Lyme disease is a multisystem disease caused by the spirochete Borrelia burgdorferi [1, 2]. Although most patients respond to two to four weeks of antibiotic therapy, a few individuals continue to have clinical manifestations after treatment. These patients do not respond to additional courses of antibiotics raising the possibility that a post infectious immunologic process is responsible for these persistent clinical manifestations.

T helper 1 responses are associated with antibiotic refractory Lyme disease [3, 4]. Outer surface protein A (OspA) from B. burgdorferi sensu stricto contains a helper T cell epitope with a similar sequence to a segment in human leukocyte function-associated antigen-1 (hLFA1). Gross et al. found that individuals with treatment resistant Lyme arthritis, but not other forms of arthritis, generated Th1-IFNγ responses to OspA, hLFA1 and their highly related peptide epitopes in synovial fluid. These authors proposed that cross-reactivity between an OspA epitope and hLFA1, allows OspA primed T cells to remain activated by continued stimulation through cells expressing hLFA1, even after elimination of the spirochetes by antibiotic therapy, initiating an autoimmune response to hLFA1 in the joint [5, 6]. Thus, the ongoing clinical manifestations in refractory Lyme disease may not depend on persistent infection, but to the immune response to a cross reactive epitope in the etiological agent [7].

Since an autoimmune cross reactivity between B. burgdorferi OspA, human LFA1 and antibiotic resistant Lyme arthritis has been suggested [6] and it is known that only a subpopulation of Lyme arthritis patients are resistant to treatment we wanted to further dissect the involvement of OspA as well a possible reactivity to the hLFA1 peptide in a population of early Lyme disease patients using healthy individuals as controls. We used borrelia whole cell sonicate and the two peptides first identified by Gross et al. [6], hLFAα_L (aa326-345) and OspAB31 (aa164-183) to measure antigen-specific T cell reactivity in peripheral blood of two groups, early Lyme disease patients without history of arthritis and healthy individuals. Th1-IFNγ production in response to the peptide hLFAα_L (aa326-345) was more frequent than Th1 reactivity to the OspAB31 (aa164-183) peptide in peripheral blood of Lyme disease patients presenting with erythema migrans as well as in a subpopulation of healthy individuals.

Samples were obtained from nineteen (19) adult patients and twenty-one (21) healthy adult individuals from endemic areas. The production of IFNγ in peripheral blood stimulated with borrelia whole cell sonicate antigen (WCS), hLFAα_L peptide (aa326-345) or OspAB31 (aa164-183) peptide was evaluated. Sixteen of the nineteen patients presented with early localized infection typified by the presence of well defined erythema migrans (EM). They had no other signs of disseminated infection and did not develop Lyme arthritis. Seven patients were evaluated within 1 month of developing EM (acute) and nine other patients were evaluated 3 months post EM presentation (convalescent). The additional three patients tested were OspA vaccine failures who presented with acute Lyme disease. These samples were not screened for anti-OspA antibodies. The 21 healthy individuals samples were used as controls.

The optimal concentration of antigen used in the assay varies for each specific antigen. The concentration of the WCS (2.5 μg/ml or 2.5 μg/~10⁶ Th cells), was previously determined at CSL Biosciences by dose response curve as the optimal concentration of antigen to be used in this T cell assay [8]. We used the peptide OspAB31 (aa164-183) at 10 μg/ml (or 10 μg/~10⁶ Th cells). This was the same concentration of OspA peptide used in Gross et al. for 3 × 10⁵ Th cells [6]. The hLFA1 peptide was used at 2.5 μg/ml (or 2.5 μg/~10⁶ Th cells), the same concentration determined for the WCS. All antigens at the proposed concentrations were tested in blood from known healthy individuals to determine the background level stimulation.

Observing the three vaccinated patients that came down with Lyme disease, we verified they were all responsive (100%) to the Borrelia WCS, 2/3 were positive for hLFA peptide (67%) and none (0%) was positive for the OspA peptide. This last result was surprising since these patients had been previously vaccinated with OspA. However, this could be explained by the obvious vaccine failure which is supported by the fact that all three patients reacted with borrelia whole cell sonicate.

Our results show that the Th1 proinflamatory cytokine IFNγ is produced in peripheral blood of patients with early Lyme disease without any signs of arthritis when stimulated with the peptide hLFAα_L(aa326-345) but not when stimulated with the homologous OspAB31 (aa164-183) peptide. This observation was also verified for healthy individuals.

It has been postulated that hLFA1 autoreactive T cells play a major role in the pathogenesis of chronic Lyme arthritis [6]. However, the hLFAα_L (aa326-345) peptide was not recognized by any of the OspA (aa164-175)-specific T cell hybridomas generated from a B. burgdorferi OspA immunized DR4 transgenic mouse, illustrating that individual T-cell receptor (TCR) possess different patterns of cross-reactivity [12]. In addition, analysis at the clonal level demonstrated that hLFAα_L (aa326-345) behaves as a partial agonist for OspA-reactive T cells, not as a full agonist [13].

Antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism. This demonstrates that flexibility in T cell recognition does not preclude specificity. If recognition were not degenerate, the immune system would be incompetent since the limited number of T-cells in an organism would allow recognition of small fraction of a mixture of randomized peptides [14]. There is ample evidence suggesting that cross-reactivity leading to T cell activation is a very frequent event [15–17, 14, 18, 12]. In fact, the presence of cross-reactive T cells is normal and required for the maintenance of memory T cell responses. T cell reactivity between a microbial peptide and a self-peptide alone is not sufficient to induce autoimmune disease [12].

TCR recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities. Itoh et al., analyzed TCR engagement and the production of multiple cytokines in a clonal Th1 CD4+ cell population. They verified that low concentrations of TCR ligand elicit only IFNγ production. Their findings provide strong support for a model of T cell activation in which different effector functions have a hierarchical arrangement of elicitation thresholds, such that the ratio of elicited effector molecules changes with antigen concentration [19]. Rogers et al., used a model of differentiation that is dependent on the initial dose and affinity of peptide presented to a naïve CD4 cell. They used this system to confirm that LFA 1 interaction can suppress differentiation of cells secreting Th2 cytokines and that this interaction is only seen over a range of peptide doses [20].

We detected a Th1 response to hLFAα_L (aa326-345) in peripheral blood of patients with Lyme disease without any signs of arthritis and in a subpopulation of healthy individuals. We observed that the reactivity to hLFA1 was significant and was dissociated from the response to OspA. The hLFA1 peptide was used at the same concentration determined for the WCS and four times less the concentration used for the OspA peptide. We anticipated that differences in data obtained for a concentration of 25 μg/ml for the hLFA antigen compared to 10 ug/ml of the OspA antigen as in Gross et al. [6] could cause skepticism. For that reason, and to minimize the amount of variables in the study, we chose a semi-empirical route.

Taking into account all previous findings, the greater response to the hLFA peptide as compared to the OspA may be explained by the different doses of hLFA peptide and OspA used. An additional, and in our opinion better explanation, is that reactivity to the hLFA1 peptide in peripheral blood is in large part the result of T cell degeneracy. This could explain the number of healthy individuals who reacted to the hLFA1 peptide. An alternative to this model could be that some individuals may have been infected with a previous pathogen that triggers activation of T cells reactive to the hLFA1 peptide. In any case, this does not rule out the possibility that primed hLFAα_L reactive Th1 cells homing to a specific milieu such as the joint could potentially be involved in the pathogenic mechanisms associated with chronic Lyme arthritis. Additional studies are required to define the role of T cell reactivity in the immunopathogenesis of antibiotic refractory Lyme disease.

We detected a significant Th1 response to the peptide hLFAα_L(aa326-345) in peripheral blood of patients with early Lyme disease without any signs of arthritis and in a subpopulation of healthy individuals. Our results suggest that reactivity to hLFA1 peptide in peripheral blood may be the result of T cell degeneracy.