Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity.

During 25 surveys of global Phytophthora diversity, conducted between 1998 and 2020, 43 new species were detected in natural ecosystems and, occasionally, in nurseries and outplantings in Europe, Southeast and East Asia and the Americas. Based on a multigene phylogeny of nine nuclear and four mitochondrial gene regions they were assigned to five of the six known subclades, 2a-c, e and f, of Phytophthora major Clade 2 and the new subclade 2g. The evolutionary history of the Clade appears to have involved the pre-Gondwanan divergence of three extant subclades, 2c, 2e and 2f, all having disjunct natural distributions on separate continents and comprising species with a soilborne and aquatic lifestyle and, in addition, a few partially aerial species in Clade 2c; and the post-Gondwanan evolution of subclades 2a and 2g in Southeast/East Asia and 2b in South America, respectively, from their common ancestor. Species in Clade 2g are soilborne whereas Clade 2b comprises both soil-inhabiting and aerial species. Clade 2a has evolved further towards an aerial lifestyle comprising only species which are predominantly or partially airborne. Based on high nuclear heterozygosity levels ca. 38 % of the taxa in Clades 2a and 2b could be some form of hybrid, and the hybridity may be favoured by an A1/A2 breeding system and an aerial life style. Circumstantial evidence suggests the now 93 described species and informally designated taxa in Clade 2 result from both allopatric non-adaptive and sympatric adaptive radiations. They represent most morphological and physiological characters, breeding systems, lifestyles and forms of host specialism found across the Phytophthora clades as a whole, demonstrating the strong biological cohesiveness of the genus. The finding of 43 previously unknown species from a single Phytophthora clade highlight a critical lack of information on the scale of the unknown pathogen threats to forests and natural ecosystems, underlining the risk of basing plant biosecurity protocols mainly on lists of named organisms. More surveys in natural ecosystems of yet unsurveyed regions in Africa, Asia, Central and South America are needed to unveil the full diversity of the clade and the factors driving diversity, speciation and adaptation in Phytophthora. Taxonomic novelties: New species: Phytophthora amamensis T. Jung, K. Kageyama, H. Masuya & S. Uematsu, Phytophthora angustata T. Jung, L. Garcia, B. Mendieta-Araica, & Y. Balci, Phytophthora balkanensis I. Milenkovic, Z. Tomic, T. Jung & M. Horta Jung, Phytophthora borneensis T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora calidophila T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora catenulata T. Jung, T.-T. Chang, N.M. Chi & M. Horta Jung, Phytophthora celeris T. Jung, L. Oliveira, M. Tarigan & I. Milenkovic, Phytophthora curvata T. Jung, A. Hieno, H. Masuya & M. Horta Jung, Phytophthora distorta T. Jung, A. Durán, E. Sanfuentes von Stowasser & M. Horta Jung, Phytophthora excentrica T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora falcata T. Jung, K. Kageyama, S. Uematsu & M. Horta Jung, Phytophthora fansipanensis T. Jung, N.M. Chi, T. Corcobado & C.M. Brasier, Phytophthora frigidophila T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora furcata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora inclinata N.M. Chi, T. Jung, M. Horta Jung & I. Milenkovic, Phytophthora indonesiensis T. Jung, M. Tarigan, L. Oliveira & I. Milenkovic, Phytophthora japonensis T. Jung, A. Hieno, H. Masuya & J.F. Webber, Phytophthora limosa T. Corcobado, T. Majek, M. Ferreira & T. Jung, Phytophthora macroglobulosa H.-C. Zeng, H.-H. Ho, F.-C. Zheng & T. Jung, Phytophthora montana T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora multipapillata T. Jung, M. Tarigan, I. Milenkovic & M. Horta Jung, Phytophthora multiplex T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora nimia T. Jung, H. Masuya, A. Hieno & C.M. Brasier, Phytophthora oblonga T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora obovoidea T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora obturata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora penetrans T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora platani T. Jung, A. Pérez-Sierra, S.O. Cacciola & M. Horta Jung, Phytophthora proliferata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora pseudocapensis T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pseudocitrophthora T. Jung, S.O. Cacciola, J. Bakonyi & M. Horta Jung, Phytophthora pseudofrigida T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora pseudoccultans T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pyriformis T. Jung, Y. Balci, K.D. Boders & M. Horta Jung, Phytophthora sumatera T. Jung, M. Tarigan, M. Junaid & A. Durán, Phytophthora transposita T. Jung, K. Kageyama, C.M. Brasier & H. Masuya, Phytophthora vacuola T. Jung, H. Masuya, K. Kageyama & J.F. Webber, Phytophthora valdiviana T. Jung, E. Sanfuentes von Stowasser, A. Durán & M. Horta Jung, Phytophthora variepedicellata T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora vietnamensis T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora ×australasiatica T. Jung, N.M. Chi, M. Tarigan & M. Horta Jung, Phytophthora ×lusitanica T. Jung, M. Horta Jung, C. Maia & I. Milenkovic, Phytophthora ×taiwanensis T. Jung, T.-T. Chang, H.-S. Fu & M. Horta Jung. Citation: Jung T, Milenkovic I, Balci Y, Janousek J, Kudlácek T, Nagy ZÁ, Baharuddin B, Bakonyi J, Broders KD, Cacciola SO, Chang T-T, Chi NM, Corcobado T, Cravador A, Dordevic B, Durán A, Ferreira M, Fu C-H, Garcia L, Hieno A, Ho H-H, Hong C, Junaid M, Kageyama K, Kuswinanti T, Maia C, Májek T, Masuya H, Magnano di San Lio G, Mendieta-Araica B, Nasri N, Oliveira LSS, Pane A, Pérez-Sierra A, Rosmana A, Sanfuentes von Stowasser E, Scanu B, Singh R, Stanivukovic Z, Tarigan M, Thu PQ, Tomic Z, Tomsovský M, Uematsu S, Webber JF, Zeng H-C, Zheng F-C, Brasier CM, Horta Jung M (2024). Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity. Studies in Mycology 107: 251-388. doi: 10.3114/sim.2024.107.04.

Extensive morphological and behavioural diversity among fourteen new and seven described species in Phytophthora Clade 10 and its evolutionary implications.

During extensive surveys of global Phytophthora diversity 14 new species detected in natural ecosystems in Chile, Indonesia, USA (Louisiana), Sweden, Ukraine and Vietnam were assigned to Phytophthora major Clade 10 based on a multigene phylogeny of nine nuclear and three mitochondrial gene regions. Clade 10 now comprises three subclades. Subclades 10a and 10b contain species with nonpapillate sporangia, a range of breeding systems and a mainly soil- and waterborne lifestyle. These include the previously described P. afrocarpa, P. gallica and P. intercalaris and eight of the new species: P. ludoviciana, P. procera, P. pseudogallica, P. scandinavica, P. subarctica, P. tenuimura, P. tonkinensis and P. ukrainensis. In contrast, all species in Subclade 10c have papillate sporangia and are self-fertile (or homothallic) with an aerial lifestyle including the known P. boehmeriae, P. gondwanensis, P. kernoviae and P. morindae and the new species P. celebensis, P. chilensis, P. javanensis, P. multiglobulosa, P. pseudochilensis and P. pseudokernoviae. All new Phytophthora species differed from each other and from related species by their unique combinations of morphological characters, breeding systems, cardinal temperatures and growth rates. The biogeography and evolutionary history of Clade 10 are discussed. We propose that the three subclades originated via the early divergence of pre-Gondwanan ancestors > 175 Mya into water- and soilborne and aerially dispersed lineages and subsequently underwent multiple allopatric and sympatric radiations during their global spread. Citation: Jung T, Milenkovic I, Corcobado T, et al. 2022. Extensive morphological and behavioural diversity among fourteen new and seven described species in Phytophthora Clade 10 and its evolutionary implications. Persoonia 49: 1-57. https://doi.org/10.3767/persoonia.2022.49.01.

Evaluating the efficiency of the Dental Teacher system as a digital preclinical teaching tool.

AIM: To investigate the use of a preparation evaluation system for enhancing the learning and performance of undergraduate dental students when cutting preparations. MATERIALS AND METHODS: Two groups of eighteen students each were randomly chosen from the fourth year of the dental programme. The task chosen for this study was to make a cavity in preparation for a mesio-occlusal ceramic onlay in a plastic tooth. The dimensions of the cavity were defined, and 2 burs of known size were used for preparation. For assessment, each tooth preparation was scanned with a digital scanner and analysed using the Dental Teacher software. In the control group, a second corrective preparation was made following the supervisor's instructions. In the test group, the second preparation was made based on Dental Teacher analysis. The final cavities were all scanned and assessed by Dental Teacher comparing the similarity of students' onlay cavity preparations to the ideal preparation. All data were recorded and analysed by the software, including cavity depth and width in the occlusal and proximal box, the extent of mesiobuccal cusp reduction and shoulder width around the mesiobuccal cusp. Finally, the data were statistically evaluated using a Wilcoxon matched pairs test and a Mann-Whitney U test. RESULTS: Three of the 6 cavity dimension parameters improved significantly in the test group whilst no improvement was found in the control group. A positive correlation was found between the improvement and the deviation measured for the first preparations, and it was stronger in the test group than in the control group. CONCLUSIONS: The use of Dental Teacher helped students to learn the preparation technique for onlay restorations more efficiently and seems to be a promising and useful method to facilitate their individual performance. Student feedback showed a great demand for digital aids in education.

Immune Class Regulation and Its Medical Significance Part II of a Report of a Workshop on Foundational Concepts of Immune Regulation.

We discussed different proposals for how the nature of the Th1/Th2 phenotype of an immune response is determined, and favoured one, the Threshold Hypothesis, as plausible and so useful as the basis for further discussions. The activation of a target CD4 T cell can be facilitated by helper CD4 T cells when the CD4 T cells interact via an antigen-presenting cell. The Threshold Hypothesis states that tentative and robust antigen-mediated CD4 T cell cooperation results in the target CD4 T cell, respectively giving rise, upon activation, to Th1 and Th2 cells. We primarily discussed four topics. We briefly discussed in the background section certain limitations of the Th1/Th2 paradigm in understanding immune class regulation, and the remarkable anti-inflammatory properties of human IgG4 antibody. Secondly, we assessed the role of class II MHC molecules in determining the number of mature CD4 T cells and so affecting the Th1/Th2 phenotype of immune responses. We also discussed the controversial role of CD8 T cells in affecting the Th1/Th2 phenotype of responses to MHC and other antigens, and the potential role of their relative scarcity in neonates in biasing responses towards an antibody, Th2 mode. Lastly, we examined the regulation of the Th1/Th2 phenotype of both primary and ongoing immune responses in the context of the intriguing proposal that antigen initially generates different classes/subclasses of immunity and then selects, by a feedback mechanism, the most effective class. We found this interesting idea difficult to reconcile with various observations.

Immunological Tolerance. Part I of a Report of a Workshop on Foundational Concepts of Immune Regulation.

This report, the first of two, arose from a one-week workshop directed at discussing concepts of immune regulation, and focuses on immunological tolerance. We first outline the major ideas we thought sufficiently plausible to provide a context for discussing more controversial issues around tolerance. We then report on our discussion of different experiments that appear paradoxical in terms of the different, contemporary models of CD4 T cell inactivation/activation, and how such observations might be resolved in terms of insights provided by these contemporary models. These discussions bear on the plausibility of the Pathogen-Associated Molecular Pattern (PAMP), Danger and Two Step, Two Signal Models for the activation of naïve CD4 T cells. Some of the observations considered appear paradoxical in terms of the PAMP and Danger Models, but not with the Two Step, Two Signal Model. For example, genetically immunodeficient mice have been given foreign, sterile ectopic grafts, and the immune system allowed to develop once these grafts were well-healed in, and so in the absence of PAMPs or danger. The grafts were rejected, unexpected on the PAMP or Danger Models. We also discussed considerations and observations bearing on the widely held idea that antigen must crosslink the membrane Ig receptors of a B cell to initiate the generation of signal 1, or the alternative possibility that monovalent binding by antigen can do so. We favored the latter possibility, and discussed a particular model, "the Elbow Model," for how this might be achieved.

First Report of Phytophthora × pelgrandis Causing Root Rot and Lower Stem Necrosis of Common Box, Lavender and Port-Orford-Cedar in Hungary.

In 2008 and 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown common box (Buxus sempervirens L.), lavender (Lavandula angustifolia Mill. 'Hidcote'), and Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Columnaris') in three ornamental nurseries in western Hungary. Number of affected plants ranged from approximately 100 (Port-Orford-cedar) to 250 (lavender). Isolations from necrotic root collars of each host plant species yielded four Phytophthora isolates developing uniform colonies on carrot agar with a maximum growth temperature of 35 to 36°C. The isolates were homothallic with smooth-walled oogonia (32.2 ± 2.3 to 35.9 ± 3.5 µm), aplerotic oospores (27.5 ± 1.8 to 32.1 ± 3.1 µm) and both amphigynous and paragynous antheridia, and produced chlamydospores (25.8 ± 3.9 to 29.1 ± 5.2 µm) and papillate sporangia (35.2 ± 2.5 to 43.5 ± 5.6 µm long and 27.6 ± 2.2 to 32.0 ± 3.8 µm wide), mostly obpyriform to nearly spherical or rarely distorted with two or three apices. In spring water, sporangia were both caducous with short pedicel and non-caducous. Multiplex ITS-PCR assay of DNA from all isolates, using primers specific for P. nicotianae (NICF1 and NICR2.1) and P. cactorum (CACTF1 and CACTR1) (1), amplified DNA fragments of the expected size for each Phytophthora species. In addition, isoenzyme analysis revealed a characteristic banding pattern of one heterodimer and two homodimer bands at both loci of the dimeric enzyme malate dehydrogenase. These bands comigrated with those of P. × pelgrandis (Gerlach et al.) (CBS 123385) and isolate PD 93/1339 (courtesy of W. A. Man in 't Veld), two natural hybrid strains of P. nicotianae and P. cactorum (2,3), proving that our four isolates can be referred to as this interspecific hybrid. Pathogenicity was tested on 1- or 3-year-old plants of the original host species and cultivars (for common box, cv. Faulkner was used). Cultures were grown for 4 to 6 weeks at 20°C on autoclaved millet grains moistened with V8 broth. Infested and uninfested grains were mixed with autoclaved soil in a ratio of 6% (w/v), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth chamber (20°C, 70% RH, 12-h photoperiod). Pots were flooded for 24 h on the 1st and 21st day after transplanting. All plants in infested potting mix showed symptoms of wilt associated with basal stem and root necrosis, similar to those observed on the plants from the field, within 2 and 3 months on lavender and both common box or Port-Orford-cedar, respectively. Additionally, a reduction of root weight ranging from 35 to 68% compared to the control was recorded. Growth reduction was significant at P ≤ 0.019 according to Mann Whitney test. Control plants remained healthy. The same Phytophthora hybrid was reisolated solely from inoculated plants. In Europe, hybrid isolates of P. nicotianae × P. cactorum have been reported on several ornamental plants, including lavender, in the Netherlands and Germany (2,3). However, to our knowledge, this is the first report of this hybrid in Hungary and as a pathogen of common box and Port-Orford-cedar in the world. References: (1) P. J. M. Bonants et al. Phytopathology 90:867, 2000. (2) W. A. Man in 't Veld et al. Phytopathology 88:922, 1998. (3) H. I. Nirenberg et al. Mycologia 101:220, 2009.

Alloreactivity: an old puzzle revisited.

Alloreactivity, defined as a strong primary T cell response against allelic variants of major histocompatibility complex (MHC) molecules in the species, has been a long-standing puzzle in immunology with some of its details remaining unclear up to now. Here I shall provide a historical overview of how our understanding of alloreactivity has evolved and propose an interpretation that considers alloreactivity to be a mixture of four mechanistically distinct prototypes of T cell response, namely, self-restricted peptide specific, allorestricted peptide specific, alloreactive peptide dependent and alloreactive peptide independent. The relative contribution of each prototype to a given alloresponse is dependent on the extent of disparity (i.e. the number and nature of amino acid substitutions in the docking surface for T cell receptor) between the MHC molecule that the T cell recognizes as self and the stimulating MHC molecule.

First Report of Phytophthora citrophthora Causing Root and Basal Stem Rot of Woody Ornamentals in Hungary.

From 2007 to 2009, necrotic bark lesions at the root collar and lower stem associated with root rot were observed on container-grown cork-bark fir (Abies lasiocarpa var. arizonica, Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Barabits Gold'), lavender (Lavandula angustifolia Mill. 'Hidcote'), flowering currant (Ribes sanguineum Pursh 'King Edward VII'), and lilac (Syringa vulgaris L. 'Belle de Nancy') in six ornamental nurseries in western Hungary. Symptoms also included reduced growth, wilting, and desiccation of branches. Mortality of affected plants ranged from a low level in flowering currant to a high frequency in cork-bark fir (1,000 plants; 50%) and lavender (2,500 plants; 50%). Isolations from necrotic tissues onto PARPB medium (1) yielded 13 isolates of a Phytophthora sp. None of the isolates produced sexual structures, sporangia, or chlamydospores but developed slightly stellate or patternless colonies with loose, aerial mycelium on nonselective carrot agar (CA) at 25°C. One isolate from each host species was further characterized and tested for pathogenicity. Growth on CA was fastest at 25°C (7.9 to 8.6 mm per day) and no growth occurred below 5°C or above 34°C. In nonsterile stream water, persistent, mono- and bipapillate, mostly ovoid, rarely distorted sporangia, measuring 40.5 to 49.4 × 29.8 to 37.3 µm were produced. In pairings on CA with A1 and A2 strains of P. cambivora and P. nicotianae, used as testers, none of our isolates produced gametangia. On the basis of these characteristics, the pathogen from ornamentals appeared to be P. citrophthora (Smith & Smith) Leon. (1). Species identity of all 13 isolates was determined in single-round PCR assays with the P. citrophthora-specific primer-pair Pc2B/Pc7 (2) and/or by sequencing the rDNA internal transcribed spacer (ITS) regions amplified with the universal ITS1/ITS4 primers. The primers Pc2B and Pc7 generated a single DNA fragment of the expected size (approximately 210 bp), and the rDNA ITS sequences (NCBI Accession Nos. GU723282, GU723284, GU723285, and GU723287) showed 99 to 100% homology with many GenBank sequences (e.g., HQ697232) of P. citrophthora as the closest match. Pathogenicity to the original host plant cultivars was tested on 2- or 3-year-old healthy plants potted in sterile soil and inoculated at the root collar (2 replicates per isolate). A 4-mm-diameter bark plug was removed and a mycelial disc of the same size from an actively growing CA culture was placed into the hole. Control plants received sterile CA plugs. Inoculation points were sealed with sterile moist cotton and Parafilm, covered with sterile soil, and then the plants were kept in a greenhouse at 24 ± 4°C. Symptoms identical to those observed on the naturally diseased hosts developed on inoculated plants within 3 months. Control plants remained healthy. P. citrophthora could be reisolated only from the infected plants. The pathogen is polyphagous, widely distributed (1), and has been associated with woody ornamentals in nurseries (3). However, to our knowledge, this is the first record of P. citrophthora in Hungary. The pathogen has to be considered as a threat to ornamental production within Hungary. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) A. Ippolito et al. Eur. J. Plant Pathol. 108:855, 2002. (3) B. W. Schwingle et al. Plant Dis. 91:97, 2007.

In vitro correlate for a clonal deletion mechanism of immune response gene-controlled nonresponsiveness.

We used T cell-antigen-presenting cell (APC) combinations from two pairs of recombinant mouse strains, B10.A(4R)-B10.A(2R) and B10.S(7R)-B10.S(9R) (abbreviated 4R, 2R, 7R, 9R, respectively), which differ from each other only in the nonexpression vs. expression of cell-surface E molecules, to study the mechanism of the Ir gene-controlled (E-restricted) response to the terpolymer poly(glu51lys34tyr15) (GLT). No response to GLT occurred when the APC were from E-nonexpressor strains 4R and 7R. When APC from E-expressor strains were used and alloreactivity against the incompatible E molecules was removed by BUdR + light treatment, 7R T cells responded to GLT presented by 9R APC, but 4R T cells failed to respond to GLT presented by 2R APC. However, 4R T cells mounted a proliferative response to GLT presented by fully allogeneic 5R or 9R APC. The latter response was completely abolished by the depletion of cells alloreactive against 2R and 5R or 2R and 9R. Since removal of alloreactivity against 5R plus 9R did not affect the response of 4R T cells to GLT presented by either 5R or 9R cells, we conclude that the 4R T cells generated in response to GLT cross-react with the additional incompatibility presented by 2R cells, that is, the Ek beta chain. In contrast, 7R T cells recognizing GLT presented by 9R APC do not cross-react with Ek beta. These results demonstrate that "blind spots" in the T cell repertoire produced by depletion of cells alloreactive against a single chain of a class II MHC molecule can render a strain nonresponsive to a synthetic polypeptide antigen, and that this nonresponsiveness corresponds to that attributed to the MHC-linked Ir genes.

Restriction molecules involved in the interaction of T cells with allogeneic antigen-presenting cells.

The proliferative responses of T cells, depleted of alloreactive cells, were tested upon stimulation by antigens presented on allogeneic antigen-presenting cells (APC). Restriction molecules involved in these responses were identified by inhibition of T cell proliferation with monoclonal antibodies against A(A alpha A beta) and E(E alpha E beta) molecules of the APC. The responses to all three antigens tested [Poly(Glu40Ala60) (GA), lactate dehydrogenase B (LDHB), and poly(Glu51, Lys34, Tyr15) (GLT)] were A plus E restricted when the allogeneic APC expressed both molecules, and only A restricted when the APC did not express cell surface E molecules. In contrast, when T cells and APC are syngeneic, the same antigens are recognized only in the context of either A molecules (GA and LDHB) or E molecules (GLT). The data indicate that the immune response gene control of these responses is not associated with either a failure of antigen presentation, or the lack of certain T cell specificities from the germ line repertoire, but probably with selective somatic elimination (tolerance) of certain clones from the T cell repertoire.

The receptor specificity of alloreactive T cells. Distinction between stimulator K, I, and D region products and degeneracy of third-party H-2 recognition by low-affinity T cells.

The specificity of binding of stimulator-derived H-2 antigens by mixed lymphocyte culture (MLC)-activated T blasts was investigated under conditions of antigen excess. We have shown that the detectable proportion of alloantigen-binding blasts from primary MLC is a function of antigen concentration, and can represent up to more than 90 percent of total blasts, when the antigen is presented in the appropriate form (on mitomycin-treated viable stimulator cells, or membrane vesicles prepared from lipopolysaccharide blasts), and at nonlimiting concentration. Thus stimulator alloantigen-binding directly parallels the proliferative response and is not restricted to a subpopulation of T blasts. However, the marked dependence of the binding on antigen concentration indicates that cells with a wide range of receptor affinities for the stimulating determinants are involved. In view of this possibility, the specificity of binding by these cells was studied. We have demonstrated that stimulator K, I, and D region products are bound by nonoverlapping subpopulations of blasts, the sum of which may represent 93 percent of total blasts. Thus, specific distinction by these cells between different H-2 region products is not affected by the putative heterogeneity in terms of receptor affinities. However, specificity with respect to unrelated H-2 haplotypes is strictly dependent on antigen concentration. A preferential binding of stimulator membrane vesicles occurs at limiting concentrations; whereas the majority of blasts bind stimulator and third- party vesicles equally well at high vesicle concentrations. The binding of both vesicle types is specific in that it can be inhibited with the relevant anti-H-2 sera. Furthermore, stimulator and third-party vesicles seem to compete for binding sites on the same cells, as shown by cold antigen inhibition. From these results, we propose that there is an imperfect distinction between stimulator and third-party H-2 antigens by the majority of primary MLC blasts. In contrast, highly selected long-term MLC blasts do not bind third-party H-2 antigens at any concentration, and seem to have high affinity for the stimulating antigens. We conclude that large numbers of clones with low-affinity (cross- reactive) receptors are generated in primary MLC, most of which become eliminated during long-term selection. This implies that the frequency of cells strictly specific for nonshared stimulating determinants must be minute.

Prevention of autoimmune diabetes in non-obese diabetic mice by treatment with a class II major histocompatibility complex-blocking peptide.

The role of antigen presentation as a possible mechanism underlying major histocompatibility complex (MHC) association of autoimmune disease has been studied in non-obese diabetic (NOD) mice. By screening for inhibition of antigen presentation to NOD T cell hybridoma, we have selected a synthetic peptide, yTYTVHAAHAYTYt (small letters denote D amino acids), that efficiently blocks antigen presentation by the NOD class II MHC molecule A alpha g7A beta g7 (Ag7) in vitro. The inhibition is MHC selective, in that it does not affect antigen presentation by the E(d) and E(k) molecules, and has only a marginal effect on presentation by the A(d) molecule. This peptide also inhibits the priming for Ag7-restricted T cell responses in vivo, and prevents the spontaneous development of diabetes in female NOD mice, when administered chronically from 3 wk of age on. Chronic treatment with a control peptide, KMKMVHAAHAKMKM, that fails to bind to Ag7 has no effect on the disease. These data indicate that antigen presentation by the Ag7 molecule plays a pivotal role in the induction of autoimmune diabetes. Furthermore, the results demonstrate that interference with antigen presentation by a class II molecule can prevent the onset of spontaneous autoimmune disease associated with the same molecule.

Mechanisms influencing the immunodominance of T cell determinants.

The preferential recognition of certain amino acid sequences from foreign protein antigens by T cells is referred to as T cell epitope immunodominance. To determine the mechanisms underlying this phenomenon, we have studied the correlation between the interaction of a series of synthetic peptides encompassing the entire hen egg-white lysozyme (HEL) sequence with class II molecules of the H-2k haplotype, and T cell responsiveness to these peptides. After HEL priming, three immunodominant T cell epitopes were found: two, included in the HEL sequences 51-61 and 112-129, were recognized in association with I-Ak molecules, and one, included in sequence 1-18, in association with I-Ek molecules. Accordingly, these peptides bound to the appropriate class II molecule, as demonstrated by competition for antigen presentation. Several other HEL peptides, although capable of associating with class II molecules, were not immunodominant. The absence of immunodominance has been shown to arise by three different mechanisms: (a) competition by an immunodominant peptide for presentation in vivo, (b) failure to generate the peptide during antigen processing, and (c) an inherently poor capacity of the T cell repertoire to respond to a particular peptide-MHC complex.

H-2-controlled suppression of T cell response to lactate dehydrogenase B. Characterization of the lactate dehydrogenase B suppressor pathway.

We characterized the cell types involved in the H-2-controlled suppression of T cell response to lactate dehydrogenase B (LDHB). The suppressor effector (Tse) was found to be an Lyt-1+2+, J+ cell that recognizes antigen together with Ek molecules of antigen-presenting cells (APC). To become functional, the Tse cell requires a second signal from a nonspecific, Lyt-1+2-, J+ suppressor-inducer (Tsi) cell. The Tsi-Tse interaction is not subject to any genetic restriction. The target cell of suppression is an Lyt-1+2-, J- (most likely T helper [Th]) cell that recognizes LDHB in the context of A molecules on APC. The suppression is manifested in inhibition of the antigen-specific, A-restricted proliferation of Th cells. The interaction between Tse and Th is restricted by the A region of the H-2 complex. Because this restriction is determined by the receptor of Th cells, the mechanism of Th-Tse interaction most likely involves a concomitant recognition of LDHB and A region-controlled molecules by Th cells on the surface of Tse cells.

Responder T cells depleted of alloreactive cells react to antigen presented on allogeneic macrophages from nonresponder strains.

T cells from strains responder to the antigen poly(Glu40 Ala60) (GA) were depleted of alloreactive cells by bromo-deoxyuridine and light treatment, and were subsequently primed in vitro in GA presented by allogeneic macrophages from nonresponder strains. Antigen-specific secondary proliferative responses restricted by allogeneic Ia molecules of the macrophages were obtained in all strain combinations tested. These data indicate that Ir gene-controlled nonresponsiveness cannot be the result of a failure of antigen presentation.

Graft-versus-host resistance induced by class II major histocompatibility complex-specific T cell clones.

Possible mechanisms of graft-vs.-host (GVH) resistance have been studied using a panel of seven class II major histocompatibility complex-specific T cell clones for elicitation and challenge. One clone recognized I-Ak,d,f, and expressed V beta 8.3 together with J beta 1.5. The remaining six clones were I-Ek specific and expressed V beta 15 rearranged to J beta 1.1 or J beta 1.3. The I-Ek-specific clones were also homologous to each other and different from the I-A-reactive one in the D and N regions. Four of the seven clones exhibited I-Ek-specific cytolytic activity. Each clone, when injected in sublethal numbers into appropriate recipients, could induce resistance to a subsequent lethal dose of any other clone in the panel. The resistance did not require sharing of either T cell receptor beta chains or antigen specificity, or MHC molecules by the eliciting and challenging clone. Cytolytic and noncytolytic clones were equally efficient in inducing GVH resistance. A prerequisite of resistance induction was the activation of eliciting clone subsequent to recognition of class II molecules in the host. Clones preactivated with high concentrations of recombinant interleukin 2, in vitro, could induce GVH resistance also in syngeneic hosts, suggesting that resistance induction was associated with the activated state of clone, rather than antigen recognition per se. In all instances of resistance, the challenging clones failed to induce vascular leakage, which was the cause of death in susceptible recipients (Lehmann, P. V., G. Schumm, D. Moon, U. Hurtenbach, F. Falcioni, S. Muller, and Z. A. Nagy. 1990. J. Exp. Med. 171:1485). Lipopolysaccharide (LPS) induced resistance to vascular leakage did not provide crossresistance to GVH and vice versa, suggesting that interleukin 1 alpha and tumor necrosis factor alpha implicated in LPS resistance are not involved in GVH resistance. Although the mechanism remains unclear, the most likely explanation for GVH resistance in this system is either the downregulation of permeability increasing effect in the challenging clone, or an induced refractoriness of blood vessels to this effect.

Flexibility of the T cell repertoire. Self tolerance causes a shift of T cell receptor gene usage in response to insulin.

Bovine insulin(BI)-specific I-Ab-restricted T cell clones have been characterized for fine specificity and TCR gene usage. We have demonstrated that mouse strains carrying H-2b on three different genetic backgrounds (C57BL, BALB, and 129) rearrange and express the V beta 6 gene in a large proportion (36%) of insulin-specific clones. In these strains, the non-MHC background did not seem to influence TCR gene usage in response to BI. The V beta 6+ clones appeared to be selected by the antigen. In contrast, no V beta 6+ clones could be isolated from (B6 x DBA/2)F1 mice, where V beta 6+ (and V beta 8.1+) T cells are deleted by self tolerance to Mls-1a. Thus, although a small proportion of residual V beta 6+ cells had been demonstrated in Mls-1a mice, these cells could not be retrieved in a response that uses V beta 6 predominantly. In functional terms, therefore, the deletion of V beta 6 by self tolerance appears to be complete. Instead of V beta 6, the majority (up to 60%) of I-Ab- as well as I-Ad-restricted insulin-specific clones from the (B6 x DBA/2)F1 mice expressed V beta 8.2 and V beta 8.3. This shift of gene usage was not accompanied by any detectable change in the fine specificity pattern of response. Thus, in the insulin-specific response, the flexibility of T cell repertoire fully compensates for deletions caused by self tolerance.

Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association.

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

Self tolerance to T cell receptor V beta sequences.

T cell tolerance to self is achieved by deletion or inactivation of clones recognizing peptides of self proteins presented by major histocompatibility complex molecules. A considerable fraction of self proteins accessible to the immune system is contributed by the system itself, for example, the receptors used for antigen recognition (antibodies and T cell receptors [TCRs]). Thus far, it has remained unclear, whether antigen receptors are subject to self tolerance, or on contrary, engage into network interactions implying immunity rather than tolerance. In this study, we demonstrate self tolerance to synthetic peptides corresponding to the first hypervariable region of the V beta 8.1 and V beta 8.2 TCR proteins. We also show that the tolerogenic synthetic peptide corresponds to a fragment produced by processing of the V beta protein, and conversely, that a V beta peptide not produced by processing is also not subject to self tolerance. Thus, the rules of tolerance seem to apply to antigen receptors, at least to their germline-encoded portions, in a similar fashion as to other self proteins. This finding has important implications for studies of natural and artificially induced immune networks.

Monoclonal suppressor factor specific for lactate dehydrogenase B. I. Mechanism of interaction between the factor and its target cells.

Hybridomas secreting a monoclonal T suppressor-effector factor (TseF) were produced by fusion of a lactate dehydrogenase B (LDHB)-specific long-term T suppressor-effector (Tse) cell line with the BW5147 thymoma. A short exposure (4 h) to TseF completely suppresses the antigen-specific and A-restricted proliferation of LDHB-primed Lyt-1+2- [possibly helper (Th)] cells. The action of TseF on Th cells, as that of the Tse cells themselves, is antigen-specific and A-restricted. The interaction of TseF with Th cells involves two binding events, of which one occurs via antigen bridge, and the other represents the recognition of a factor-derived Ak-like moiety by the anti-Ak receptor of Th cells. The Ak-like moiety of the TseF carries the determinants that serve as restriction elements for antigen recognition by Th cells, and additional determinants demonstrable by T cell-specific monoclonal "anti-Ak" antibodies, however, it lacks serologically detectable determinants of the B cell-derived A alpha A beta class II Mhc molecules.