytokine production with similar efficacy as Hepatect® CP, whereas the former Cytotect® does not. In addition, Cytotect® CP inhibits allogeneic T-cell responses better than Cytotect®. Our results advocate the use of hyperimmunoglobulins that have not been exposed to ß-propiolactone in order to benefit from their immunomodulatory properties.

Long-term restoration of the human T-cell compartment after thymectomy during infancy: a role for thymic regeneration?

Thymectomy during early childhood is generally thought to have serious consequences for the establishment of the T-cell compartment. In the present study, we investigated the composition of the T-cell pool in the first 3 decades after thymectomy during infancy due to cardiac surgery. In the first 5 years after thymectomy, naive and total CD4(+) and CD8(+) T-cell numbers in the blood and T-cell receptor excision circle (TREC) levels in CD4(+) T cells were significantly lower than in healthy age-matched controls. In the first years after thymectomy, plasma IL-7 levels were significantly elevated and peripheral T-cell proliferation levels were increased by ∼ 2-fold. From 5 years after thymectomy onward, naive CD4(+) and CD8(+) T-cell counts and TRECs were within the normal range. Because TREC levels are expected to decline continuously in the absence of thymic output, we investigated whether normalization of the naive T-cell pool could be due to regeneration of thymic tissue. In the majority of individuals who had been thymectomized during infancy, thymic tissue could indeed be identified on magnetic resonance imaging scans. Whereas thymectomy has severe effects on the establishment of the naive T-cell compartment during early childhood, our data suggest that functional regrowth of thymic tissue can limit its effects in subsequent years.

Immune reconstitution in children following chemotherapy for haematological malignancies: a long-term follow-up.

Modern intensive chemotherapy for childhood haematological malignancies has led to high cure rates, but has detrimental effects on the immune system. There is little knowledge concerning long-term recovery of the adaptive immune system. Here we studied the long-term reconstitution of the adaptive immune system in 31 children treated for haematological malignancies between July 2000 and October 2006. We performed detailed phenotypical and functional analyses of the various B and T cell subpopulations until 5 years after chemotherapy. We show that recovery of newly-developed transitional B cells and naive B and T cells occurred rapidly, within months, whereas recovery of the different memory B and T cell subpopulations was slower and incomplete, even after 5 years post-chemotherapy. The speed of B and T cell recovery was age-independent, despite a significant contribution of the thymus to T cell recovery. Plasmablast B cell levels remained above normal and immunoglobulin levels normalised within 1 week. Functional T cell responses were normal, even within the first year post-chemotherapy. This study shows that after intensive chemotherapy for haematological malignancies in children, numbers of several memory B and T cell subpopulations were decreased on the long term, while functional T cell responses were not compromised.

Lymphocyte characteristics in children with common variable immunodeficiency.

The diagnosis of common variable immunodeficiency (CVID) is reserved for patients who suffer from undefined B cell dysfunction. Division of the CVID population into subgroups enables research for underlying disease causes. We studied clinical features and lymphocyte characteristics in 38 children with CVID and compared them to 30 children with less severe antibody deficiencies (e.g. specific antibody deficiency combined with IgG subclass deficiency) and with 65 pediatric controls. Most pediatric immune phenotypes were comparable to adult CVID phenotypes, including a selective increase in newly formed B cells and a decrease in memory B cells and CD4(+) T cells. Eighteen percent of pediatric patients had a mutation in the TNFRSF13B gene, which requires further investigation. Finally, pediatric patients with decreased class-switched memory B cells had significantly more complications. A pediatric classification for CVID may enable prediction and early diagnosis of disease related complications and provide a framework for further etiologic research.

In vivo dynamics of stable chronic lymphocytic leukemia inversely correlate with somatic hypermutation levels and suggest no major leukemic turnover in bone marrow.

Although accumulating evidence indicates that chronic lymphocytic leukemia (CLL) is a disease with appreciable cell dynamics, it remains uncertain whether this also applies to patients with stable disease. In this study, (2)H(2)O was administered to a clinically homogeneous cohort of nine stable, untreated CLL patients. CLL dynamics in blood and bone marrow were determined and compared with normal B-cell dynamics in blood from five healthy individuals who underwent a similar (2)H(2)O labeling protocol. Average CLL turnover rates (0.08-0.35% of the clone per day) were approximately 2-fold lower than average B-cell turnover rates from healthy individuals (0.34-0.89%), whereas the rate at which labeled CLL cells in blood disappeared (0.00-0.39% of B cells per day) was approximately 10-fold lower compared with labeled B cells from healthy individuals (1.57-4.24% per day). Leukemic cell turnover variables inversely correlated with the level of somatic hypermutation of the CLL clone (IgVH mutations). Although CLL cells in bone marrow had a higher level of label enrichment than CLL cells in blood, no difference between proliferation rates and proapoptotic and antiapoptotic profiles of CLL cells from these compartments was observed. These data suggest that, in stable disease, there is a biological relationship between the degree of somatic hypermutation of the CLL clone and its dynamics in vivo. Furthermore, in contrast to lymph nodes, the bone marrow does not seem to be a major CLL proliferation site.

Restoration of the CD4 T cell compartment after long-term highly active antiretroviral therapy without phenotypical signs of accelerated immunological aging.

It remains uncertain whether full T cell reconstitution can be established in HIV-infected children and adults with long-term sustained virological control by highly active antiretroviral therapy (HAART). In this study, we comprehensively analyzed various phenotypical markers of CD4 T cell recovery. In addition to measuring T cell activation and proliferation markers, CD4 T cell generation and aging of the CD4 T cell compartment were assessed by measuring TCR excision circles and the fraction of CD31-expressing naive CD4 T cells. In all children and in adults with relatively high CD4 T cell counts at start of therapy (>200 cells/microl), total CD4 T cell numbers normalized within 1 year of therapy. After long-term HAART (4.4-9.6 years), naive CD4 T cell counts had normalized in both groups. Although in adults with low baseline CD4 T cell counts (<200 cells/microl) total CD4 T cell numbers normalized eventually after at least 7 years of HAART, naive CD4 T cell counts had still not recovered. TCR excision circle data showed that thymic T cell production contributed to naive T cell recovery at all ages. The fraction of CD31-expressing naive CD4 T cells was found to be normal, suggesting that the CD4 T cell repertoire was diverse after long-term HAART. Hence, under sustained viral suppression during long-term HAART, the T cell compartment has the potential to fully recover by generating new naive T cells both in children and in adults with high baseline CD4 T cells counts. Irrespective of baseline CD4 T cell counts, reconstitution occurred without a significant effect on T cell aging as reflected by markers for replicative history.

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing.

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5' untranslated region (UTR) through the 3'-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5' and 3'-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.