AbstractIn this global phase 2 study in patients with relapsed/refractory follicular lymphoma (FL), zandelisib was administered on intermittent dosing to mitigate immune‐related adverse events and infections that have been reported with oral PI3Kδ inhibitors administered daily continuously. Eligible patients with measurable disease and progression after at least two prior therapies were administered zandelisib until disease progression or intolerability. The primary efficacy endpoint was objective response rate (ORR) and the key secondary efficacy endpoint was duration of response (DOR). We report on 121 patients with FL administered zandelisib on intermittent dosing after 8 weeks of daily dosing for tumor debulking. The median number of prior therapies was 3 (range, 2–8) and 45% of patients had refractory disease. The ORR was 73% (95% confidence interval [CI], 63.9–80.4), the complete response (CR) rate was 38% (95% CI, 29.3–47.3), and the median DOR was 16.4 months (95% CI, 9.5–not reached). With a median follow‐up of 14.3 months (range, 1–30.5), the median progression‐free survival was 11.6 months (95% CI, 8.3–not reached). Twenty‐one patients (17%) discontinued therapy due to an adverse event. Grade 3–4 class‐related toxicities included 6% diarrhea, 5% lung infections, 3% colitis (confirmed by biopsy or imaging), 3% rash, 2% AST elevation, and 1% non‐infectious pneumonitis. Zandelisib achieved a high rate of durable responses in heavily pretreated patients with relapsed/refractory FL. The intermittent dosing resulted in a relatively low incidence of severe class‐related toxicities, which supports the evaluation of zandelisib as a single agent and in combination with indolent B‐cell malignancies.

PURPOSE To evaluate patient-specific immunotherapy with mitumprotimut-T (idiotype keyhole limpet hemocyanin [Id-KLH]) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in CD20(+) follicular lymphoma. PATIENTS AND METHODS Patients with treatment-naive or relapsed/refractory disease achieving a complete response (CR), partial response (PR), or stable disease (SD) with four weekly rituximab infusions were randomly assigned to mitumprotimut-T/GM-CSF or placebo/GM-CSF, with doses given monthly for six doses, every 2 months for six doses, and then every 3 months until disease progression (PD). Randomization was stratified by prior therapy (treatment-naive or relapsed/refractory) and response to rituximab (CR/PR or SD). The primary end point was time to progression (TTP) from randomization. RESULTS A total of 349 patients were randomly assigned; median age was 54 years, 79% were treatment naive, and 86% had stage III/IV disease. Median TTP was 9.0 months for mitumprotimut-T/GM-CSF and 12.6 months for placebo/GM-CSF (hazard ratio [HR] = 1.384; P = .019). TTP was comparable between the two arms in treatment-naive patients (HR = 1.196; P = .258) and shorter with mitumprotimut-T/GM-CSF in relapsed/refractory disease (HR = 2.265; P = .004). After adjusting for Follicular Lymphoma International Prognostic Index (FLIPI) scores, the difference in TTP between the two arms was no longer significant. Overall objective response rate, rate of response improvement, and duration of response were comparable between the two arms. Toxicity was similar in the two arms; 76% of adverse events were mild or moderate, and 94% of patients had injection site reactions. CONCLUSION TTP was shorter with mitumprotimut-T/GM-CSF compared with placebo/GM-CSF. This difference was possibly due to the imbalance in FLIPI scores.

Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources.

Background : Early studies demonstrated that patients with indolent B-cell lymphomas have the capacity to mount anti-idiotype (Id) B-cell immune responses following active immunization with patient-specific Id proteins. Durable clinical remissions were observed in patients undergoing vaccination in chemotherapy-induced first remissions. This study evaluated the efficacy and safety of active immunotherapy with mitumprotimut-T (Id- KLH, SPECIFID™, Favrille, San Diego, CA) and GM-CSF (sargramostim, LEUKINE®) in patients with CD20+ follicular lymphoma. Mitumprotimut-T is a patient-specific therapeutic vaccine composed of the Id protein produced by proprietary recombinant technology from the malignant lymphoma biopsy specimen and conjugated to KLH, a potent immunogenic protein. Patients and Methods : Patients with treatment-naive (T-N) or relapsed/refractory (R/R) WHO Grade 1–3 CD20+ follicular lymphoma received rituximab infusions at 375 mg/m2 weekly for 4 weeks, and those achieving a complete response (CR), partial response (PR), or stable disease (SD) at Week 11 were randomized to mitumprotimut-T (1 mg subcutaneously on Day 1) and GM-CSF (250 mcg subcutaneously daily on Days 1–4) or placebo and GM-CSF. Randomization was stratified by prior therapy (T-N vs. R/R) and response to rituximab (CR/PR vs. SD). Patients were immunized monthly × 6, every other month × 6, and then every 3 months until disease progression (PD). The primary efficacy endpoint was time-to-progression (TTP), which was assessed by an independent central radiology review. Results : 349 patients were randomized; their median age was 54 years (range, 21–86 years), 79% were T-N, 85% had an ECOG performance status of 0, and 86% had Stage III-IV disease. Thirty-four randomized patients (10%) did not receive blinded study drug: 28 because mitumprotimut-T could not be produced, 5 due to PD prior to start of blinded study drug, and 1 who withdrew for personal reasons. The mean number of courses was 10.6 (range, 1–21), and was comparable in the 2 groups. After a median follow-up of 40 months, 215 patients (62%) had progressed, 113 who were randomized to mitumprotimut-T and 102 who had received placebo. Median TTP from randomization was 9.0 months for patients randomized to mitumprotimut-T/GM-CSF and 12.6 months for placebo/GM-CSF (hazard ratio = 1.384, p = 0.019). Significantly more patients with high-risk FLIPI and fewer patients with low-risk FLIPI were unexpectedly randomized to mitumprotimut-T (p = 0.0042). After adjusting for FLIPI risk group in a Cox regression model, there was no significant difference in TTP between the two arms in the intent-to-treat population (p = 0.128), in patients with high risk FLIPI (p = 0.891), in patients with intermediate/low risk FLIPI (p = 0.143), in the 315 patients treated with blinded study drug, or in any of the patient subsets based on stratification factors. Comparisons of TTP between the two treatment arms using the investigators assessment of response were consistent with those obtained from central radiology review. There were no significant differences between the two treatment arms in objective responses (CR+PR) to rituximab at Week 11 (57.6%, combined data from both arms) or in objective responses any time on study (64.7%, combined data). Treatment was usually well tolerated, with 76% of adverse events graded as mild or moderate. The most common side effect was local injection site reaction, reported in 94% of patients. Conclusion : This Phase 3 trial showed no improvement in TTP with mitumprotimut-T and GM-CSF following rituximab in CD20+ follicular lymphoma.

Rituximab (Rtx) has shown significant therapeutic activity in follicular lymphoma (FL) patients, yet it's exact mechanism of action has not been fully defined. Although killing of FL cells through complement dependent cytolysis, antibody dependent cellular cytotoxicity or direct induction of apoptosis may contribute to its effectiveness, these mechanisms are unlikely to be the only ones as;

Background: Patient specific active idiotype immunotherapy with immunoglobulin idiotype is a promising new therapy for follicular NHL. Response to therapy may include both humoral and cellular anti-idiotypic immunity, but it is not clear which is most important. Prior studies have suggested that immunoglobulin FCgammaRIIIa (FCgRIIIa) polymorphisms at position 158 valine (V) or phenylalanine (F) effect the response to treatment with rituximab as well as outcomes from idiotype immunotherapy following objective response to chemotherapy. Here we present data assessing the correlation of FCgRIIIa polymorphisms and outcomes from idiotype immunotherapy following treatment with rituximab.

Treatment of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH) has shown promising results in phase 2 clinical trials. However, vaccines fail to elicit anti-Id immune responses in some patients, thus prompting the search for ways to improve the immunogenicity of Id-KLH vaccines. Current Id vaccine trials utilize tumor-specific Id proteins secreted by tumor-myeloma hybridomas, or recombinant Id proteins produced in mammalian lymphoid cells, bacteria, or insect cells. We now provide evidence that terminal mannose carbohydrate structures, characteristic of recombinant proteins produced in insect cells, lead to Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian sources. Monocyte-derived human dendritic cells (DCs) were incubated with fluorescently labeled Id proteins produced in insect or mammalian cell cultures. Insect cell-derived Id demonstrated substantially higher binding to DCs compared to the Id from a mammalian source by flow cytometry, and only the insect cell Id showed reduced binding when the DCs were preincubated with mannose receptor inhibitors. These results demonstrated that the insect cell-derived Id resulted in better targeting to DCs compared to the mammalian Id. When insect cell-derived Id proteins were coupled to KLH using glutaraldehyde and co-cultured with immature human DCs, increased expression of CD80 and CCR7 was observed by flow cytometry, indicating DC maturation. Upregulation of these markers was blocked by pre-treatment with anti-mannose receptor antibody. In tumor therapy studies, mice with 4-day established A20 murine B cell lymphoma were treated with 3 weekly injections of Id-KLH plus GM-CSF and followed for survival. A20 bearing mice treated with Id-KLH containing insect cell-derived A20 Id displayed improved survival compared with mice treated with hybridoma-derived A20 Id-KLH (61% vs. 46%, respectively). Anti-Id antibodies against A20 murine B cell lymphoma were assessed by ELISA following Id-KLH immunization. Both insect and mammalian sources of Id generated similar levels of anti-Id antibodies, showing no impairment in antibody responses due to the differences in glycoslylation. Anti-A20 cytotoxic T lymphocyte (CTL) activity was measured in splenic T cells from Id-KLH-immunized mice. Here, induction of CD8+ CTLs by insect cell-derived A20 Id-KLH was significantly greater than CTL induction following immunization with hybridoma-derived A20 Id-KLH (P=0.0061). To determine the importance of the T cell response in A20 tumor killing in vivo, mice were depleted of CD4+ and CD8+ T cell subsets, challenged with A20 tumor, and then vaccinated 4 days later as in the experiment above. Mice depleted of CD8+ T cells all succumbed to tumor demonstrating a critical role for CD8+ T cells in A20 tumor eradication. In conclusion, our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines suggest that post-translational modifications, namely terminal mannose residues, can significantly influence the immunological properties and eventual therapeutic efficacy of the product.

PURPOSE:To evaluate idiotype (Id) vaccination as a single agent in previously treated patients with indolent non-Hodgkin's lymphoma.PATIENTS AND METHODS:Patients underwent biopsy for determination of their lymphoma-specific Id sequence. Recombinant Id protein was manufactured and covalently linked with keyhole limpet hemocyanin (KLH) to generate Id/KLH. Patients received Id/KLH 1 mg on day 1 subcutaneously, with granulocyte-macrophage colony-stimulating factor 250 mug on days 1 to 4, monthly for 6 months. Booster injections were administered until progression. Both clinical and immune responses were evaluated.RESULTS:Thirty-two previously treated patients received at least one injection of Id/KLH, and 31 were assessed for efficacy. Responses were observed in four patients (one complete response and three partial responses). Median time to onset of response was 5.9 months (range, 2.3 to 14.1 months). Median duration of response has not been reached but should be at least 19.4 months (range, 10.4 to 27.2+ months). Median time to progression is 13.5 months. The most common adverse events were mild to moderate injection site reactions. Six (67%) of nine patients tested demonstrated a cellular immune response, and four (20%) of 20 patients demonstrated an antibody response against their Id.CONCLUSION:This trial demonstrates that Id/KLH alone can induce tumor regression and durable objective responses. Further study of Id/KLH is recommended in other settings where efficacy may be further enhanced as in first-line therapy or after cytoreductive therapy.

Background: Antibody diversity is generated by recombination of individual immunoglobulin (Ig) gene segments and subsequent somatic diversification driven by antigen recognition. In the repertoire of expressed B cell receptors (BCR) among normal peripheral B cells, variable heavy (V H ) gene segments are not equally represented. The ratio of kappa to lambda light chain usage is also skewed; the normal κ/λ is 1.5. Investigation of the BCR repertoire may provide clues to the genesis of B cell malignancies, as suggested in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). BCR V gene identification during the production of recombinant antibodies used in our ongoing PhII and PhIII FavId® (idiotype/KLH) immunotherapy studies has enabled us to analyze V gene usage from 475 B cell follicular lymphoma (FL) tissue samples. This study reports the results of V H gene and κ/λ gene expression in this FL sample collection. Methods: Ig heavy chain (HC) and light chain (LC) isotypes from B cell FL samples were identified by flow cytometry. V H and V L regions were sequenced from gene specific cDNA libraries prepared from these samples. V H gene usage and κ/λ ratios were compared to frequencies determined for normal peripheral B cells isolated from six healthy volunteers as well as published reports for normal peripheral B cells and other B cell malignancies. Results: Compared to V H gene family usage determined for normal B cells, VH3 usage is higher (68% vs. 42%), VH1 usage is lower (7.8% vs. 22%) and VH4 usage is equivalent (22% vs. 26%) in our cohort of FL patients while VH2, 5, 6 and 7 are infrequently used in both populations. Usage of the VH3 genes within FL derived sequences also depends upon isotype, in that this gene family is preferentially associated with the IgM HC isotype relative to IgG (76% and 57% respectively). Additionally, the combined usage of the specific genes VH3-23 and VH3-48 in our patient collection accounts for over 29% of all VH genes - compared to 9% among normal B cells. These V H gene usages also differ from reports of V H gene expression among CLL and MCL patients. With respect to LC usage, VH3 isolates are associated with a normal κ/λ ratio of 1.6 while VH4 gene isolates are preferentially associated with λ light chains with a κ/λ ratio of 0.9. Finally, FL B cells expressing the IgM HC isotype preferentially co-express κ light chains (κ/λ ratio of 2.4) while IgG expressing cells preferentially utilize λ chains (κ/λ ratio of 0.6). Conclusions: Non-random V gene and LC expression among patients with FL is noted. These distortions in Ig gene expression suggest that lymphomagenesis in FL may be associated with B cell stimulation by common antigens. A program to investigate the epitopes recognized by FL derived BCRs via binding of recombinant FL derived antibodies to protein arrays containing common auto-antigens is currently underway.

This phase 1 clinical trial was conducted to evaluate the safety and to determine the maximum tolerated dose (MTD) of the immunocytokine EMD 273066 huKS-IL2 and, secondarily, to assess its pharmacokinetics, immunogenic potential, and immunologic activity in patients with androgen-independent prostate cancer (n = 22). EMD 273066 was administered in 3-day cycles (separated by 4 weeks) of once-daily, 4-hour intravenous infusions at a dose determined by an escalation protocol (0.4, 0.7, 1.4, 2.8, 4.3, 6.4, or 8.5 mg/m/d). Approximately 2/3 of patients received a second cycle of treatment. The results show that the MTD of EMD 273066 [ie, one dose level below that producing dose-limiting toxicity (DLT) in at least 33% of patients in a dosing group] was 6.4 mg/m/d. EMD 273066 was generally well tolerated up to a dose of 4.3 mg/m/d. No DLTs, defined as drug-related toxicities >OR= Grade 3 occurring during the first treatment cycle, were observed among patients in the 0.4-, 0.7-, 1.4-, or 4.3-mg/m/d dosing groups. Four patients treated with 2.8, 6.4, or 8.5 mg/m/d EMD 273066 experienced DLTs. Titers of both antiimmunocytokine and anti-FcIL-2 antibody responses were observed after the first dose cycle and either decreased or remained stable during a second course of treatment. No hypersensitivity reactions were observed. EMD 273066 exhibited immunologic activity as demonstrated by increases in lymphocyte counts, natural killer cell number and specific activity, and antibody-dependent cellular cytotoxicity activity. On average, Cmax, which was dose-dependent, was achieved within 1 hour after infusion. Mean t(1/2) which was independent of dose, ranged from 4.0 to 6.7 hours across doses. A zero-compartment body model with one-order kinetics best described the concentration-time profiles. These data demonstrate that the novel immunocytokine EMD 273066 is well tolerated at doses above a level of observed systemic biologic activity in patients with androgen-independent prostate cancer.

Here, we demonstrate that the Wistar Kyoto (WKY/NHsd) rat, which bears the same RT1(1) haplotype as the experimental autoimmune encephalomyelitis (EAE)-susceptible Lewis rat strain, is highly resistant to myelin basic protein (MBP)-induced EAE. No differences between Lewis and WKY strains were found in T cell proliferative specificity or the use of Vbeta8.2 T cell receptors in response to MBP. A Th2 cytokine bias correlated with WKY's EAE resistance. MBP challenge of WKY-into-Lewis adoptive transfer recipients produced a novel biepisodic EAE. The WKY strain should be useful in studies of many tissue-specific autoimmune diseases to which the Lewis rat is susceptible. (C) 2002 Elsevier Science B.V. All rights reserved.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. It is reported that preconditioning sublethally irradiated immunodeficient NOD/SCID (nonobese diabetic/X-linked severe combined immunodeficient) mice with human cord blood mononuclear cells facilitates the engraftment, expansion, and dissemination in these mice of primary T-ALL cells obtained from patients at the time of diagnosis. Cells recovered from mouse bone marrow or spleen resembled the original leukemia cells from patients with respect to surface lineage markers and T-cell receptor Vbeta gene rearrangements. Moreover, the pattern of leukemia dissemination in mouse tissues, resulting in universally fatal leukemia, is reminiscent of the human clinical disease. In addition, the fidelity of the model to the human disease is documented with regard to the presence of morphologically identifiable human leukemia cells in mouse bone marrow and blood and the maintenance of leukemia-initiating capacity within the leukemia-engrafted mouse. Therefore, several lines of independent approaches are used to suggest that the engrafted cells are of human leukemia origin and are not derived from cord blood. The in vivo model described here should enable the study of the growth properties of primary T-ALL cells obtained from patients and should prove useful in evaluating the potential efficacy of therapeutic strategies directed toward T-ALL.

Inflammatory Th1 cells reacting to tissue/myelin derived antigens likely contribute to the pathogenesis of diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis. One regulatory mechanism that may be useful for treating autoimmune diseases involves an innate second set of Th2 cells specific for portions of the T cell receptor of clonally expanded pathogenic Th1 cells. These Th2 cells are programmed to respond to internally modified V region peptides from the T cell receptor (TCR) that are expressed on the Th1 cell surface in association with major histocompatibility molecules. Once the regulatory Th2 cells are specifically activated, they may inhibit inflammatory Th1 cells through a non-specific bystander mechanism. A variety of strategies have been used by us to identify candidate disease-associated TCR V genes present on pathogenic Th1 cells, including BV5S2, BV6S5, and BV13S1 in MS, BV3, BV14, and BV17 in RA, and BV3 and BV13S1 in psoriasis. TCR peptides corresponding to the mid region of these BV genes were found to be consistently immunogenic in vivo when administered either i.d. in saline or i.m. in incomplete Freund's adjuvant (IFA). In MS patients, repeated injection of low doses of peptides (100-300 μg) significantly boosted the number of TCR-reactive Th2 cells. These activated cells secreted cytokines, including IL-10, that are known to inhibit inflammatory Th1 cells. Cytokine release could also be induced in TCR-reactive Th2 cells by direct cell-cell contact with Th1 cells expressing the target V gene. These findings indicate the potential of regulatory Th2 cells to inhibit not only the target Th1 cells, but also bystander Th1 cells expressing different V genes specific for other autoantigens. TCR peptide vaccines have been used in our studies to treat a total of 171 MS patients (6 trials), 484 RA patients (7 trials), and 177 psoriasis patients (2 trials). Based on this experience in 824 patients with autoimmune diseases, TCR peptide vaccination is safe and well tolerated, and can produce significant clinical improvement in a subset of patients that respond to immunization. TCR peptide vaccination represents a promising approach that is well-suited for treating complex autoimmune diseases.

Rodents immunized with complete Freund's adjuvant (CFA) are resistant to subsequent attempts to induce autoimmune disease, while animals immunized with incomplete Freund's adjuvant (IFA) remain susceptible. Mycobacterial extracts can stimulate inducible nitric oxide synthase (NOS2) gene transcription. Robust expression of NOS2 has been linked to suppression of T cell proliferation and alterations in immune responses. Our studies investigated the hypothesis that the immunoprotective effect of CFA before immunization requires functional NOS2. NOS2 gene expression is chronically elevated in lymph nodes and spleens of CFA-immunized mice. Maximal expression of NOS2 after CFA immunization requires the presence of functional type I tumor necrosis factor α receptor (TNFR1) and interferon γ. Groups of nontreated and CFA-preimmunized male C57BL/6J or C57BL/6NOS2−/− mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 in CFA to induce experimental allergic encephalomyelitis (EAE). Wild-type C57BL/6J mice were protected from the development of symptoms of EAE, while the NOS2−/− mice failed to be protected. NOS2-dependent effects of CFA included an augmentation of the MOG-specific IgG1 response, a decrease in interleukin 6 production by MOG-reactive lymphocytes, and a marked decrease in mononuclear cell infiltrates in the central nervous system. These studies support the hypothesis that CFA immunization modulates immune responses through a nitric oxide–dependent mechanism.

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68 – 86. The TCR β chains of these disease-causing T cells show three distinct features: they are almost exclusively Vβ8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68 – 86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery – i.e., that such β chains are conformationally the most appropriate for triggering by an epitope of 68 – 86 complexed to class II RT1.Bl MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vβ8.2+ , CDR3 AspSer+ and use few N region additions. We conclude that T cells with these TCR β chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.

Cells expressing markers of both natural killer and T lymphocytes (NK T cells) in humans and mice express a restricted T‐cell receptor (TCR) repertoire, are of CD4− CD8− or CD4+ CD8− phenotype, and upon anti‐CD3 stimulation secrete large amounts of interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ). NK T cells may be the primary source of IL‐4‐promoting T helper type 2 (Th2) responses and/or they might be involved in regulating the balance between Th1‐ and Th2‐type immune responses, and may consequently affect susceptibility to autoimmune diseases associated with a skewed Th phenotype. We show that rat NK T cells selectively proliferate to IL‐2, and use this fact to analyse cytokine production by NK T cells in two rat strains differentially susceptible to Th1‐ or Th2‐type autoimmune diseases. Analysis by reverse transcription–polymerase chain reaction revealed that, in contrast to mouse, rat NK T cells secrete exclusively IFN‐γ and not IL‐4 after anti‐CD3 stimulation, and use a wider TCR‐Vβ repertoire, suggesting that rat NK T cells are not essential for the development of Th2‐type CD4+ T‐cell responses.

The initiation of T cell dependent immune responses requires the T cell to utilize a heterodimeric cell surface antigen receptor to recognize an antigenic peptide in association with major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs). This is a necessary but insufficient signal for T cell activation. T cells must additionally receive a costimulatory signal that can be delivered through a variety of receptor-ligand pairs. Of these, the best characterized is the CD28-B7 (CD80 and CD86) couple. It has been appreciated for decades that the outcome of the T cell-APC interaction is highly variable and can lead to different forms of immune responses. The past decade has witnessed major advances in the definition of how distinct lymphocyte functions are dictated by expression of distinct cytokines, activation of defined signal transduction pathways, and, most recently, expression of distinct transcription factors. The production of ever increasing numbers of induced mutant mouse strains has created new reagents in which to analyze the role of particular cytokines or other proteins in defined immune responses. While the temptation remains to accomodate emerging information into reductionist models of T cell dependent immunopathology, it is our view that in vivo immune responses are highly complex and regulated events that defy simple categorization. The goal of this review is to provide an overview of current information relevant to how different types of T cell responses develop and how such responses relate to the expression of disease. Our review will focus in depth on the concept of a "Th1-Th2" paradigm and how that relates to harmful versus protective immune responses. We will briefly touch on the outcome of costimulation of T cells via distinct receptor-ligand couples, and the effect of a number of other defined cell surface glycoproteins and cytokines on T cell responses. Relevant work in model systems of inflammatory renal injury will be discussed where appropriate. Over a decade has passed since initial studies by Mosmann et al defined two distinct subsets of murine CD4+ T cell clones based on their ability to synthesize nonoverlapping profiles of cytokines1. Since that time, there has been an enormous amount of work examining the cellular and molecular basis for this polarization of CD4+ T cell responses as well as investigations into the relationship between polarized CD4+ T cell responses and immune responses to infections and self antigens. In this section, we will summarize some of this information. Figure 1 provides a schematic outline for the information discussed in this section. Since the original description of Th1 and Th2 clones, additional characterization of these populations has been performed. A third subset, denoted "Th0" T cells, has been defined that produces a mixture of the Th1 and Th2 cytokine patterns6. IL-6, IL-10, and IL-13 have been added to the group of Th2 cytokines7. Although several cell-surface markers, including CD45 isoforms, have been proposed as candidates to distinguish the Th1 and Th2 subsets, the stable definition continues to be based on cytokine profiles and effector function. Two recent publications, however, have provided evidence that Th1 and Th2 subsets additionally exhibit distinct migratory capacities into tissues. These capacities map with distinct cell surface receptor expression. Murine Th1 cells, but not Th2 cells, can bind to P- and E-selectin8. The migration of these Th1 cells into inflamed sites can be blocked by antibodies to E- and P-selectin. Human Th2 cells, on the other hand, express a high affinity receptor for the CC-chemokine eotaxin, called CCR39. Eotaxin is a potent chemoattractant for eosinophils and basophils. It is produced by phagocytes and epithelial cells. Since Th2-like cytokines like IL-4 and IL-5 are critical growth factors for eosinophils and basophils, the expression of CCR3 on Th2 cells provides a mechanism for the recruitment of this CD4+ subset into sites of allergic inflammation. IL-12 and -IFN promote the expansion of Th1 cells11,12, and -IFN additionally blocks differentiation along the Th2 pathway. Studies supporting these conclusions have been performed using polyclonal stimuli in vitro followed by restimulation to induce cytokine expression and with TCR transgenic T cells stimulated with their relevant antigen. They have additionally been performed by administering IL-12 and -IFN to intact animals and studying the resultant populations. IL-4 is the cytokine that has consistently demonstrated powerful effects in driving differentiation of CD4+ T cells to the Th2 phenotype13. Studies utilizing mice deficient in specific cytokines, be they genetic knockout mice or animals receiving neutralizing antibodies to cytokines, have demonstrated profound influences on Th phenotypes14,15,16. This latter observation has led to the hypothesis that one of the in vivo functions of NK1+ T cells may be to provide the early IL-4 burst that subsequently biases the immune response to differentiation down the Th2 pathway. This hypothesis is supported indirectly by the fact that some mouse strains (such as, SJL) that express low numbers of NK1+ cells do not demonstrate the early IL-4 burst following anti-CD3 administration in vivo, nor do they produce IgE in response to anti-IgD antibodies24. SJL and NOD mice (the latter also have low numbers of NK1+ T cells25) both display increased susceptibility to a variety of autoimmune diseases. Reports in both experimental and clinical autoimmune diseases have described decreased numbers of NK1+ T cells with disease progression26,27. Finally, in vivo treatment with an antibody to the dominant NK1+ T cell TCR alpha chain (V14) accelerates murine lupus26. However, in mice made genetically deficient in CD1, NK1+ T cells are severely reduced in numbers, and early IL-4 burst is missing, yet the CD1-/- mice generate IgE normally in response to anti-IgD challenge, and they can mount Th2-like recall responses19,28. Since the impact of the mixed genetic background of the knockout mice on these read-out assays is as yet unclear, it is too soon to definitively rule out an important role for NK1+ T cells in driving Th2 responses. It should additionally be noted that NK1+ T cells can also express -IFN, but their role in Th1 responses is unexplored29. An alternative pathway to early IL-4 expression has been recently described and involves the stimulation of naive T cells by APC-derived IL-6 to make IL-430. Mast cells and basophils can additionally be induced to make IL-4 following cross-linking of Fc receptors by IgG or IgE31,32. Finally, a recent study investigating the Th2-polarized response of CD4+ T cells from Balb/c mice infected with Leishmania major, has strongly implicated a subset of CD4+ T cells in the generation of an early IL-4 "burst" resulting in commitment down the Th2 effector pathway. These cells are of additional interest since they express a highly restricted TCR (V4, V8)33. Th2 polarization in the Leishmania major system additionally appears to be independent of NK1.1+ T cells34. In the aggregate, these studies underscore the complexities of polarization of the CD4+ Th response and the potential for distinct mechanisms of regulation in different model systems. Classic antigen-presenting cells are the likely source of IL-12, which initiates Th1 effector responses. IL-12 is a 70 kDa heterodimer comprised of two covalently linked proteins (p40 and p35). The genes encoding both components must be expressed within the same cell to produce the heterodimer. Although the p35 gene is transcribed in many cell types, p40 gene expression is more highly restricted and tightly regulated. B cells, adherent monocytes, dendritic cells and skin Langerhans cells produce amounts of IL-12, which are likely important in directing the differentiation of CD4+ T cells. A variety of other cell types, including some cells not traditionally regarded as classical APCs or as involved in the innate immune response, likely can also express IL-12 in culture under defined conditions. The in vivo significance of these observations has not yet been fully clarified. IL-12 acts directly on undifferentiated T cells and provides an early stimulus for natural killer cells to produce IFN35. In complementary studies, recent work employed the combination of the T cell receptor transgenic system with mice lacking the expression of the transcription factor interferon regulatory factor-1 (IRF-1) to determine the importance of IRF-1 in Th1 versus Th2 differentiation40,41. IRF-1 is a transcription factor induced by interferon (IFN)-//. The studies performed in this system suggested that IRF-1 is essential for the development of the Th1 response and that it functions in multiple cell types which participate in the differentiation of Th1 CD4+ T cells. T cells from these mice exclusively undergo Th2 differentiation in vitro. The animals demonstrate defects in macrophage function characterized by impaired IL-12 production. Their CD4+ T cells are not responsive to IL-12 and they have abnormal natural killer cell activity41. A dominant paradigm for infectious disease models, in particular the immune response to Leishmania major, has been that cell-mediated immunity critical for the defense against intracellular microorganisms is mediated by Th1-like T cells42. Resistance to extracellular pathogens is generated most effectively by activation of Th2-like cells. In some model systems of organ-specific autoimmunity, there is a good correlation between the preferential induction of Th1 T cells and the development of autoimmune pathology with impaired organ function. Some investigators have tested the relevance of polarized CD4+ T cells to autoimmunity by employing maneuvers that lead to a polarized response and examining whether such a maneuver results in directionally consistent changes in disease expression. Examples of this could include the administration of polarizing cytokines, such as IL-12, -IFN, or IL-4, or the administration of neutralizing antibodies to such cytokines. Difficulties in the interpretation of such studies include the obvious problem that cytokine administration may trigger effector or regulatory events relevant to disease expression in conjunction with, but unrelated to, the observed polarization of the Th response. The major technical problems with antibody administration center around the duration of therapy required, the timing of the administration, and the development of immune responses targeted to the neutralizing antibody. Genetically altered mice can be used theoretically to avoid some of the above-described problems with either cytokine or antibody infusion. For example, the incidence and severity of collagen-induced arthritis are significantly reduced in IL-12 knockout mice. These animals also display decreases in IgG2a responses to type II collagen (the immunogen) and depressed collagen-induced secretion of -IFN by splenocytes in vitro43. Yet it is frequently unclear what definitive conclusions can be drawn from studies of autoimmunity in cytokine knockout mice. The majority of cytokine knockouts generated to date are animals in which the cytokine(s) is deleted from birth in all cells of the animal. Given the well recognized redundancy in cytokine function, the failure of cytokine deletion to affect the course of an autoimmune process does not establish that in the intact organism that cytokine has no important role in the disease process. This generic issue has been well summarized recently44. The course of experimental allergic encephalitis (EAE) in various cytokine knockout animals provides instructive examples. There is little controversy that Th1-like T cells are important to the pathogenesis of EAE. The CD4+ T cells that differentiate following immunization to produce disease in both mice and rats are Th1-like in cytokine expression. Th1-like T cell clones can adoptively transfer disease into naive hosts. Multiple studies have provided both direct and correlative evidence implicating TNF- and lymphotoxin (LT)- in the immunopathogenesis of EAE. Despite this, EAE develops normally in animals in whom both TNF- and LT- are inactivated45. Gamma-IFN knockout mice display heightened mortality when immunized to produce EAE, rather than protection46. Such results may be related to other cytokines substituting for the roles typically played by TNF-, LT-, and -IFN. Alternatively, cytokines such as -IFN may be subserving protective, rather than pathogenic roles that have been underappreciated47. In the case of -IFN, a strong argument can be made for the ability of this cytokine to induce nitric oxide synthesis through the cytokine inducible isoform and thereby dampen pathogenic T cell responses (see below)48. In the murine model of crescentic glomerulonephritis described above, treatment of C57BL/6 mice with IL-4 and/or IL-10, either prior to or following the induction of the immune response resulted in an improved functional outcome, diminished DTH responses to sheep globulin, and diminished glomerular crescent formation62,63. The mechanism underlying this protective effect correlated with selective inhibition of the Th1 response to the antigen, when IL-4/IL-10 treatment was initiated prior to induction of the immune response62. With initiation of treatment after the establishment of injury, the mechanism of protection was less clear, as an analysis of the levels of IgG isotypes between experimental groups and splenic production of -IFN did not support a deviation of the induced immune response to a Th2-predominant form63. Although treatment with IL-4 and IL-10 was protective in a mouse strain selected on the basis of its propensity to display Th1-predominant immune responses, IL-10 alone was ineffective in a related model of accelerated anti-glomerular basement membrane (GBM) disease and crescentic glomerulonephritis in rats64. The differences in outcome may relate to differing protocols of administration (including the use of a single cytokine, rather than IL-4 and IL-10), but may also reflect the genetic background. The outcome of the above described murine model is strikingly different in Th2-biased (Balb/c) mice65. When Balb/c mice sensitized to sheep globulin are given a subnephritogenic intravenous dose of anti-mouse GBM globulin, they develop glomerular deposition of Ig and complement, a significant neutrophil influx in the glomeruli, and significant proteinuria. There are no significant crescents or infiltrating T cells and macrophages in contrast to the C57BL/6 mice. The lesion in the Balb/c mice can be attenuated by complement depletion but not CD4+ T cell depletion. The reverse is true for C57BL/6 mice65. These studies support the notion that the polarized Th responses may not map phenotypically with injury or protection but rather different forms of injury. Another well studied model system in which autoimmune glomerulonephritis develops in conjunction with a strong induced polyclonal Th2-like response is the HgCl2 induced model of glomerulonephritis in the Brown Norway (BN) rat66,67. Finally, in some transgenic mice that overexpress IL-4, there is demonstrable B cell hyperactivity, elevated IgG1 and IgE levels, and spontaneous glomerulonephritis with complement and antibody deposition within the glomerulus68. This is in contrast to the protective effect that constitutive expression of IL-4 has on the lupus-like glomerulonephritis in (NZW x C57BL/6. Yaa)F1 mice69. While the argument can be made that many studies in transgenic and knockout mice "muddy the waters" by creating highly artificial and unphysiologic scenarios, the bulk of emerging evidence suggests that the Th1-Th2 (pathogenic-protective) paradigm is too simplistic a model for understanding autoimmunity. T cell proliferation is critically dependent on costimulation. This section briefly reviews the process and recent studies on costimulation in the context of autoimmunity are discussed. The studies summarized above support the conclusion that the outcome of the T cell-APC interaction will be critically dependent on the nature of the costimulatory interaction, and not simply the absence or presence of costimulation, as was previously hypothesized. The cues that underlie a dominant B7-CD28 versus a B7-CTLA-4 interaction are being intensively investigated. The issues are complex given the potential for interaction between both B7-1 and B7-2 with either CD28 or CTLA-4. In general, the differences in B7-1 and B7-2 expression appear to be largely temporal and in part cell specific. B7-2 is induced earlier than B7-1 in most immune responses. Both molecules have been described to be inducible on dendritic cells, Langerhans cells, B cells, macrophages, and T cells. They are typically up-regulated following macrophage activation and are up-regulated by -IFN, LPS, GM-CSF reviewed in81. The relative role of the B7-1 versus B7-2 interaction with CD28 and CTLA-4 has been investigated in a variety of autoimmune model systems. The variety of results obtained underscores the complexities of these interactions. When murine CTLA4Ig is injected into NZB/W F1 lupus prone mice, it blocks the production of anti-DNA antibodies and prolongs life82. The role, however, of B7-1 versus B7-2 is harder to generalize between autoimmune models. For example in the lupus prone mice, treatment with anti-B7-2 antibodies blocks production of some anti-DNA antibodies, particularly IgG1 anti-DNA antibodies, but was without effect on nephritis. Injection of anti-B7-1 had no significant effect on anti-DNA antibodies or nephritis. However, the injection of both anti-B7-1 and anti-B7-2 prevented production of anti-DNA antibodies of all subclasses, blocked the development of nephritis and resulted in a longer lifespan83. In the spontaneous model of insulin dependent diabetes in non-obese diabetic mice, treatment with CTLA4-Ig or anti-B7-2 blocked the development of diabetes. Antibodies to B7-1 had no protective effect84. The timing of this treatment was critical in that only animals treated between 5 to 7 weeks of age were protected. It is most intriguing that using NOD mice bred to the CD28 knockout mouse, or NOD mice bred to a transgenic mouse expressing CTLA-4Ig driven by a skin-specific keratin promoter, the development and progression of spontaneous autoimmune diabetes is promoted, rather than inhibited85. The authors propose that this may be attributable to a preferential activation of Th1-like rather than Th2-like autoreactive T cells early during life. This unexpected experimental result raises another caution flag regarding the interruption of this costimulatory pathway as a treatment for autoimmune disease. In murine experimental EAE, injection with anti-B7-2 antibodies does not affect the severity of disease while injections with anti-B7-1 do86,87. CTLA4-Ig can prevent the onset of collagen induced arthritis in BB rats88. These seemingly discrepant results in different model systems would mitigate against being able to broadly generalize the role of these interactive molecules in autoimmune diseases and the types of immune response that are generated. It seems possible that in each particular autoimmune disease the outcome of blocking both B7-1 and B7-2, or either independently, may additionally depend on the types of co-stimulatory molecules expressed on both professional and non-professional antigen presenting cells within the target organ of interest. Diversity in antigen recognition by T cells is calculated to be as high as for immunoglobulin due to the inherent diversity accomplished in the process of DNA rearrangements of the TCR variable element genes during T cell development and due to the random sorting of assembled TCR and chains97. The fact that T cell recognition occurs through clonally distributed TCR chains interacting with MHC-associated peptide fragments of foreign or self proteins, raises the possibility that either expression of unique TCR chains or presentation of unique peptide fragments of self antigens could be factors in determining susceptibility or resistance to autoimmune disease. Support for the idea that unique TCR expression was associated with autoimmunity initially came from observations in both the mouse and rat models of experimental allergic encephalomyelitis (EAE). In both rodent models, it was observed that the majority of T cells responsive to the major encephalitogenic epitopes of myelin basic protein (MBP) used a very limited repertoire of V and V genes98,99,100,101,102. Furthermore, strategies aimed at deleting or inactivating cells utilizing these families of TCR chains resulted in protection from disease98,102,103,104. Prompted by these studies, several groups have analyzed T cell responses and TCR usage in a number of clinical autoimmune disease settings. As an example, when studies were performed to assess human T cell responses to MBP, no measurable differences were observed in the frequency of MBP reactive T cells among peripheral blood lymphocytes isolated from normal individuals and patients with multiple sclerosis. Furthermore, there does not appear to be any convincing differences in TCR usage among normal individuals and patients with multiple sclerosis. There are no convincing similarities in TCR usage among MBP reactive T cell clones, isolated from multiple sclerosis (MS) patients, responding to the same epitope of MBP presented by the same HLA-DR or -DQ molecule. One exception of note, however, is the finding of Oksenberg and colleagues who described the presence of a common TCR rearrangement in demyelinating plaques isolated from CNS tissue of deceased MS patients105. These unique rearrangements were previously observed in human and rat T cell clones recognizing similar regions of MBP. These results suggest to the authors that certain epitopes of MBP are being recognized in the CNS of patients with MS and perhaps by T cells expressing a limited repertoire of TCR chains. Overall, the conclusion from these studies and those in other autoimmune settings is that the TCR per se is quite likely not going to be a major susceptibility factor with respect to T cell recognition of autoantigens106. However, it is equally likely that the diversity of pathologic T cell responses in any individual or in different individuals of similar HLA type and responding to similar epitopes of an autoantigen, may in fact be limited. If the TCR itself is not a determining factor in the selection of a limited autoaggressive repertoire, then perhaps the repertoire of HLA chains expressed by the individual predisposes towards autoreative T cell development. Inheritance of particular alleles of HLA genes has been associated with several autoimmune diseases over the years. In addition, more recently it has become appreciated that certain alleles are in fact protective towards the development of certain autoimmune diseases. The best example of this phenomenon is seen in the case of juvenile onset diabetes107. Because HLA-associated peptide presentation to T cells is critical for both T cell development in the thymus and T cell activation in the periphery, several models have been proposed to account for the apparent HLA linkage to autoimmune disease. It is possible that certain peptide/MHC combinations can cause the deletion of potentially autoreactive T cells maturing in the thymus to a lesser or greater extent depending on amino acid differences in the peptide binding domains of the HLA proteins. Since autoreactivity to many self antigens can be demonstrated in healthy individuals, it is unlikely that thymic deletion per se can explain HLA linkage and autoimmune reactivity. Certainly, the overall affinities of allowable self reactive antigen receptors could differ depending on the nature of the peptide/MHC combinations encountered during thymic T cell development and this difference in affinity could be a determining factor in whether a potential autoreactive T cell clone will be triggered or not. An alternative hypothesis has suggested that certain alleles of HLA may have higher affinities for certain self antigens and act as a "peptide sink," thereby removing or competing for particular self antigen epitopes that if presented on a permissive HLA proteins would evoke autoaggressive T cell responses108. This model could help account for the observation of protective HLA alleles. A slightly different version of this model would suggest that certain HLA alleles could bind particular regions of an autoantigen with high affinity, thereby altering the proteolytic processing of other epitopes for which autoreactive T cells may exist109. Undoubtedly, all of these possibilities as well as others not mentioned will prove to be true, in some form contributing to the overall complexity of autoantigen TCR repertoire development and T cell autoreactive responsiveness. There is a growing interest in the role of chemokines in the phenotypic expression of autoimmune kidney disease115,116,117,118,119. With the recent discovery of genetic polymorphisms in chemokine receptors and the biologic importance of this trait in susceptibility to HIV infection, it is possible that further work will identify these chemokine receptor polymorphisms as additional susceptibility factors for autoimmunity. The immune system has evolved to deal with infectious organisms. Major studies over the past ten years have provided an enormous amount of information regarding the various phenotypes of activated T cells, the cytokines they produce, the functions they mediate, their requirements for activation, and how they are differentially regulated. The characteristics which result in a particular T cell response being "host beneficial" in the context of infectious disease can be similar to those resulting in autoimmune pathology. Autoimmunity is a complex process, however, and it is clear that a simple Th1/Th2 paradigm does not fully explain all forms of autoimmune tissue damage. Moreover, although some rodent models of autoimmunity are more widely studied than others, each model system has unique characteristics that preclude generalizing conclusions from investigations into disease pathogenesis or treatment in one or two model systems. Given the available model systems, reagents, and major advances in understanding T cell biology, the next decade will undoubtedly witness innovative approaches to the therapy of autoimmune disease. We appreciate the assistance of David Wolfe in the design of Figure 1. In writing this review, we could not, because of space concerns, reference all publications relevant to the areas of discussed. We have referenced recent review articles where appropriate.