Innate adaptive immune cell dynamics in tonsillar tissues during chronic SIV infection.

HIV-infected patients are at higher risk of developing oral mucosal infection and Epstein-Barr virus (EBV)-associated B cell malignancies. However, the potential role of oral immunity in the pathogenesis of oral lesions is unknown. Tonsils are oral-pharyngeal mucosal-associated lymphoid tissues that play an important role in oral mucosal immunity. In this study, we investigated the changes of innate and adaptive immune cells in macaque tonsils during chronic SIV infection. We found significantly higher frequencies of classical monocytes, CD3+CD56+ (NKT-like) cells, CD3+CD4+CD8+ (DP), and CD161+ CD4 T cells in tonsils from chronic infected compared to naïve animals. On the contrary, intermediate monocytes and CD3+CD4-CD8- (DN) cells were lower in chronic SIV-infected macaques. We further confirmed a recently described small B-cell subset, NKB cells, were higher during chronic infection. Furthermore, both adaptive and innate cells showed significantly higher TNF-α and cytotoxic marker CD107a, while IL-22 production was significantly reduced in innate and adaptive immune cells in chronic SIV-infected animals. A dramatic reduction of IFN-γ production by innate immune cells might indicate enhanced susceptibility to EBV infection and potential transformation of B cells in the tonsils. In summary, our observation shows that the SIV-associated immune responses are distinct in the tonsils compared to other mucosal tissues. Our data extends our understanding of the oral innate immune system during SIV infection and could aid future studies in evaluating the role of tonsillar immune cells during HIV-associated oral mucosal infections.

CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells.

CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact of CD3e-IT treatment on these local cells, however, remains poorly understood. Here, using a new murine testing model, we demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment. Differential surface expression of CD3e among T-cell subsets appears to be a main driver of Treg enrichment in CD3e-IT treatment. The surviving Tregs in CD3e-IT-treated mice were mostly the CD3edimCD62Llo effector phenotype, but the levels of this phenotype markedly varied among different lymphoid and nonlymphoid organs. We also found notable variations in surface CD3e levels among tissue-resident T cells of different organs, and these variations drive CD3e-IT to uniquely reshape T-cell compositions in local organs. The functions of organs and anatomic locations (lymph nodes) also affected the efficacy of CD3e-IT. The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Estrogen May Enhance Toll-Like Receptor 4-Induced Inflammatory Pathways in People With HIV: Implications for Transgender Women on Hormone Therapy.

Background: Transgender women (TW) are at increased risk for both human immunodeficiency virus (HIV) and cardiovascular disease (CVD). Antiretroviral therapy-treated HIV has been associated with a two-fold increased risk of CVD, potentially due to dysregulated Toll-like receptor (TLR)-induced immune activation. Use of estrogens in feminizing hormone therapy (FHT) may enhance inflammatory responses and the risk of cardiovascular mortality in TW. Despite this, the immunomodulatory effects of estrogen use in TW with HIV have been inadequately explored. Methods: As an in vitro model for FHT, cryopreserved PBMCs (cryoPBMCs) from HIV negative (HIV-), HIV+ ART-suppressed (HIV+SP), and HIV+ ART-unsuppressed (HIV+USP) cisgender men were cultured overnight in the presence of 17-ß estradiol or 17-α ethinylestradiol with and without the TLR4 agonist LPS or the TLR8 agonist ssPolyU. Monocyte activation (CD69, HLA-DR, CD38) was assessed by flow cytometry. Cytokine levels (IL-6, TNF-α, IL-1ß, and IL-10) were measured in cell culture supernatants by Legendplex. Levels of phosphorylated TLR signaling molecules (JNK, MAPK p38) were assessed by Phosflow. Plasma levels of immune activation biomarkers (LPS-binding protein, monocyte activation markers sCD14 and sCD163, and inflammatory molecules IL-6 and TNF-α receptor I) were measured by ELISA. Results: PBMCs from people with HIV (PWH) produced greater levels of inflammatory cytokines following exposure to LPS or ssPolyU compared to levels from cells of HIV- individuals. While estrogen exposure alone induced mild changes in immune activation, LPS-induced TLR4 activation was elevated with estrogen in cisgender men (CM) with HIV, increasing monocyte activation and inflammatory cytokine production (IL-6, TNF-α). Interestingly, testosterone inhibited LPS-induced cytokine production in CM regardless of HIV status. Plasma markers of immune activation and microbial translocation (e.g., sCD14, sCD163, LPS-binding protein) were generally higher in PWH compared to HIV- CM, and these markers were positively associated with in vitro responsiveness to estrogen and LPS in CM with HIV. Conclusions: Our in vitro data suggest that estrogen exposure may enhance innate immune activation in PWH. Further examination is needed to fully understand the complex interactions of FHT, HIV, and CVD in TW, and determine optimal FHT regimens or supplementary treatments aimed at reducing excess immune activation.

Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. METHODS: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. RESULTS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. CONCLUSIONS: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

Expression of CD40L by the ALVAC-Simian Immunodeficiency Virus Vector Abrogates T Cell Responses in Macaques.

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.IMPORTANCE CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective cytotoxic and humoral responses against pathogens. Because of its immunomodulatory function, CD40L has been used to enhance immune responses to vaccines, including candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a strategy combining canarypox virus-based vector (ALVAC) together with an envelope protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing HIV-1/SIV acquisition in humans and macaques. In both species, protection was associated with vaccine-induced antibodies against the HIV envelope and CD4+ T cell responses, including type 1 antiviral responses. In this study, we tested whether augmenting CD40L expression by coexpressing it with the ALVAC vector could increase the protective immune responses. Although coexpression of CD40L did increase humoral responses, it blunted type 1 CD4+ T cell responses against the SIV envelope protein and failed to protect macaques from viral infection.

Lung T-Cell Profile Alterations are Associated with Bronchiolitis Obliterans Syndrome in Cystic Fibrosis Lung Transplant Recipients.

The contribution of T-cells after lung transplant (LTx) remains controversial with no current consensus of their role concerning chronic lung allograft dysfunction. Using flow cytometry to assess T-cell subsets of bronchoalveolar lavage fluid (BALF) in 16 cystic fibrosis (CF) LTx recipients, we identified a decline in CD4+ T-cell frequency and an increase in CD8+ T-cell frequency in patients who developed severe bronchiolitis obliterans syndrome (BOS) (N = 10) when comparing baseline (6 months post-LTx) and follow-up (most recent bronchoscopy-clinical or surveillance per protocol). Comparing BOS to No BOS cohorts, significant differences were found in CD4+ T-cell frequency [17.4 (12.5, 28.2) vs 46.6 (44.4, 48.4), p = 0.003] and CD8+ T-cell frequency [65.6 (62.8, 75.3) vs 39.2 (32.2, 43.3), p = 0.014], respectively. The mean difference of the CD4:CD8 ratio was 0.87 units lower (95% CI - 1.44 to - 0.30, p = 0.006) than patients without BOS, while the median difference of the CD4:CD8 ratio was 0.92 units lower (95% CI - 1.83 to - 0.009, p = 0.048). Therefore, our results suggest that T-cell profiles measured through flow cytometry of BALF in the CF LTx population are associated with the development of severe BOS. Further work is needed in larger patient populations to validate our findings and to determine if this is useful for recipients who underwent LTx for other indications.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer.

Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition.

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

Glucocorticoid treatment at moderate doses of SIVmac251-infected rhesus macaques decreases the frequency of circulating CD14+CD16++ monocytes but does not alter the tissue virus reservoir.

Subsets of CD16-positive monocytes produce proinflammatory cytokines and expand during chronic infection with the human immunodeficiency virus type 1 (HIV). HIV-infected macrophage in tissues may be long lived and contribute to the establishment and maintenance of the HIV reservoir. We found that the (intermediate) CD14(++)CD16(+) and (nonclassical) CD14(+)CD16(++) monocyte subsets are significantly expanded during infection of Rhesus macaques with pathogenic SIV(mac251) but not during infection of sooty mangabeys with the nonpathogenic isolate SIVSM. In vitro glucocorticoid (GC) treatment of peripheral blood mononuclear cells (PBMCs) from uninfected or SIV(mac251)-infected Rhesus macaques and HIV-infected patients treated or not with antiretroviral therapy (ART) resulted in a significant decrease in the frequency of both CD16-positive monocyte subsets. Short-term in vivo treatment with high doses of GC of chronically SIV(mac251)-infected macaques resulted in a significant decrease in the CD14(+)CD16(++) population and, to a lesser extent, in the CD14(++)CD16(+) monocytes, as well as a significant decrease in the number of macrophages in tissues. Surprisingly, treatment of SIV(mac251)-infected macaques with ART significantly increased the CD14(++)CD16(+) population and the addition of GC resulted in a significant decrease in only the CD14(+)CD16(++) subset. No difference in SIV DNA levels in blood, lymph nodes, gut, and spleen was found between the groups treated with ART or ART plus GC. Thus, it appears that high doses of GC treatment in the absence of ART could affect both CD16-positive populations in vivo. Whether the efficacy of this treatment at higher doses to decrease virus levels outweighs its risks remains to be determined.

Antibody to the gp120 V1/V2 loops and CD4+ and CD8+ T cell responses in protection from SIVmac251 vaginal acquisition and persistent viremia.

The human papillomavirus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an immune response in the vaginal tract of nonhuman primates and mice. Intravaginal vaccination with HPV-PsVs expressing SIV genes, combined with an i.m. gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming systemic immune responses with i.m. immunization with ALVAC-SIV vaccines, followed by intravaginal HPV-PsV-SIV/gp120 boosting, expanded and/or recruited T cells in the female genital tract. Using a stringent repeated low-dose intravaginal challenge with the highly pathogenic SIVmac251, we show that although these regimens did not demonstrate significant protection from virus acquisition, they provided control of viremia in a number of animals. High-avidity Ab responses to the envelope gp120 V1/V2 region correlated with delayed SIVmac251 acquisition, whereas virus levels in mucosal tissues were inversely correlated with antienvelope CD4(+) T cell responses. CD8(+) T cell depletion in animals with controlled viremia caused an increase in tissue virus load in some animals, suggesting a role for CD8(+) T cells in virus control. This study highlights the importance of CD8(+) cells and antienvelope CD4(+) T cells in curtailing virus replication and antienvelope V1/V2 Abs in preventing SIVmac251 acquisition.

Comparative analysis of SIV-specific cellular immune responses induced by different vaccine platforms in rhesus macaques.

To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.

Humoral immunity induced by mucosal and/or systemic SIV-specific vaccine platforms suggests novel combinatorial approaches for enhancing responses.

Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in the bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest that combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.

Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

BACKGROUND: Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated. MATERIALS AND METHODS: The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72 hours post-treatment by flow cytometry. RESULTS: Similar time course of HhepCDT-induced G2 /M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72 hours. The presence of all three HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2 /M arrest and apoptotic death. CONCLUSION: All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.

Helicobacter hepaticus cytolethal distending toxin causes cell death in intestinal epithelial cells via mitochondrial apoptotic pathway.

BACKGROUND: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram-negative bacteria is associated with double-stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells. MATERIALS AND METHODS: INT407 cells were incubated with or without recombinant HhCDT for 0-72 hours. H2AX phosphorylation and apoptotic parameters were analyzed. RESULTS: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro-apoptotic Bax protein was upregulated after 24 hours, while Bcl(2) expression decreased. Cytochrome c was released from mitochondria after 12-24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z-VAD-FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT-treated cells. CONCLUSION: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.

Regulation of the bioavailability of thioredoxin in the lens by a specific thioredoxin-binding protein (TBP-2).

Thioredoxin (TRx) is known to control redox homeostasis in cells. In recent years, a specific TRx binding protein called thioredoxin binding protein-2 (TBP-2) was found in other cell types and it appeared to negatively regulate TRx bioavailability and thereby control TRx biological function. In view of the sensitivity of lens transparency to redox status, proper regulation of TRx bioavailability is of the utmost importance. This study was conducted to examine the presence and function of TBP-2 in human lens epithelial cells (HLE B3). We cloned human lens TBP-2 from a human cDNA library (GenBank accession number AY 594328) and showed that it is fully homologous to the human brain TBP-2 gene. The recombinant TBP-2 protein was partially purified and mass spectrometric analysis confirmed its sequence homology to that of brain TBP-2. Immunoprecipitates obtained from HLE B3 cells using anti-TRx and anti-TBP-2 antibodies showed the presence of TRx and TBP-2 in immunoprecipitates indicating the formation of a TRx-TBP-2 complex in vivo. Furthermore, under H(2)O(2)-stress conditions, TRx gene expression was transiently up-regulated while TBP-2 gene expression was inversely down-regulated as seen in both HLE B3 cells and in the epithelial cell layers from cultured pig lenses. Cells with overexpressed TBP-2 showed lower TRx activity, grew slower and were more susceptible to oxidative stress-induced apoptosis. This is the first report of the presence of a TRx-specific binding protein in the lens. Our data suggest that TBP-2 is likely a negative regulator for the bioavailability, and therefore, the overall function of TRx in the lens.