Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

Single-chain VαVß T-cell receptors function without mispairing with endogenous TCR chains.

Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vß single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

Development of a novel strategy for engineering high-affinity proteins by yeast display.

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells.

Adoptive T cell therapy, involving the ex vivo selection and expansion of antigen-specific T cell clones, provides a means of augmenting antigen-specific immunity without the in vivo constraints that can accompany vaccine-based strategies. A phase I study was performed to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8+ T cell clones targeting the tumor-associated antigens, MART1MelanA and gp100 for the treatment of patients with metastatic melanoma. Four infusions of autologous T cell clones were administered, the first without IL-2 and subsequent infusions with low-dose IL-2 (at 0.25, 0.50, and 1.0 x 10(6) unitsm(2) twice daily for the second, third, and fourth infusions, respectively). Forty-three infusions of MART1MelanA-specific or gp100-specific CD8+ T cell clones were administered to 10 patients. No serious toxicity was observed. We demonstrate that the adoptively transferred T cell clones persist in vivo in response to low-dose IL-2, preferentially localize to tumor sites and mediate an antigen-specific immune response characterized by the elimination of antigen-positive tumor cells, regression of individual metastases, and minor, mixed or stable responses in 8 of 10 patients with refractory, metastatic disease for up to 21 mo.

A transcriptional block in the IL-2 promoter at the -150 AP-1 site in effector CD8+ T cells.

Both CD4+ and CD8+ T cells that produce IL-2 in response to Ag recognition have been isolated. However, most effector CD8+ T cells recovered after exposure to Ag do not produce sufficient IL-2 to sustain growth, and depend on CD4+ T helper cells for this obligate growth factor. IL-2 expression in CD4+ T cells is primarily controlled at the level of transcription, but mechanisms restricting IL-2 production in CD8+ T cells have not been elucidated. To evaluate transcriptional regulation of the IL-2 gene in CD8+ T cells, we stably transfected reporter genes into Ag-specific CD8+ T cell clones. CD28+ CD8(+) T cells unable to transcribe the IL-2 gene in response to antigenic stimulation had a block in transactivation of the -150 CD28 response element (CD28RE)/AP-1 site of the IL-2 promoter, but did transactivate the composite NFAT/AP-1 and OCT/AP-1 sites, and a consensus AP-1 motif. Mutation of the nonconsensus -150 AP-1 site to a consensus AP-1 site, or insertion of a CD28RE/AP-1 consensus site upstream of the native -150 CD28RE/AP-1 site restored transactivation of the altered promoter. These results suggest that the defect at the -150 site may reflect the absence or inactivity of a required factor rather than repression of the IL-2 promoter.

In vivo tracking of tumor-specific T cells.

Novel immunologic assays now enable visualization of the antigen-specific response to an extent not previously possible. Assessment of not only the numeric frequency but also the functional properties of individual tumor-specific T cells in the endogenous and manipulated immune response has provided insights that will facilitate the development of immunotherapeutic strategies.

Expression of a tolerizing tumor antigen in peripheral tissue does not preclude recovery of high-affinity CD8+ T cells or CTL immunotherapy of tumors expressing the antigen.

Transgenic (TG) mice were generated selectively expressing the gag protein of Friend murine leukemia virus (FMuLV) in the liver. FMuLV(gag) is also expressed by the FBL leukemia, and is the immunodominant tumor Ag of the CD8(+) T cell response in C57BL/6 mice. gag-TG mice expressing FMuLV(gag) in the liver were tolerant to the protein and failed to generate a CTL response to either FBL or FMuLV(gag). This tolerance reflected anergy rather than deletion, as CTL responsiveness could be recovered after four cycles of in vitro stimulation. Adoptively transferred gag-specific T cells were not anergized in gag-TG recipients, as revealed by antitumor activity in vivo. Also, such T cells did not induce detectable autoimmune injury in gag-TG liver cells. These results suggest that the requirements for a tissue Ag to provide a tolerizing stimulus are distinct from those for being the target of a T cell-mediated autoimmune response and that the requirements for induction and maintenance of peripheral tolerance are distinct for naive and primed T cells. That anergic T cells reactive with tumor-associated Ags can be recovered by repetitive in vitro stimulation and can mediate tumor therapy suggests strategies that use such Ags to generate CTL for adoptive immunotherapy should be further developed.

Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of gene-modified autologous T cells in a nonhuman primate model.

The in vivo persistence of gene-modified cells can be limited by host immune responses to transgene-encoded proteins. In this study we evaluated in a nonhuman primate model whether the administration of a nonmyeloablative regimen consisting of low-dose total-body irradiation with 200 cGy followed by immunosuppression with mycophenolate mofetil and cyclosporin A for 28 and 35 days, respectively, could be used to facilitate persistence of autologous gene-modified T cells when a transgene-specific immune response had already been established or to induce long-lasting tolerance in unprimed recipients. Two macaques (Macaca nemestrina) received infusions of T cells transduced to express either the enhanced green fluorescent protein and neomycin phosphotransferase genes or the hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes. In the absence of immunosuppression, both macaques developed potent class I major histocompatibility complex-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses that rapidly eliminated the gene-modified T cells and that persisted long term as memory CTL. Treatment with the nonmyeloablative regimen failed to abrogate preexisting memory CTL responses but interfered with the induction of transgene-specific CTL and facilitated in vivo persistence of gene-modified cells in an unprimed host. However, sustained tolerance to gene-modified T cells was not achieved with this regimen, indicating that further modifications will be required to permit sustained persistence of gene-modified T cells.

Melanocyte destruction after antigen-specific immunotherapy of melanoma: direct evidence of t cell-mediated vitiligo.

Current strategies for the immunotherapy of melanoma include augmentation of the immune response to tumor antigens represented by melanosomal proteins such as tyrosinase, gp100, and MART-1. The possibility that intentional targeting of tumor antigens representing normal proteins can result in autoimmune toxicity has been postulated but never demonstrated previously in humans. In this study, we describe a patient with metastatic melanoma who developed inflammatory lesions circumscribing pigmented areas of skin after an infusion of MART-1-specific CD8(+) T cell clones. Analysis of the infiltrating lymphocytes in skin and tumor biopsies using T cell-specific peptide-major histocompatibility complex tetramers demonstrated a localized predominance of MART-1-specific CD8(+) T cells (>28% of all CD8 T cells) that was identical to the infused clones (as confirmed by sequencing of the complementarity-determining region 3). In contrast to skin biopsies obtained from the patient before T cell infusion, postinfusion biopsies demonstrated loss of MART-1 expression, evidence of melanocyte damage, and the complete absence of melanocytes in affected regions of the skin. This study provides, for the first time, direct evidence in humans that antigen-specific immunotherapy can target not only antigen-positive tumor cells in vivo but also normal tissues expressing the shared tumor antigen.

T-cell therapy of cytomegalovirus and human immunodeficiency virus infection.

Acute and persistent virus infections are limited by the development and maintenance of host T-cell responses to viral antigens. Individuals with congenital or acquired immunodeficiencies are at risk of progressive and often life-threatening infection. Recent studies have provided insight into the nature of protective T-cell responses to viruses and advances in T-cell culture technology have made it possible to evaluate the adoptive transfer of T-cell clones of defined antigen specificity and function to restore deficient responses in immunocompromised hosts. The progress of these studies in cytomegalovirus and human immunodeficiency virus infection is the subject of this review.

A prospective multicenter evaluation of new fecal occult blood tests in patients undergoing colonoscopy.

OBJECTIVE: Guaiac-based fecal occult blood (FOB) tests, in particular, Hemoccult II (HO), are commonly used to detect colorectal neoplasia. Because the sensitivity and specificity of these tests are critical to cost-effective screening programs, we aimed to investigate the improved performance characteristics of new FOB tests for known colonic lesions. METHODS: Nine centers worldwide performed FOB testing with guaiac-based tests (Hemoccult II [HO] and Hemoccult II SENSA [SENSA]) and immunochemical tests (HemeSelect [HS] and FlexSure OBT [FS]) on 554 patients referred for colonoscopy for predetermined indications. A combination testing strategy consisting of SENSA followed by HS or FS (which was considered positive only when both tests were positive) was also evaluated. Results of FOB tests were compared to findings on colonoscopy. RESULTS: Cancers were identified in 2.9% of subjects, whereas adenomas > or =10 mm were found in 39 patients. Small adenomas, colitis, and other lesions were identified in 141 patients. The positivity rate of HO for adenomas > or =10 mm was less than for SENSA (20.5% vs 35.9%, p < 0.05), whereas the positivity rate of HO, SENSA, FS, HS, or the combination tests for cancers was not statistically different. The overall positivity rates were significantly greater for FS (15.9%, p = 0.0002) and significantly lower using the combination tests (SENSA/FS 6.0%, p = 0.01; SENSA/HS 6.2%, p = 0.02) compared to HO (9.4%). In this study population, the relative specificity (i.e., true-negative tests/true-negatives + false-positives in patients without adenomas > or =10 mm or cancers) of HO (93.9%; 95% CI, 91.7-96.1) was similar to that of SENSA (92.8%; 95% CI, 90.4-95.2) and HS (90.1%; 95% CI, 87.4-92.8), and greater than FS (88.0%; 95% CI, 85.1-90.9, p < 0.001). When considering adenomas > or =10 mm, cancers alone or cancers and adenomas combined, the combination test using SENSA/FS was associated with significantly fewer false-positive tests than any of the individual tests. CONCLUSIONS: Compared to single tests, the combination test with the highly sensitive SENSA and an immunochemical test had slightly reduced sensitivity but significantly fewer false-positive tests than any single test. These data raise the possibility that a combination test (i.e., highly sensitive guaiac plus immunochemical) could reduce the costs of screening for colon cancer, and suggest that further study of combination test strategies is warranted.

HIV-specific cytotoxic T lymphocytes traffic to lymph nodes and localize at sites of HIV replication and cell death.

We have tracked the in vivo migration and have identified in vivo correlates of cytotoxic T-lymphocyte (CTL) activity in HIV-seropositive subjects infused with autologous gene-marked CD8(+) HIV-specific CTL. The number of circulating gene-marked CTL ranged from 1.6 to 3.5% shortly after infusion to less than 0.5% 2 weeks later. Gene-marked CTL were present in the lymph node at 4.5- to 11-fold excess and colocalized within parafollicular regions of the lymph node adjacent to cells expressing HIV tat fusion transcripts, a correlate of virus replication. The CTL clones expressed the CCR5 receptor and localized among HIV-infected cells expressing the ligands MIP-1alpha and MIP-1beta, CC-chemokines produced at sites of virus replication. Aggregates of apoptotic cells and cells expressing granzyme-B localized within these same sites. In contrast, lymph node sections from untreated HIV-seropositive subjects, all with significant viral burden (> 50,000 HIV RNA copies/mL plasma), showed no CC-chemokine expression and exhibited only sporadic and randomly distributed cells expressing granzymes and/or apoptotic cells. These studies show that the infused CTL specifically migrate to sites of HIV replication and retain their antigen-specific cytolytic potential. Moreover, these studies provide a methodology that will facilitate studies of both the magnitude and functional phenotype of Ag-specific CD8(+) T cells in vivo.

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes.

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.

The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5.

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R beta-chain (IL-2Rbeta). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rbeta except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.

Lack of effect of treatment for Helicobacter pylori on symptoms of nonulcer dyspepsia.

BACKGROUND: Prior studies have yielded conflicting results on whether or not Helicobacter pylori causes nonulcer dyspepsia. PATIENTS AND METHODS: We enrolled 100 consecutive patients with nonulcer dyspepsia into a randomized, double-blind, placebo-controlled trial. Patients with peptic ulcer disease, esophagitis, hepatobiliary disease, irritable bowel disease, or predominantly reflux-related symptoms were excluded by history and upper endoscopy. Helicobacter pylori infection was determined by biopsy and histologic examination. Serum H. pylori IgG antibodies and CagA status were determined by Western blot. Enrolled patients were randomized to a 14-day regimen of omeprazole (20 mg twice daily) and clarithromycin (500 mg three times daily) or placebo. Dyspeptic symptoms were assessed by use of a visual analog scale at baseline and at 1, 3, 6, and 12 months after treatment. Follow-up upper endoscopy with biopsy was performed 4 weeks after treatment. Compliance was measured by tablet counts. RESULTS: At 1 year, the change in dyspeptic symptoms was -24.0 (95% confidence interval, -69.0 to 21.0) in the omeprazole and clarithromycin group and -24.2 in the placebo group (95% confidence interval, -70.0 to 21.6). Furthermore, patients with persistent H. pylori infection demonstrated a greater, but not significant, improvement in symptoms (-40 +/- 144 [mean +/- SD], -65 +/- 142, -45 +/- 138, and -39 +/- 163) than those with successful eradication (-26 +/- 126, -26 +/- 148, -12 +/- 126, and -25 +/- 151) at months 1, 3, 6, and 12, respectively. CONCLUSION: Patients with nonulcer dyspepsia should not routinely be treated for H. pylori, since it is not a cause of this condition in most patients.

Relationship of low-dose aspirin to GI injury and occult bleeding: a pilot study.

BACKGROUND: Low-dose aspirin is commonly used in patients with cardiovascular disease. However, little is known about the effect of aspirin on occult blood loss caused by gastrointestinal injury. Therefore, we studied endoscopic injury and fecal occult blood loss in patients ingesting different quantities of low-dose aspirin. METHODS: Forty healthy volunteers were enrolled in a randomized, double-blind, prospective, pilot endoscopic study. Each volunteer underwent 30 days of treatment with daily aspirin 30 mg, 81 mg, 325 mg, or placebo. Subjects completed fecal occult blood test cards before and at the end of treatment of two types: guaiac impregnated (Hemoccult and Hemoccult SENSA) and immunochemical (HemeSelect and FlexSure OBT). Each volunteer underwent upper endoscopy at baseline and after completing 30 days of study medication. RESULTS: Aspirin did not induce significant injury as determined by endoscopy when compared with placebo. Six of 30 volunteers taking aspirin developed erosions, whereas 1 of 10 subjects on placebo developed a new erosion (p = 0.66). Aspirin (325 mg) was associated with a higher mean symptom score than the lower aspirin dosages and the placebo group (p = 0.12). Only one subject taking aspirin (325 mg) had fecal occult blood on a single HemeSelect card. No subject had a positive fecal occult blood test with Hemoccult II, Hemoccult II SENSA, or FlexSure OBT cards. CONCLUSIONS: Aspirin in dosages commonly used for cardiovascular prophylaxis does not generally cause significant gastric or duodenal mucosal endoscopic lesions. In the absence of frank ulceration, low-dose aspirin does not result in positive fecal occult blood tests. Low-dose aspirin should not interfere with fecal occult blood testing and probably should not be stopped during stool collection.