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CD8 T cells play a critical role in immunity against intracellular pathogens and cancer. A primary objective of T cell-based vaccine strategies is the induction of durable and effective immune responses. Achieving this goal involves more than simply boosting the numbers of responding T cells. Of particular interest is the induction of CD8 T cells with polycytokine capability, specifically with the ability of CD8 T cells to co-produce IFNγ, TNFα and IL-2. The presence of these polycytokine-producing CD8 T cells correlates strongly with protection against foreign pathogens and cancer. Therefore, approaches capable of inducing such polyfunctional responses are needed. NKG2D engagement on CD8 T cells has been shown to result in increased effector response. However, the manner in which NKG2D engagement results in improved CD8 T cell effector response is unclear. Here we demonstrate in vitro and in vivo that NKG2D engagement by its natural ligand, Rae-1ε, shifts the balance from single cytokine to polycytokine (IL-2, IFNγ, and TFNα) production. These data define a previously unrecognized process in which NKG2D costimulation on CD8 T cells results in improved effector responses.

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells., Graphical Abstract, Nishimura and colleagues demonstrate that transgene expression in gene-modified anti-tumor T cells is reversibly downregulated over time in vivo and in vitro. Treatment with IL-15 or with HDAC inhibitors (sodium butyrate or vorinostat) reduced this downregulation and maintained T cell functional responses to tumor cells.

Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.

Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.

Immunotherapy is a promising method of treatment for a number of cancers. Many of the curative results have been seen specifically in advanced-stage melanoma. Despite this, single-agent therapies are only successful in a small percentage of patients, and relapse is very common. As chemotherapy is becoming a thing of the past for treatment of melanoma, the combination of cellular therapies with immunotherapies appears to be on the rise in in-vivo models and in clinical trials. These forms of therapies include tumor-infiltrating lymphocytes, T-cell receptor, or chimeric antigen receptor-modified T cells, cytokines [interleukin (IL-2), IL-15, IL-12, granulocyte-macrophage colony stimulating factor, tumor necrosis factor-α, interferon-α, interferon-γ], antibodies (αPD-1, αPD-L1, αTIM-3, αOX40, αCTLA-4, αLAG-3), dendritic cell-based vaccines, and chemokines (CXCR2). There are a substantial number of ongoing clinical trials using two or more of these combination therapies. Preliminary results indicate that these combination therapies are a promising area to focus on for cancer treatments, especially melanoma. The main challenges with the combination of cellular and immunotherapies are adverse events due to toxicities and autoimmunity. Identifying mechanisms for reducing or eliminating these adverse events remains a critical area of research. Many important questions still need to be elucidated in regard to combination cellular therapies and immunotherapies, but with the number of ongoing clinical trials, the future of curative melanoma therapies is promising.

Numerous neoepitope-based vaccination strategies are in testing for clinical use in the treatment of cancer. Rapid identification of immunostimulatory neoantigen targets hastens neoantigen vaccine development. Papers recently published in Nature Biotechnology describe two independent machine-learning-based algorithms that demonstrate improved identification of MHC class II-binding peptides. Herein, we outline the benefits of these algorithms and their implications for future immunotherapies.

Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.

Gata5 is a transcription factor expressed in the lung, but its physiological role is unknown. To test whether and how Gata5 regulates airway constrictor responsiveness, we studied Gata5(-/-), Gata5(+/-), and wild-type mice on the C57BL/6J background. Cholinergic airway constrictor responsiveness was assessed invasively in mice without and with induction of allergic airway inflammation through ovalbumin sensitization and aerosol exposure. Gata5-deficient mice displayed native airway constrictor hyperresponsiveness (AHR) in the absence of allergen-induced inflammation. Gata5-deficient mice retained their relatively greater constrictor responsiveness even in ovalbumin-induced experimental asthma. Gata5 deficiency did not alter the distribution of cell types in bronchoalveolar lavage fluid, but bronchial epithelial mucus metaplasia was more prominent in Gata5(-/-) mice after allergen challenge. Gene expression profiles revealed that apolipoprotein E (apoE) was the fifth most down-regulated transcript in Gata5-deficient lungs, and quantitative RT-PCR and immunostaining confirmed reduced apoE expression in Gata5(-/-) mice. Quantitative RT-PCR also revealed increased IL-13 mRNA in the lungs of Gata5-deficient mice. These findings for the first time show that Gata5 regulates apoE and IL-13 expression in vivo and that its deletion causes AHR. Gata5-deficient mice exhibit an airway phenotype that closely resembles that previously reported for apoE(-/-) mice: both exhibit cholinergic AHR in native and experimental asthma states, and there is excessive goblet cell metaplasia after allergen sensitization and challenge. The Gata5-deficient phenotype also shares features that were previously reported for IL-13-treated mice. Together, these results indicate that Gata5 deficiency induces AHR, at least in part, by blunting apoE and increasing IL-13 expression.

T cell migration toward sites of antigen exposure is mediated by G protein signaling and is a key function in the development of immune responses. Regulators of G protein signaling (RGS) proteins modulate G protein signaling; however, their role in the regulation of adaptive immune responses has not been thoroughly explored. Herein we demonstrated abundant expression of the Gi/Gq-specific RGS3 in activated T cells, and that diminished RGS3 expression in a T cell thymoma increased cytokine-induced migration. To examine the role of endogenous RGS3 in vivo, mice deficient in the RGS domain (RGS3ΔRGS) were generated and tested in an experimental model of asthma. Compared with littermate controls, the inflammation in the RGS3ΔRGSmice was characterized by increased T cell numbers and the striking development of perivascular lymphoid structures. Surprisingly, while innate inflammatory cells were also increased in the lungs of RGS3ΔRGSmice, eosinophil numbers and Th2 cytokine production were equivalent to control mice. In contrast, T cell numbers in the draining lymph nodes (dLN) were reduced in the RGS3ΔRGS, demonstrating a redistribution of T cells from the dLN to the lungs via increased RGS3ΔRGST cell migration. Together these novel findings show a nonredundant role for endogenous RGS3 in controlling T cell migration in vitro and in an in vivo model of inflammation.

CD8+ T cell responses have been shown to be regulated by dendritic cells (DCs) and CD4+ T cells, leading to the tenet that CD8+ T cells play a passive role in their own differentiation. In contrast, by using a DNA vaccination model, to separate the events of vaccination from those of CD8+ T cell priming, we demonstrate that CD8+ T cells, themselves, actively limit their own memory potential through CD8+ T cell-derived IFN-γ–dependent modification of the IL-12/IL-15Rα axis on DCs. Such CD8+ T cell-driven cytokine alterations result in increased T-bet and decreased Bcl-2 expression, and thus decreased memory progenitor formation. These results identify an unrecognized role for CD8+ T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation and memory formation.

CD4-unhelped CD8(+) T cells are functionally defective T cells primed in the absence of CD4(+) T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8(+) T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8(+) T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8(+) T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4(+) T cells in a CD4-dependent influenza model and rescues HIV-specific CD8(+) T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8(+) T cells from their pathophysiological fate and may provide therapeutic benefits.

While the effects of TCR affinity and TGFβ on CD8(+) T-cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFβ-mediated suppression remains unknown. To address this issue, we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8(+) T cells primed with lower-affinity TCR ligands are more effectively regulated by TGFβ. Susceptibility to TGFβ-mediated suppression is associated with downregulation of RGS3, a recently recognized negative regulator of TGFβ signaling, but not expression of TGFβ receptors I/II. These results suggest a novel tolerance mechanism whereby CD8(+) T cells are discriminately regulated by TGFβ according to the affinity of the ligand on which they were initially primed. In addition, because of the major role played by TGFβ in tumor-induced immune suppression, these results identify the affinity of the priming ligand as a primary concern in CD8(+) T-cell-mediated cancer immunotherapeutic strategies.

CD43 interaction with ERM proteins regulates CD43 phosphorylation and T-cell migration. CD43 phosphorylation can also drive CD43 localization in T-cells independently of ERM association., Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase Cθ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation–dependent mechanism.

Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28(-/-) mice, Th2-mediated airway inflammation was not induced in CD28(-/-) mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS(-/-) mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.

The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS−/− mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.

The CD8 coreceptor on T cells has two functions. Namely, CD8 acts to stabilize the binding of the T-cell receptor (TCR) to the peptide-MHC complex while localizing p56lck (lck) to the TCR/CD3 complex to facilitate early signaling events. Although both functions may be critical for efficient activation of a CTL, little is known about how the structural versus signaling roles of CD8, together with the relative strength of the TCR, influences T-cell function. We have addressed these issues by introducing full-length and truncated versions of the CD8α and CD8β chains into CD8− Jurkat cell clones expressing cloned TCRs with known antigen specificity and relative affinities. Using a combination of antigen recognition and tetramer-binding assays, we find that the intracellular lck-binding domain of CD8 is critical for enhanced T-cell activation regardless of the relative strength of the TCR. In contrast, the extracellular domain of CD8 seems to be critical for TCRs with lower affinity but not those with higher affinity. Based on our results, we conclude that there are different requirements for CD8 to enhance T-cell function depending on the strength of its TCR. (Cancer Res 2006; 66(23): 11455-61)

In an open clinical trial for Stage IV melanoma patients, we are testing a promising new therapy in which the patient’s own T cells are genetically engineered with a T cell receptor (TIL 1383I TCR) targeting the melanoma antigen tyrosinase. Included in the TIL 1383I TCR vector is a nonfunctional CD34 marker that can be readily tracked by flow cytometry. These transgenic T cells are infused into the patient with the goals of killing tumor cells and promoting anti-tumor inflammatory responses. We find that the transgenic T cells persist at low levels for at least twenty months in vivo. Many of the transgenic T cells are activated in the patient, indicating that they are responding to antigen. A subset of activated cells express inhibitory receptors, including PD-1 and CTLA-4. Upon further analysis of patient samples, we found that the percent of transgene-expressing T cells can be increased up to three-fold with either cytokine stimulation and/or CD3/CD28-bead stimulation. When cultured in vitro, transduced T cells lose transgene expression over time, and culturing in the absence of cytokines dramatically increases the rate of transgene expression loss. As seen ex vivo, restimulation of in vitro cultured T cells restores transgene expression. Cells undergoing division express substantially more transgene than resting cells. These data suggest that mitogenic factors causing cell division may promote transgene expression. We next plan to test mitogenic (high dose IL-2, IL-15) and anti-inhibition (PD-1 and CTLA-4 blockade) therapies with T cell transfer utilizing murine mouse models and potentially amend our protocol in the future to reduce the effects of exhaustion and transgene loss on transgenic anti-tumor T cells.

In this study, we demonstrate that engagement of two different natural killer receptors (NKRs) can lead to contrasting effects in the development of self-reactive CD8+T cells and autoimmune vitiligo. Specifically, using a mouse model, we show that CD8+T-cell targeting of a melanocyte antigen, tyrosinase-related protein-1 (TRP-1) in combination with delivery of the NKG2D ligands (Rae-1ϵ or H60), results in strong CD8+T-cell responses against TRP-1 and in the development of autoimmune vitiligo. In contrast, targeting of TRP-1 in combination with delivery of CD48, the natural ligand for the NKR 2B4, leads to reduced formation of TRP-1-reactive CD8+T-cell responses and decreased development of vitiligo. These data indicate that autoimmune vitiligo is limited by insufficient signals, despite plentiful self-reactive T cells in the peripheral immune system. To our knowledge, this is the first experimental evidence supporting the role of NKRs in modulating CD8+T-cell autoimmune vitiligo. This study supports the utilization of NKR signaling as a therapeutic avenue toward prevention of vitiligo and other autoimmune diseases.

We and others reported that inducible costimulator-deficient (ICOS(-/-)) mice manifest a defect in Th2-mediated airway inflammation, which was attributed to reduced Th2 differentiation in the absence of ICOS signaling. Interestingly, the number of CD4 T cells present in the airways and lungs after sensitization and challenge is significantly reduced in ICOS(-/-) mice. We now show that this reduction is not attributable simply to a reduced proliferation of ICOS(-/-) cells, because significantly more ICOS(-/-) than wild-type activated CD4 T cells are present in the lymph nodes, suggesting that more ICOS(-/-) CD4 T cells than wild-type CD4 T cells migrated into the lymph nodes. Further investigation revealed that activated ICOS(-/-) CD4 T cells express higher concentrations of the lymph node homing receptors, CCR7 and CD62L, than do wild-type CD4 T cells, leading to a preferential return of ICOS(-/-) cells to the nondraining lymph nodes rather than the lungs. Blocking reentry into the lymph nodes after the initiation of Th2-mediated airway inflammation equalized the levels of CD4 and granulocyte infiltration in the lungs of wild-type and ICOS(-/-) mice. Our results demonstrate that in wild-type CD4 T cells, co-stimulation with ICOS promotes the down-regulation of CCR7 and CD62L after activation, leading to a reduced return of activated CD4 T cells to the lymph nodes and a more efficient entry into the lungs.

Our previous studies revealed that, in a murine model of asthma, mice that received Fas-deficient T cells developed a prolonged phase of airway inflammation, mucus production, and airway hyperreactivity that failed to resolve even 6 weeks after the last challenge. To investigate how Fas-Fas ligand (FasL) interaction occurs between T cells and other cells in vivo, Gld mice with abnormalities of the FasL signaling pathway were used. The reconstituted mice were made by transferring T cells from B6 or Gld mice to Rag(-/-) or FasL-deficient Rag(-/-) mice. We found that Rag(-/-) mice that received B6 T cells resolved the airway inflammation, whereas FasL-deficient Rag(-/-) mice that received Gld T cells developed a prolonged airway inflammation at Day 28, with decreased IFN-gamma production. Both FasL-deficient Rag(-/-) mice that received B6 T cells and Rag(-/-) mice that received Gld T cells also had completely resolved their airway inflammation by Day 28 after challenge. Interestingly, FasL-deficient Rag(-/-) mice that received Gld T cells eventually resolved airway inflammation at Day 42, with a similar level of IFN-gamma production to that of control group. These results demonstrate that FasL expression on either T cells only or non-T cells only was sufficient for the eventual resolution of airway inflammation, and the prolonged airway inflammation in FasL-deficient Rag(-/-) mice that received Gld T cells was correlated with decreased IFN-gamma production by Gld T cells.

Background Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5′ promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology. Methodology/Principal Findings We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/− mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/− mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/− mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/− is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4. Conclusion These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.

Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones, was CD4- CD8- indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8- Jurkat cells, the resulting Jurkat cells recognized gp100:209-217 peptide loaded T2 cells and had high functional avidity, but could not recognize HLA-A2+ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously loading the tumor cells with the native gp100:209-217 peptide. These results indicate that functional avidity of a T cell does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate their functional avidity independent of the affinity of their TCRs.

The immune attack against malignant tumors require the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR) alphabeta+ CD4- CD8- double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-gamma and TNF. Although lacking the CD8 molecule the gp100-specific DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.

Despite the wealth of information that has been acquired regarding the way T cells recognize their targets, we are left with far more questions than answers regarding how to manipulate the immune response to better treat cancer patients. Clearly, most patients have a broad repertoire of T cells capable of recognizing their tumor cells. Despite the presence of these tumor reactive T cells and our ability to increase their frequency though vaccination or adoptive transfer, patients still progress. From the T cell side, defects in T cell signaling may account for much of our failure to achieve significant numbers of objective clinical responses. In spite of these negatives, the horizon does remain bright for T cell based immune therapy of cancer. The periodic objective clinical response tells us that immune therapy can work. Now that we know that cancer patients have the capacity to mount immune responses against their tumors, current and future investigations with agents which alter T cell function combined with vaccination or adoptive T cell transfer may help tip the balance towards effective immune therapies.

Hepatitis C virus (HCV) has been reported to elicit B and T cell immunity in infected patients. Despite the presence of antiviral immunity, many patients develop chronic infections leading to cirrhosis, hepatocellular carcinoma, and liver failure that can require transplantation. We have previously described the presence of HLA-A2–restricted, HCV NS3–reactive cytotoxic T lymphocytes (CTL) in the blood of HLA-A2− liver transplantation patients that received an HLA-A2+ liver allograft. These T cells are analogous to the “allospecific” T cells that have been described in hematopoietic stem cell transplantation patients. It has been speculated that allospecific T cells express high-affinity T cell receptors (TCRs). To determine if our HCV-reactive T cells expressed TCRs with relatively high affinity for antigen, we identified and cloned a TCR from an allospecific HLA-A2–restricted, HCV:NS3:1406-1415–reactive CD8+ T cell clone and expressed this HCV TCR in Jurkat cells. Tetramer binding to HCV TCR–transduced Jurkat cells required CD8 expression, whereas antigen recognition did not. In conclusion, based on the reactivity of the TCR-transduced Jurkat cells, we have identified a TCR that transfers anti-HCV reactivity to alternate effectors. These data suggest this high affinity HCV-specific TCR might have potential new immunotherapic implications. (HEPATOLOGY 2006;43:973–981.)

Despite the wealth of information that has been acquired regarding the way T cells recognize their targets, we are left with far more questions than answers regarding how to manipulate the immune response to better treat cancer patients. Clearly, most patients have a broad repertoire of T cells capable of recognizing their tumor cells. Despite the presence of these tumor reactive T cells and our ability to increase their frequency though vaccination or adoptive transfer, patients still progress. From the T cell side, defects in T cell signaling may account for much of our failure to achieve significant numbers of objective clinical responses. In spite of these negatives, the horizon does remain bright for T cell based immune therapy of cancer. The periodic objective clinical response tells us that immune therapy can work. Now that we know that cancer patients have the capacity to mount immune responses against their tumors, current and future investigations with agents which alter T cell function combined with vaccination or adoptive T cell transfer may help tip the balance towards effective immune therapies.

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+) T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+) T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/-) mice, we found that CD4(+) T cells and ICOS(+/+) T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+)Foxp3 (+) ICOS(+) T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+) Tregs promoted survival in animals that received ICOS(-/-) T cells. Direct comparison of ICOS(-/-) Tregs to ICOS(+/+) Tregs found defects in vitro but no differences in the ability of ICOS(-/-) Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

Inducible costimulator (ICOS) belongs to the CD28 family of costimulatory receptors. Although ICOS has been found to augment CD4 T cell differentiation and cytokine production, its effects on proliferation and survival are controversial. To dissect the effects of ICOS on T cell survival and proliferation, ICOS-/- or wild-type TCR-transgenic T cells were adoptively transferred and followed after immunization. ICOS-/- cells divided significantly less than wild-type CD4 T cells by CFSE dilution. Unexpectedly, when the total number of cells was examined, more ICOS-/- cells were found in the lymph nodes and fewer in the spleen. We then shifted our focus to the effects of ICOS on CCR7 and CD62L, receptors known to retain cells in the lymph nodes. Interestingly, ICOS-/- TCR-transgenic cells had a defect in down-regulation of CD62L and CCR7 compared to wild-type cells. In a model of Th2-mediated airway inflammation, ICOS-/- mice had significantly fewer CD4 T cells in their airways than wild-type mice, and the CD4 T cells in the draining lymph node expressed higher levels of CCR7. Finally, ICOS-/- cells migrated more to the draining lymph nodes than the spleen compared to wild-type cells in inflamed mice. Together, these data suggest that ICOS-/- CD4 T cells have defective down-regulation of CCR7 and CD62L, leading to a defect in cell egress from lymph nodes and migration to sites of inflammation.

The immune attack against malignant tumors requires the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR)αβ+ CD4− CD8− double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.