CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy targeting solid tumors has stagnated as a result of tumor heterogeneity, immunosuppressive microenvironments, and inadequate intratumoral T cell trafficking and persistence. Early (≤3 days) intratumoral presentation of CAR T cells post-treatment is a superior predictor of survival than peripheral persistence. Therefore, we have co-opted IL-8 release from tumors to enhance intratumoral T-cell trafficking through a CAR design for maximal antitumor activity in solid tumors. Here, we demonstrate that IL-8 receptor, CXCR1 or CXCR2, modified CARs markedly enhance migration and persistence of T cells in the tumor, which induce complete tumor regression and long-lasting immunologic memory in pre-clinical models of aggressive tumors such as glioblastoma, ovarian and pancreatic cancer.

CD70, a novel target of CAR T-cell therapy for gliomas.

Background: Cancer immunotherapy represents a promising treatment approach for malignant gliomas but is hampered by the limited number of ubiquitously expressed tumor antigens and the profoundly immunosuppressive tumor microenvironment. We identified cluster of differentiation (CD)70 as a novel immunosuppressive ligand and glioma target. Methods: Normal tissues derived from 52 different organs and primary and recurrent low-grade gliomas (LGGs) and glioblastomas (GBMs) were thoroughly evaluated for CD70 gene and protein expression. The association between CD70 and patients' overall survival and its impact on T-cell death was also evaluated. Human and mouse CD70-specific chimeric antigen receptors (CARs) were tested respectively against human primary GBMs and murine glioma lines. The antitumor efficacies of these CARs were also examined in orthotopic xenograft and syngeneic models. Results: CD70 was not detected in peripheral and brain normal tissues but was constitutively overexpressed by isocitrate dehydrogenase (IDH) wild-type primary LGGs and GBMs in the mesenchymal subgroup and recurrent tumors. CD70 was also associated with poor survival in these subgroups, which may link to its direct involvement in glioma chemokine productions and selective induction of CD8+ T-cell death. To explore the potential for therapeutic targeting of this newly identified immunosuppressive axis in GBM tumors, we demonstrate that both human and mouse CD70-specific CAR T cells recognize primary CD70+ GBM tumors in vitro and mediate the regression of established GBM in xenograft and syngeneic models without illicit effect. Conclusion: These studies identify a previously uncharacterized and ubiquitously expressed immunosuppressive ligand CD70 in GBMs that also holds potential for serving as a novel CAR target for cancer immunotherapy in gliomas.

Preclinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers.

Purpose: CD70 expression in normal tissues is restricted to activated lymphoid tissues. Targeting CD70 on CD70-expressing tumors could mediate "on-target, off-tumor" toxicity. This study was to evaluate the feasibility and safety of using anti-human CD70 CARs to treat cancer patients whose tumors express CD70.Experimental Design: Seven anti-human CD70 CARs with binding moieties from human CD27 combined with CD3-zeta and different costimulatory domains from CD28 and/or 41BB were constructed. In vitro functionality of these receptors was compared and in vivo treatment efficacy was evaluated in a xenograft mouse model. A homologous, all murine anti-CD70 CAR model was also used to assess treatment-related toxicities.Results: The CAR consisting of the extracellular binding portion of CD27 fused with 41BB and CD3-zeta (trCD27-41BB-zeta) conferred the highest IFNγ production against CD70-expressing tumors in vitro, and NSG mice bearing established CD70-expressing human tumors could be cured by human lymphocytes transduced with this CAR. In the murine CD27-CD3-zeta CAR model, significant reduction of established tumors and prolonged survival were achieved using CAR-transduced splenocytes in a dose-dependent manner. Host preirradiation enhanced treatment efficacy but increased treatment-related toxicities such as transient weight loss and hematopoetic suppression. The treatment did not appear to block adaptive host immune responses.Conclusions: Preclinical testing supports the safety and efficacy of a CD27-containing CAR targeting CD70-expressing tumors. Clin Cancer Res; 23(9); 2267-76. ©2016 AACR.

Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors.

KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60% to 70% of pancreatic cancers and 20% to 30% of colorectal cancers. The consistency, frequency, and tumor specificity of these "neoantigens" make them attractive therapeutic targets. Recent data associate T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCR) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBL) transduced with these TCRs could recognize multiple HLA-A*11:01(+) tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers supports clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types.

Distinctive features of the differentiated phenotype and infiltration of tumor-reactive lymphocytes in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (RCC) is considered an immunogenic tumor, but it has been difficult to identify tumor-infiltrating lymphocytes (TIL) that show in vitro tumor recognition. We compared the characteristics of fresh RCC TIL to peripheral blood lymphocytes (PBL) or melanoma TIL. Our results showed that RCC TIL contained fewer CD27(+) T cells, and fewer naïve and central memory (CM) T cells, but more effector memory (EM) T cells than melanoma TIL or renal PBL. We hypothesized that factors in the RCC microenvironment were skewing TIL phenotype toward EM. One possibility was the expression of CD70 on nearly all human RCCs, but not melanomas. Differentiation of naïve T cells to EM cells only occurred from CD70 costimulation in concert with T-cell receptor (TCR) stimulation (signal one), suggesting that EM TIL responding to CD70 would be enriched for T cells reactive with local antigens, including those associated with RCC. Clonotypic analysis of TCRs in fresh RCCs showed that EM T cells were more clonally expanded than CM or naïve T cells, and the clonal expansion occurred at the tumor site as oligoclonal TCRs were distinct from PBL TCRs from the same patient. In addition, we found that 2 TCRs from the highly represented EM TIL clones, when reexpressed in fresh PBL, recognized an MHC-class II or MHC-class I-restricted antigens shared by multiple RCC lines. Our results suggest that RCC-reactive TIL do exist in situ, but may be difficult to recover and study because of proliferative exhaustion, driven by tumor-expressed CD70.

The shedding of CD62L (L-selectin) regulates the acquisition of lytic activity in human tumor reactive T lymphocytes.

CD62L/L-selectin is a marker found on naïve T cells and further distinguishes central memory (Tcm, CD62L+) from effector memory (Tem, CD62L-) T cells. The regulation of CD62L plays a pivotal role in controlling the traffic of T lymphocytes to and from peripheral lymph nodes. CD62L is shed from the cell membrane following T cell activation, however, the physiological significance of this event remains to be elucidated. In this study, we utilized in vitro generated anti-tumor antigen T cells and melanoma lines as a model to evaluate the dynamics of CD62L shedding and expression of CD107a as a marker of lytic activity. Upon encounter, with matched tumor lines, antigen reactive T cells rapidly lose CD62L expression and this was associated with the acquisition of CD107a. By CD62L ELISA, we confirmed that this transition was mediated by the shedding of CD62L when T cells encountered specific tumor antigen. The introduction of a shedding resistant mutant of CD62L into the tumor antigen-reactive T cell line JKF6 impaired CD107a acquisition following antigen recognition and this was correlated with decreased lytic activity as measured by (51)Cr release assays. The linkage of the shedding of CD62L from the surface of anti-tumor T cells and acquisition of lytic activity, suggests a new function for CD62L in T cell effector functions and anti-tumor activity.

Irradiation enhances human T-cell function by upregulating CD70 expression on antigen-presenting cells in vitro.

In addition to the direct killing of tumor cells, radiation therapy can alter the balance of immune cells in vivo due to the differential radiosensitivity of different cell types. The addition of adjuvant radiation therapy before adoptive cell transfer therapy has been shown to enhance antitumor responses in both mouse models and clinical trials. This study examines the effects of in vitro irradiation on the phenotype and function of human antigen-presenting cells. The results indicated that irradiation upregulated CD70 expression on both B cells and mature dendritic cells (DCs). Expression of CD70 on mature DCs was enhanced in a dose-dependent manner, whereas under the same conditions, no significant upregulation of CD80, CD86, or CD40 was observed. The levels of expression of CD70 induced on mature DC by irradiation correlated highly with the ability of those cells to stimulate T-cell proliferation and interferon-γ production. Furthermore, significant reductions in T-cell proliferation and interferon-γ production were seen when CD70 expression on DCs was partially reduced using shRNA, as well as when DCs were incubated with a blocking anti-CD70 antibody. Radiation therapy may therefore enhance T-cell activation in vivo through the CD27 pathway by virtue of its ability to upregulate the expression of CD70 on antigen-presenting cells.

Molecular identification of an MHC-independent ligand recognized by a human {alpha}/{beta} T-cell receptor.

During an analysis of T-cell responses against human renal cell carcinoma (RCC), we identified a CD4(+) T-cell line that showed TCR-mediated recognition and lysis of nearly all RCC lines regardless of MHC type. We have now elucidated the nature of the ligand for this α/ß TCR, and it contains no MHC-related moiety and does not involve classic peptide processing. First, matrix metalloproteinase 14 (MMP14) expressed on RCC cells releases membrane-bound TRAIL expressed by the T cell; then, soluble TRAIL binds to its receptor DR4 (TRAIL-R1), which is expressed on tumor cells, and this TRAIL-DR4 complex is recognized by the TCR through a complementarity-determining region 3α (CDR3α)-mediated interaction. Direct and specific antigen-TCR interaction was demonstrated when the immobilized recombinant TRAIL/DR4 complex stimulated the TCR. In addition, amino acid substitutions in the CDR3α of the TCR either obliterated or enhanced target-specific recognition. This description of the molecular nature of a non-MHC target structure recognized by a naturally occurring α/ß TCR not only broadens our concept of what the TCR can recognize, but also raises the question of whether such a T cell could be of clinical utility against RCC.

Development of a genetically-modified novel T-cell receptor for adoptive cell transfer against renal cell carcinoma.

A novel α/ß T-cell clone with broad reactivity against human clear cell renal cell carcinomas (RCC) was generated from a patient with renal cancer. The T-cell receptor (TCR) from this clone recognizes soluble TNF-related apoptosis inducing ligand bound to death receptor 4, a complex found on the surface of nearly all RCC. In this study, we modified this novel TCR by introducing amino acid (AA) substitutions in its complementarity determining region 2 (CDR2) and CDR3 regions of both chains, to increase its activity. We demonstrated that tumor recognition by PBL, retrovirally-transduced with these TCRs, was decreased or unchanged by substitutions in the TCR beta chain, and in the CDR2α region. Yet some AA substitutions in the CDR3α region at positions 109 and 112 could augment tumor recognition. Specifically, substituting phenylalanine for tyrosine at AA109 (109Y-F) and alanine or lysine for serine at AA112 (112S-K or 112 S-A) augmented tumor recognition. Increased benefit was seen on combining both AA substitutions and a retrovirus encoding the modified TCR 109Y-F/112S-K conferred the best tumor recognition to transduced PBL. This modified TCR retained the recognition pattern of parental clone HC/2G-1 against RCC lines, other tumors and normal tissues. These results document that CDR3α plays an important role in the interaction of the HC/2G-1 TCR and its novel ligand. A phase I/II clinical trial, adoptively transferring autologous PBL transduced with this modified TCR has just begun in patients with metastatic RCC.

Selection of CD8+PD-1+ lymphocytes in fresh human melanomas enriches for tumor-reactive T cells.

CD8+ tumor-infiltrating lymphocytes (TILs) in human melanomas express high levels of PD-1 and are functionally impaired. However, adoptive cell therapy using in vitro-expanded TIL can be a highly effective therapy for patients with advanced melanoma. This discrepancy led us to further analyze the CD8+PD-1+ TILs. We found that the percentage of PD-1-expressing CD8+ T cells was higher in the tumor digests that generate tumor-reactive TILs after in vitro culture in interleukin-2 (P=0.0007). Also sorted and expanded CD8+PD-1+ T cells in tumor digests showed much higher tumor-specific interferon-γ production compared with CD8+PD-1⁻ T cells. These results suggested that tumor-specific CD8+ T cells in melanoma tumor digests are largely PD-1, and this population can recover function after culturing in interleukin-2. PD-1 has been reported as an inhibitory receptor on T cells. We found that the in vitro functional suppression of cultured-TILs from native levels of PD-L1 expression on melanomas was minimal, and moreover expression level of PD-1 on CD8+ tumor-specific TILs decreased during the culture. As a consequence, the PD-1 receptor can be a useful biomarker for enriching tumor-specific T cells from fresh melanomas.

A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity.

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3zeta to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2(+) tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3zeta signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3zeta signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3zeta ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3zeta signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

IL-17 secreted by tumor reactive T cells induces IL-8 release by human renal cancer cells.

TH17 is a newly identified pathogenic memory CD4 T-cell lineage with potent agonist effects in some murine experimental autoimmunity models, however, its role in tumor immunology is still unclear. Clinical experience with interleukin (IL)-2 and ipilumumab [anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) antibody], particularly in treating immunogenic malignancies such as melanoma and renal cell carcinoma (RCC), has suggested an association between a variety of autoimmune phenomena and tumor regression. To investigate this issue in patients with RCC, we isolated T-cell clones from peripheral blood that released IL-17 on stimulation with their autologous RCC tumor line. Clones were generated from 1 patient before any systemic treatment by in vitro stimulation with dendritic cells, autologous tumor, and IL-2 in the presence of anti-CTLA4 antibody. That patient subsequently received treatment with ipilimumab and showed both objective tumor regression and immune-mediated colitis. Limiting dilution cloning of his tumor-dendritic cell-stimulated T cells produced the 3G8D CD4+ clone, which secreted both IL-17 and interferon-gamma when cocultured with the autologous RCC line (transduced with class II transactivation molecule to induce major histocompatibility complex-class II expression). Broader analysis of its cytokine secretion profile showed large amounts of IL-8 when cocultured with RCC, but not when triggered with phorbol 12-myristate 13-acetate and ionomycin. This led to the discovery that IL-8 was being produced by the RCC cells in response to T-cell-derived IL-17. This effect of exogenous IL-17 on IL-8 release from tumor was seen in 5 of 8 RCCs, but not in other tumors tested. Preliminary data on the frequency of IL-17-secreting T cells in the lymphocytes infiltrating RCCs suggest that there may be a positive correlation between this frequency and IL-8 production by nonlymphoid cells as determined by quantitative reverse transcription-polymerase chain reaction. This report extends the known bidirectional interactions between immune cells and malignant cells in the tumor microenvironment that can shape and modulate the host immune response to cancer.

Characterization of a novel nonclassical T cell clone with broad reactivity against human renal cell carcinomas.

A CD4(+) T cell clone (HC/2G-1) was established by stimulating peripheral blood T cells from a patient with renal cell carcinoma (RCC) with dendritic cells preincubated with the autologous apoptotic renal tumor line in the presence of IFN-alpha. It recognizes the autologous RCC and most allogeneic RCC lines by IFN-gamma release (10 of 11 lines) and lysis (9 of 10 lines), but does not recognize multiple EBV B cells or fibroblasts. It shows little or no recognition of a panel of melanomas, breast cancers and non-small-cell lung cancers. Phenotypically, HC/2G-1 is CD3(+)CD4(+) TCR alphabeta(+), but CD161(-)CD16(-)NKG2D(-). Tumor recognition by clone HC/2G-1 was not blocked by Abs to HLA class I or class II, but was significantly reduced by anti-TCR alphabeta Ab. Furthermore, tumor recognition was beta(2)-microglobulin-independent. HC/2G-1 does not use a Valpha or Vbeta described for classical NKT cells, but rather Valpha14 and Vbeta2.1. Allogeneic T cells cotransfected with mRNAs encoding the alpha and beta chains of the HC/2G-1 TCR recognized renal tumor lines, demonstrating that tumor recognition is TCR-mediated. Interestingly, TRAIL appears to play a role in tumor recognition by HC/2G-1 in that reactivity was blocked by anti-TRAIL Ab, and soluble TRAIL could enhance IFN-gamma secretion by HC/2G-1 in response to renal tumors. Our findings suggest that clone HC/2G-1 represents a novel type of CD4(+) cell that has broad TCR-mediated recognition of a determinant widely expressed by RCC.

Consistency of initial antiretroviral therapy with HIV treatment guidelines in a US cohort of HIV-infected women.

BACKGROUND: HIV treatment guidelines define optimal initial antiretroviral therapy (ART). OBJECTIVE: To characterize initial ART used by a cohort of HIV-infected women according to US HIV treatment guidelines and determine whether regimen characteristics predict short-term outcomes. METHODS: Initial ART self-reported by Women's Interagency HIV Study (WIHS) participants. Regimens were classified as guideline consistent (GC), guideline not recommended (GNR), or unlisted. Univariate and multivariate logistic regression was used to analyze factors associated with guideline category. RESULTS: Two hundred seventeen WIHS participants initiated ART during the study period. Fifty-three percent reported use ofGC ART, 17% reported GNR ART, and 30% reported ART unlisted in guidelines. Study site, higher pretreatment CD4 cell count, lower HIV RNA level, and initiation before 2001 were associated with use of GNR regimens. GC ART users had a higher rise in CD4 cell counts and more frequent undetectable HIV-1 RNA levels 2 years after initiation compared with those GNR (P = 0.0003) or unlisted initial ART. CONCLUSIONS: A higher than expected proportion of WIHS participants reported using initial ART not recommended by HIV treatment guidelines, although this decreased over time. Use of such regimens was associated with a higher incidence of switching and poorer short-term immunologic and virologic outcomes.

High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines.

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 microM to 26 pM). CD8(+) T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (K(D) value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, K(D) 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range-with K(D) values of 450 nM and 4 microM-demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8(+) T cells, introduction of these TCR dramatically increased the reactivity of CD4(+) T cells to tumor cell lines.

Generating renal cancer-reactive T cells using dendritic cells (DCs) to present autologous tumor.

Dendritic cells (DCs) have been used as professional antigen-presenting cells in vitro to prime T-cell responses. In this study, we generated both CD8+ and CD4+ renal cell carcinoma (RCC)-reactive T cells using a completely autologous system of DCs presenting engulfed whole-tumor cells. We compared DCs presenting RCC tumor cells in different preparations and found ultraviolet-irradiated apoptotic tumor cells to be more immunogenic than necrotic tumor cells or live untreated tumor cells in generating tumor-reactive T cells. In analyzing the T cells generated in this fashion, a CD8+ RCC-reactive T-cell clone generated from a patient recognized an epitope derived from fibroblast growth factor 5 in the context of human leukocyte antigen (HLA) B44*02. CD4+ T cells generated from another patient recognized multiple allogeneic RCC lines expressing HLA-DRbeta1*04, indicating a common shared tumor antigen presented by HLA-DRbeta1*04. The technique of using DCs to present whole-tumor cells can consistently generate both CD4+ and CD8+ RCC-reactive T cells for use in both antigen identification and therapeutic protocols.

Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women.

Bacterial vaginosis (BV) is a clinical syndrome presenting with a malodorous vaginal discharge and increased vaginal pH. Diagnosis has been based on clinical Amsel criteria and direct Gram stain of vaginal secretions. Human immunodeficiency virus (HIV)-infected participants in the Women's Interagency HIV Study contributed cervicovaginal lavage (CVL) samples. Lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage samples were quantified by PCR. Gynecologic evaluation included Nugent score and Amsel criterion assessment. We compared the gold standard Nugent score to Amsel criteria and quantitative bacterial PCR for diagnosing BV in 203 CVL samples from women with Nugent scores of 7 to 10 (BV group) and 203 samples from women with BV Nugent scores of 0 to 3 ("No-BV" group). Only 75 of the 203 CVL samples from women with Nugent scores of 7 to 10 met positive Amsel criteria. Increasing levels of G. vaginalis and M. hominis and decreasing levels of lactobacilli were significantly associated with BV by Nugent score. Of the group with Nugent scores of 7 to 10, 83% and 81% had log(10) G. vaginalis counts and log(10) M. hominis counts greater than 6.81 and 4.82, respectively, while only 30% and 31% of the group with Nugent scores of 0 to 3 were above these thresholds, respectively. There was significant overlap in the log(10) lactobacillus counts between the two groups. Utilizing all three log(10) bacterial counts (G. vaginalis, M. hominis, and lactobacilli) in our model improved the sensitivity and specificity to 83% and 78%, respectively, in comparison with Nugent score. In this cohort, Amsel criteria were poorly predictive of BV. PCR quantification of G. vaginalis and M. hominis from CVL is significantly more sensitive than Amsel criteria for diagnosing BV.

Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis.

BACKGROUND: Bacterial vaginosis (BV) is associated with human immunodeficiency virus (HIV) acquisition. We examined the association between BV and BV-associated bacteria and expression of HIV in the female genital tract. METHODS: HIV RNA, lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage (CVL) samples were quantified by polymerase chain reaction (PCR). Gynecologic evaluation included Nugent score assessment, Amsel criteria assessment, detection of other genital-tract infections, and dysplasia grading. CD4 cell count, plasma HIV RNA level, and antiretroviral history were obtained. RESULTS: A total of 203 CVL samples from women with Nugent scores of 7-10 (BV group) and 203 samples from women with Nugent scores of 0-3 (no-BV group) were matched by plasma HIV RNA level and analyzed. After controlling for plasma HIV RNA level and Nugent score in univariate analyses, we found that G. vaginalis and M. hominis bacterial counts, Candida vaginitis, and herpes simplex virus (HSV) were positively associated with CVL HIV RNA levels. In multivariate analysis, only lactobacilli bacterial counts (P=.006; inverse association), M. hominis bacterial counts (P=.0001; positive association), Candida vaginitis (P=.007), and HSV (P=.03) were significantly associated with CVL HIV RNA levels. CONCLUSION: Bacteria associated with BV increase genital-tract HIV RNA levels. Quantitative bacterial counts for lactobacilli and M. hominis are better correlates of CVL HIV RNA than are Nugent score or Amsel criteria. Since plasma virus and CD4 cell levels did not differ between the BV and no-BV groups, these data suggest that the bacterial flora associated with BV influence genital-tract HIV shedding.

Relationship of U1 cell HIV-stimulatory activity to bacterial vaginosis and HIV genital tract virus load.

Bacterial vaginosis (BV) has been associated with HIV sexual transmission and increased levels of genital tract HIV RNA. We postulated that BV induces the appearance of substances in the genital tract that stimulate HIV expression locally. To test this, we measured HIV RNA levels in genital mucosal fluid from women with or without BV (defined by Nugent score) and compared them with the ability of those fluids to stimulate HIV expression in the chronically HIV-infected monocytic line U1. The U1 activity was significantly higher in women with BV (median = 1320 pg/ml p24) than in women with normal flora (median = 103 pg/ml p24, p = 0.0001). However, levels of the U1 activity were not significantly associated with levels in the genital tract of HIV RNA. Levels of the U1 activity were also not associated with levels of Gardnerella vaginalis or Mycoplasma hominis in genital fluids, suggesting these bacteria were not the source of the activity. Thus, while these data show a strong association of U1 stimulatory activity with BV, no influence of the U1 activity on genital tract HIV expression was observed.

Identification of multiple antigens recognized by tumor-infiltrating lymphocytes from a single patient: tumor escape by antigen loss and loss of MHC expression.

The authors describe a patient who experienced recurrence of metastatic melanoma after an initial dramatic response to immunotherapy using peptides derived from gp100, MART-1, and tyrosinase emulsified in incomplete Freund's adjuvant, and present data to support the hypothesis that the progression of disease in this patient was due to in vivo immunoselection for immunoresistant tumor variants. The authors previously demonstrated the existence of T-cell clones in this patient's peripheral blood and tumor-infiltrating lymphocytes (TILs) reactive against multiple antigens, including gp100, the tyrosinase-related protein (TRP)-2, a novel TRP-2 isoform-TRP-2-6b, SOX10, and the melanoma antigen NY-ESO-1. In addition to the multiple HLA-A2 restricted T-cell clones, the authors have now identified additional HLA-B/C-restricted as well as class II (HLA-DP)-restricted anti-melanoma antigen T-cell clones from this patient's TIL. One recurrent tumor showed loss of expression of multiple tumor antigens but retention of HLA class I expression. The other recurrent lesion showed total loss of HLA class I expression even though the tumor cells still expressed many melanoma antigens. This paper thus provides evidence for both the effectiveness of the immune destruction of cancer as well as problems associated with antigen-loss tumor escape mechanisms.