Gene complementation in the T lymphocyte proliferative response to poly(Glu56Lys35Phe9)nFunctional evidence for a restriction element coded for by both the I-A and I-E subregions

The T lymphocyte proliferative response to poly(Glu⁵⁶Lys³⁵Phe⁹)_n (GLΦ) is under the control of two immune response genes, Ir-GLΦ-β and Ir-GLΦ-α, mapping in the I-A and I-E/C subregions of the major histocompatibility complex, respectively. Previous studies have demonstrated that in order to generate a response to GLΦ, both gene products must be expressed in the antigen-presenting cell (APC) but that neither responder allele has to be present in the responding T lymphocyte, provided that the T cell has matured in a responder environment. These results suggested that both gene products function as restricting elements in GLΦ presentation by APC. In this report, we provide further evidence for this model from experiments designed to test histocompatibility restrictions in antigen presentation at the I-E/C subregion. Genetic identity at the I-A subregion between T cells and APC was required for GLΦ presentation. To assess the requirements at I-E/C, B10. A(5R) T cells (I-A^b, I-E^k) primed to GLΦ were stimulated in vitro with GLΦ-pulsed spleen cells from F₁ hybrids between C57BL/10 (B10: I-A^b, I-E^b) which made the cells compatible at I-A, and a variety of B10 congenics bearing other H-2 haplotypes. Although none of the parental spleen cells could present GLΦ to B10.A(5R) T cells, spleen cells from F₁ hybrids between B10 and strains possessing H-2I of k, d, p and r presented GLΦ, whereas hybrids with strains possessing H-2I of f, q and s failed to present. This pattern of complementation for GLΦ presentation could not be explained on the basis of the responder status of the I-E/C donating parental haplotypes nor by invoking inhibitory stimuli from mixed lymphocyte reactions induced by the F_l APC. Rather, the pattern correlated with the presence of the serologic marker Ia.7 coded for by the I-E subregion of the complementing parental haplotype and the possession of an I-E-encoded a chain which has been shown by peptide mapping to be very similar in strains bearing the k, d, p and r haplotypes. These results suggest that the restriction element involved in the presentation of GLΦ to B10.A(5R) T cells is composed of a β chain encoded in I-A^b and an a chain encoded in I-E for which the allelic products of the k, d, p and r haplotypes are functionally equivalent. This correlation between structure and function represents the strongest evidence so far that Ia antigen-bearing molecules are the Ir gene products.     

The murine T-lymphocyte response to tyrosine-azobenzenearsonate. Characteristics of a low responder haplotype T-cell clone

An I-Ab-restricted, L3T4+ Ly2- T-cell clone, 5R-4F3, specific for ABAtyr was established in culture from a B10.A(5R) mouse. Since b haplotype mice respond weakly to ABAtyr compared to other haplotypes, this is a candidate clone of low responder phenotype. In support of this contention, 5R-4F3 grew very poorly under conditions that supported the vigorous growth of E beta bE alpha k-restricted T-cell clones from the same mouse. The I-A (low responder) and I-E (high responder) restricted T-cell clones also differed in their responses to apc pre-pulsed with antigen, compared to apc with antigen present continuously during culture. The low and high responder clones responded comparably to IL-2. Attempts to elevate the response of C57BL/6 mice to ABAtyr in vivo by injecting them with human recombinant IL-2 and antigen together were only partially successful: C57BL/6 mice treated in this way showed a 3-5-fold increase in their proliferative responses to ABAtyr, which was at best only one quarter of the level of response shown by high responder A/J mice to the same antigen dose.     

Epitope maps of the Escherichia coli heat-labile toxin B subunit for development of a synthetic oral vaccine.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum IgG anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When a 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-A(b), and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.     

Immunogenetic aspects of human thyroglobulin-reactive T cell lines and hybridomas

The in vitro proliferative response to T cells primed with human thyroglobulin (Tg) was compared in 11 independent haplotypes on B10 background. B10.K and B10.S mice were the most responsive, whereas, with the exception of B10.PL (H-2u), all other B10 congenics were intermediate responders. The two best responders to in vitro challenge with human Tg, of the k and s haplotype, are the same as those showing H-2-linked susceptibility to induction of experimental autoimmune thyroiditis (EAT) with mouse Tg. Since shared epitopes on human and mouse Tgs have been shown to be thyroiditogenic by adoptive transfer studies in CBA (H2k) mice, the findings indicate that shared epitopes may be studied in appropriate (i.e. EAT-susceptible) strains of mice. Therefore, we proceeded to develop methods to produce T-cell lines and hybridomas to human Tg in B10.K and B10.S mice, test their cross-reactivity to heterologous Tgs and their Ia restriction patterns. By using antigen-presenting cells from recombinant strains, we identified restriction elements encoded by the I-A subregion alone and a combinatorial molecule from the I-A/I-E subregions.     

Does staphylococcal enterotoxin B bind directly to murine T cells?

We have investigated the binding potential of staphylococcal enterotoxin B (SEB) to murine T cells using the induction of early activation events in Th1 and Th2 T-cell clones in the absence of antigen-presenting cells (APC) as indicators of direct interactions between SEB and the T-cell receptor (TcR). We consistently found that concanavalin A (Con A) induced rises in intracellular free calcium as well as inositol phosphate accumulation in APC-free T-cell clones. However, SEB uniformly failed to induce either calcium fluxes or inositol phosphate turnover in Th1 and Th2 T-cell clones in the absence of APC. In addition, we have used proliferation assays to show that (i) T-cell clones prepulsed with SEB did not respond when APC were added, (ii) APC-independent T-cell clones responded to soluble anti-TcR antibodies but not to SEB in the absence of APC, and (iii) SEB coupled to Sepharose beads did not stimulate T-cell clones in the absence of APC. Taken together our results argue against SEB binding to the TcR without the participation of MHC class II molecules.     

Adoptive transfer of islet antigen-autoreactive T cell clones to transgenic NOD.Ea(d)mice induces diabetes indicating a lack of I-E mediated protection against activated effector T cells

Transgenic insertion of the MHC class II Ea(d)gene in NOD mice restores I-E expression and prevents T-cell-mediated autoimmune diabetes (IDDM). The specific molecular and cellular mechanisms responsible for the diabetes resistance of transgenic NOD.Ea(d)mice remain unclear. We adoptively transferred islet antigen-specific T cell clones into NOD and transgenic NOD.Ea(d)mice to evaluate the level of protection provided by I-E expression against activated effector T cells. We have found that neither neonatal or 3-5-week-old I-E-expressing NOD.Ea(d)mice can completely inhibit the diabetogenic activities of activated islet antigen-specific T cell clones. These data indicate that Ealpha protein expression in NOD antigen presenting cells (APC) does not reduce islet autoantigen presentation in the context of I-A(g7)below the threshold required for stimulation of effector/memory diabetogenic T cells. Our results suggest that the mechanism of Ealpha protein-mediated diabetes resistance in NOD mice may be "antigen ignorance," in which the quantity of islet autoantigens presented in the context of I-A(g7)by APC is reduced below the threshold required to activate nai;ve islet antigen-specific T cells.     

I-E expression and susceptibility to parasite infection

In most inbred strains of mice, antigen-presenting cells express I-A and I-E antigens (class II major histocompatibility complex antigens), and these antigens are involved in antigen-recognition by T cells. In some strains I-E products are not expressed or aberrantly expressed, yet these mice seem to be immunologically normal. In this article, Don Wassom and his colleagues discuss reports that antigen presented in the context of I-E produces a response which suppresses I-A restricted T-cell proliferation, in relation to their own findings that mice which do express I-E molecules are more susceptible to certain nematode infections than mice which do not express I-E.     

I-E/I-C region-associated induction of murine gamma interferon by a haplotype-restricted polyclonal T-cell mitogen derived from Mycoplasma arthritidis

Cell-free supernatants from cultures of Mycoplasma arthritidis induced significant levels of interferon when cocultured with murine splenic cells. On the basis of physicochemical characteristics and antibody neutralization studies, the antiviral substance was identified as gamma interferon. Use of inbred and congenic mouse strains established that splenic cells from mice expressing the H2k and H2d haplotypes produced interferon in response to M. arthritidis culture supernatants, but those from mice with H2b and H2q haplotypes did not. Further studies with recombinant mouse strains established that interferon induction by the mycoplasma supernatant was associated with the haplotype expressed at the I-E/I-C subregion of the murine major histocompatibility complex. The specificity seen for interferon induction was identical with that reported earlier for induction of cytotoxic lymphocytes and for lymphocyte proliferation in response to the mitogen. All of these reactions appear to be dependent upon binding of the mitogen to specific I-E/I-C region-coded products present on splenic cell surfaces. The observations presented introduce the concept that microbial mitogens or their lymphokine products might modify immune responses and defense mechanisms of the naive host in a genetically restricted manner.     

B lymphocyte activation upon exclusive recognition of major histocompatibility antigens by T helper cells

This study has investigated whether exclusive recognition of I-A or I-E molecules on the B cell surface by T helper cells is sufficient to activate resting B cells. Lines and clones of long-term-cultured T helper cells with specificity for I-A or I-E antigens have been derived from mixed lymphocyte cultures between spleen cells from major histocompatibility complex (MHC)-congenic mouse strains. These cells were tested for helper activity and proved competent to induce resting B lymphocytes expressing the specific MHC antigens to polyclonal expansion and maturation to Ig secretion. B cell activation was shown to require direct recognition of I-A/E antigens by the helper cells on the responding B lymphocyte surface and it could not be achieved by soluble factors released by "third-party" helper cell activity ongoing in the same cultures. Since B lymphocyte activation occurs in the absence of antigen recognition by the responding B cells, these observations suggest that I-A and I-E molecules expressed on the B cell surface participate in the functional reception of T helper cell-derived induction signals.     

Differential pattern of T cell recognition of the 65-kDa mycobacterial antigen following immunization with the whole protein or peptides

The 65-kDa stress protein from Mycobacterium bovis (Bacillus Calmette Guérin) elicited T cell proliferation and antibody responses in seven B10 congenic mouse strains with different H-2 haplotypes. To analyze T cell determinants on this antigen, seven peptides corresponding to six predicted T cell epitopes, and one defined B cell epitope were synthesized. Mice were either immunized with the whole antigen and the specificity of the response was ascertained in respect of the six peptides, or mice were immunized with seven of the peptides and tested for proliferative responses to the whole molecule. The results showed that three peptides carried epitopes to which mice responded following injection of the whole molecule and that immunization with two additional peptides could prime for in vitro stimulation with the native antigen. The latter result indicates the feasibility of generating T cell responses to "cryptic" epitopes on proteins by immunizing with peptides. The peptide-specific T cell responses were distinctly influenced by the H-2 haplotype of mouse strains. However, two peptides were recognized by several H-2-disparate mouse strains, and one peptide could be presented by both I-A and I-E molecules. Immunization with several peptides induced a cross-reactive T cell proliferative response to the homologous GroEL protein isolated from E. coli. The amount of cross-reactivity was influenced by the extent of sequence homology between mycobacterial and E. coli proteins and the major histocompatibility complex class II molecule used to present the peptide.     

Ir gene control of the murine secretory IgA response to cholera toxin

In these experiments we examined the genetic control of the secretory IgA (sIgA) response to cholera toxin (CT) after CT feeding. Inbred, congenic and intra-H-2I region recombinant mouse strains were immunized with intragastric application of 10 micrograms CT on days 0 and 14. Samples of intestinal secretions and plasma were collected 1 week after the second dose and antibodies to CT measured in them by antigen- and isotype-specific enzyme-linked immunosorbent assay. In three different sets of H-2-congenic strains the intestinal IgA anti-CT response clearly depended on the H-2 haplotype rather than on background or IgH genes. H-2b (B10, A.BY/SnJ, C3H.SW) and H-2q (B10.T(6R), DBA/1J) strains were high responders, H-2k (B10.BR, C3H/He), H-2s (A.SW/SnJ) and H-2d (B10.D2) strains were low responders. Within the H-2 complex the intestinal IgA anti-CT response was mapped to the I-A subregion with the use of congenic intra-H-2I region recombinant strains: B10.A(3R) and B10.A(5R) were high responders and B10.A(4R), B10.MBR and B10.GD were low responders. Plasma IgG anti-CT after CT feeding paralleled the sIgA results. Surprisingly, the sIgA and plasma IgG anti-CT responses in individual mice of the various strains tested showed a highly significant positive correlation. We conclude that both the sIgA response and plasma IgG anti-CT response after CT feeding is controlled by the I-A subregion of H-2.     

Genetic control of immune responses in mice to synthetic peptides of a Streptococcus mutans surface protein antigen.

The immune responses to a cell surface protein antigen (PAc) of Streptococcus mutans and a peptide corresponding to residues 301 to 319 of the protein antigen [PAc(301-319)] in various strains of mice were studied, with attention being given to the haplotype of major histocompatibility complex (MHC) class II genes. Subcutaneous immunization of mice carrying the MHC class II I-Ad gene [BALB/c, B10.D2, B10.GD, and (B10.D2 x B10.G)F1 mice] with the peptide induced strong serum immunoglobulin G (IgG) responses to recombinant PAc (rPAc) and the peptide. Subcutaneous immunization of mice carrying the haplotype k or b of the H-2 I-A gene (C3H/HeN, C57BL/6, B10.BR, B10.A, or B10 mice) with the peptide induced intermediate serum IgG responses to rPAc and the peptide, and subcutaneous immunization of mice carrying the haplotype s or q of the H-2 I-A gene (DBA/1, B10.S, or B10.G mice) induced weak serum IgG responses to rPAc and the peptide compared with the responses of mice carrying the I-Ad gene. PAc(301-319) strongly induced PAc(301-319)-specific T-cell proliferation in B10.D2 mice but not in B10.G mice. The T-cell proliferation in B10.D2 mice was inhibited by treatment of antigen-presenting cells with anti-I-Ad monoclonal antibody but not with anti-I-Ab monoclonal antibody. These results indicate that the immune responses to the peptide in mice are genetically restricted or dominated by the MHC class II gene (I-Ad). To map antigenic epitopes in PAc(301-319) and PAc in mice bearing different H-2 haplotypes, 10 overlapping decapeptides covering PAc(301-319) and 153 decapeptides covering the entire mature PAc were synthesized. Of 10 decapeptides covering PAc(301-319), 6, 7, 1, and 1 decapeptides showed strong reactions with anti-PAc(301-319) sera from B10.D2 (H-2d), B10.GD (H-2g2), B10.BR (H-2k), and B10.A (H-2a) mice, respectively. None of these overlapping decapeptides reacted with anti-PAc(301-319) sera from B10.S (H-2s) and B10.G (H-2q) mice. Epitope-scanning analyses of the mature PAc molecule showed that antigenic epitopes scattered throughout the molecule and that antigenic epitope patterns differed in mice with different H-2 haplotypes. In addition, there was little overlap of immunogenic peptides among the mice with different haplotypes.     

Prevention of murine lupus by an I-E α chain transgene: Protective role of I-E α chain-derived peptides with a high affinity to I-Ab molecules

The expression of a transgene encoding the I-E α chain prevents a lupus-like autoimmune syndrome in BXSB mice. However, it had not been elucidated whether the Eα^d transgene-mediated protective effect results from I-E expression or from the generation of I-E α chain-derived peptides (Eα peptide) displaying high affinity for the I-A^b molecule. To address this question, two different BXSB lines expressing the transgene at low or high levels were crossed with lupus-prone MRL mice; this resulted in three types of (MRL × BXSB)F₁ mice, differing in the expression levels of I-E molecules and of Eα peptides presented by I-A^b molecules. Comparative analysis of these three (MRL × BXSB)F₁ mice as well as several BXSB transgenic lines showed that the Eα^d transgene-mediated protection paralleled the expression levels of Eα peptide presented by I-A^b molecules, but not of I-E molecules on B cells. In addition, use of transgenic and nontransgenic double bone marrow chimeras showed a selective activation of nontransgenic B cells during I-A^b-restricted T cell-dependent immune responses, while both transgenic and nontransgenic B cells were comparably activated during T cell-independent responses. These results favor a model of autoimmunity prevention based on competition for antigen presentation, in which excessive generation of Eα peptides prevents, because of their high affinity to the I-A molecules, activation of potential autoreactive T and B cells.     

H-2-linked control of the Yaa gene-induced acceleration of lupus-like autoimmune disease in BXSB mice.

The accelerated development of lupus-like autoimmune disease in male BXSB mice (H-2b, I-E-) is associated to the presence of a mutant gene, designated Yaa, located on their Y chromosome. To investigate whether the H-2b haplotype and/or the lack of expression of I-E molecules play any role in the Yaa-linked acceleration of autoimmune disease, an I-E+ BXSB.H-2d congenic strain was created by backcross procedures. We compared the development of autoimmune disease in the novel BXSB.H-2d (I-E+) strain to that of BXSB.H-2b (I-E-) and BXSB.H-2b/d (I-E+) heterozygous mice. Male BXSB.H-2d (I-E+) mice exhibited only a limited production of autoantibodies and a lower incidence of glomerulonephritis with a markedly prolonged survival rate, which were essentially identical to those of female BXSB mice of both-H-2b and H-2d haplotypes. However, BXSB.H-2b/d (I-E+) heterozygous males developed an accelerated disease comparable to that of conventional BXSB.H-2b (I-E-) male mice. These results indicate that the expression of I-E molecules and consequent clonal deletion or anergy of I-E reactive T cells does not appear to be responsible for the prevention of accelerated autoimmune disease in BXSB.H-2d (I-E+) male mice. The finding that the Yaa gene-induced acceleration of lupus-like autoimmune disease is modulated by gene(s) within or closely linked to the H-2 complex underlines the crucial role of the major histocompatibility complex and the polygenetic nature of autoimmune disease in BXSB mice.     

Vβ11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vβ11⁺ T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality. Interestingly, Vβ11⁺ T-cell mediated joint pathology varies in different mouse strains. In this study, we characterized the in vitro pattern of Vβ11⁺ T-cell expansion by TSST-1 in mice with various genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class II I-E molecules did not expand Vβ11⁺ T cells upon stimulation with TSST-1. Using B10 congeneic I-E negative mouse strains, we found that the TSST-1-expanded Vβ11⁺ T cells in B10Q (H-2^q) and B10M (H-2^f) mice but not in B10B (H-2^b) mice. Antigen-presenting cells (APC) from B10Q mice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vβ11⁺ T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-A^b APC were able to stimulate Vβ11⁺ T cells from H-2^q mice. Furthermore, Vβ11⁺ T cells in H-2^b mice did expand when exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cell repertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for the different magnitude of Vβ11⁺ T-cell expansion among the different mouse strains.     

Role of the first external domain of I-A beta chain in immune responses and diabetes in non-obese diabetic (NOD) mice.

Diabetes in the non-obese diabetic (NOD) mouse is a multigenic autoimmune disease and is possibly controlled by three recessive loci, including one that is linked to the major histocompatibility complex (MHC). The first external domain of the Class II MHC I-A beta chain in these mice is unique and has been suggested as being responsible for autoimmunity. The I-A alpha chain in these mice is I-A alpha d, and they lack the expression of I-E molecules. We have investigated immune responses to various Ir gene control antigens in NOD mice to determine the influence of the NOD Ia and particularly the I-A beta chain. We find that sheep insulin is highly immunogenic while other insulins are weakly immunogenic in these mice. Hen egg lysozyme, pigeon cytochrome C and the synthetic polypeptide Poly 18, Poly EYK(EYA)5 antigen produce good antibody responses. Apart from H-2d, NOD are the only mice where Poly 18 antigen is immunogenic. In these mice Poly 18 induced good T-cell proliferative response, which was inhibited by anti-Ia antibody, and the mice were able to respond to tyrosine-containing polypeptide Poly EYA but not to the phenylalanine-containing antigen Poly EFA. We also found that synthetic peptide 48-60 of the NOD I-A beta chain is highly immunogenic in syngeneic NOD mice both for T cells and B cells. Using an I-A beta chain-specific monoclonal antibody, we are able to prevent induction of diabetes when the antibody was administrated in prediabetic, young mice. Our results suggest that the immune response to various antigens and autoimmune diabetes in NOD mice is directly influenced by the I-A beta chain.     

Class-II HLA Restriction of Antigen-Specific Human T-Lymphocyte Clones

Blast-enriched suspensions of T cells primed for Chlamydia trachomatis antigen were cloned by a limiting dilution technique. The class-II HLA restriction of T-lymphocyte clones (TLC) was studied by using allogeneic antigen-presenting cells (APC) carrying foreign class-II HLA antigens. Most of the TLC were restricted by one or the other of the D/DR determinants of the T-cell donor; that is, they did not respond when antigen was presented by APC expressing foreign D/DR determinants. Furthermore, heterogeneity of the DR4-expressing molecule could be demonstrated by T-cell clones from one person; APC from family members expressing DR4 gave high proliferative responses, whereas no proliferation was observed with most APC from unrelated persons expressing DR4. This heterogeneity of DR4 was confirmed by mixed lymphocyte culture (MLC) experiments, indicating a close relationship between restriction epitopes and those that activate allogeneic T cells. Other clones seemed to be restricted by other class-II HLA determinants, most probably MT determinants of the T-cell donor. The restriction specificities were confirmed by subcloning experiments.     

Control by H-2 genes of the Th1 response induced against a foreign antigen expressed by attenuated Salmonella typhimurium.

Attenuated salmonellae represent an attractive vehicle for the delivery of heterologous protective antigens to the immune system. Here, we have investigated the influence of the genetic background of the host which regulates the growth and elimination of Salmonella cells on the cellular response induced against a foreign antigen delivered by an aroA Salmonella strain. We have tested CD4+ T-cell responses (cell proliferation and cytokine production) in various mouse strains following immunization with Salmonella typhimurium SL3261 expressing a high level of the recombinant Escherichia coli MalE protein. We were able to detect a CD4+ T-cell response against the recombinant MalE protein only in a restricted number of mouse strains, whereas all mice produced good levels of anti-MalE immunoglobulin G antibodies. The Ity gene did not play a major role in these differences in T-cell responses, since both Ity-resistant and -susceptible strains of mice were found to be unresponsive to MalE delivered by recombinant salmonellae. In contrast, when B10 congenic mice were used, a correlation was established between MalE-specific T-cell unresponsiveness and H-2 genes. The discrepancies described in this paper in the ability of various strains of mice to develop an efficient Th1 response against a recombinant antigen displayed by a live Salmonella vaccine underscore the difficulties that can be encountered in the vaccination of human populations by such a strategy.     

Effects of major histocompatibility genes and antigen delivery on induction of protective mucosal immunity to E. acervulina following immunization with a recombinant merozoite antigen.

Intramuscular immunization with the recombinant p250 surface antigen of Eimeria acervulina merozoite (rEAMZp250) or oral inoculation with live recombinant Escherichia coli expressing the rEAMZp250 protein resulted in antigen-specific T-cell and humoral responses and conferred a significant reduction in mucosal parasitism compared to immunization with the negative control antigen preparation. Among the major histocompatibility complex (MHC) (B)-congenic chickens receiving intramuscular immunization, strain .6-2 (B2-B2) showed significant (P less than 0.05) protection to live E. acervulina challenge compared to the other strains examined. In contrast, strains .C-12(B12B12) and .P-13 (B13B13) showed significant protection among the groups given live recombinant E. coli. In general, strains showing enhanced T-cell responses to the rEAMZp250 protein were better protected compared to those showing minimal or no T-cell responses. Thus the results suggest that the B haplotypes of the host and the mode of antigen presentation influence the outcome of protection following an immunization of chickens with recombinant coccidial antigen.